The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology (Frontiers in Neuroscience)

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THE DYNAMIC SYNAPSE MOLECULAR METHODS IN IONOTROPIC RECEPTOR BI0LOGY

© 2006 by Taylor & Francis Group, LLC

FRONTIERS IN NEUROSCIENCE Series Editors Sidney A. Simon, Ph.D. Miguel A.L. Nicolelis, M.D., Ph.D.

Published Titles Apoptosis in Neurobiology Yusuf A. Hannun, M.D., Professor of Biomedical Research and Chairman/Department of Biochemistry and Molecular Biology, Medical University of South Carolina Rose-Mary Boustany, M.D., tenured Associate Professor of Pediatrics and Neurobiology, Duke University Medical Center Methods for Neural Ensemble Recordings Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering, Duke University Medical Center Methods of Behavioral Analysis in Neuroscience Jerry J. Buccafusco, Ph.D., Alzheimer’s Research Center, Professor of Pharmacology and Toxicology, Professor of Psychiatry and Health Behavior, Medical College of Georgia Neural Prostheses for Restoration of Sensory and Motor Function John K. Chapin, Ph.D., Professor of Physiology and Pharmacology, State University of New York Health Science Center Karen A. Moxon, Ph.D., Assistant Professor/School of Biomedical Engineering, Science, and Health Systems, Drexel University Computational Neuroscience: Realistic Modeling for Experimentalists Eric DeSchutter, M.D., Ph.D., Professor/Department of Medicine, University of Antwerp Methods in Pain Research Lawrence Kruger, Ph.D., Professor of Neurobiology (Emeritus), UCLA School of Medicine and Brain Research Institute Motor Neurobiology of the Spinal Cord Timothy C. Cope, Ph.D., Professor of Physiology, Emory University School of Medicine Nicotinic Receptors in the Nervous System Edward D. Levin, Ph.D., Associate Professor/Department of Psychiatry and Pharmacology and Molecular Cancer Biology and Department of Psychiatry and Behavioral Sciences, Duke University School of Medicine Methods in Genomic Neuroscience Helmin R. Chin, Ph.D., Genetics Research Branch, NIMH, NIH Steven O. Moldin, Ph.D, Genetics Research Branch, NIMH, NIH Methods in Chemosensory Research Sidney A. Simon, Ph.D., Professor of Neurobiology, Biomedical Engineering, and Anesthesiology, Duke University Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering, Duke University The Somatosensory System: Deciphering the Brain’s Own Body Image Randall J. Nelson, Ph.D., Professor of Anatomy and Neurobiology, University of Tennessee Health Sciences Center

© 2006 by Taylor & Francis Group, LLC

The Superior Colliculus: New Approaches for Studying Sensorimotor Integration William C. Hall, Ph.D., Department of Neuroscience, Duke University Adonis Moschovakis, Ph.D., Institute of Applied and Computational Mathematics, Crete New Concepts in Cerebral Ischemia Rick C. S. Lin, Ph.D., Professor of Anatomy, University of Mississippi Medical Center DNA Arrays: Technologies and Experimental Strategies Elena Grigorenko, Ph.D., Technology Development Group, Millennium Pharmaceuticals Methods for Alcohol-Related Neuroscience Research Yuan Liu, Ph.D., National Institute of Neurological Disorders and Stroke, National Institutes of Health David M. Lovinger, Ph.D., Laboratory of Integrative Neuroscience, NIAAA In Vivo Optical Imaging of Brain Function Ron Frostig, Ph.D., Associate Professor/Department of Psychobiology, University of California, Irvine Primate Audition: Behavior and Neurobiology Asif A. Ghazanfar, Ph.D., Primate Cognitive Neuroscience Lab, Harvard University Methods in Drug Abuse Research: Cellular and Circuit Level Analyses Dr. Barry D. Waterhouse, Ph.D., MCP-Hahnemann University Functional and Neural Mechanisms of Interval Timing Warren H. Meck, Ph.D., Professor of Psychology, Duke University Biomedical Imaging in Experimental Neuroscience Nick Van Bruggen, Ph.D., Department of Neuroscience Genentech, Inc., South San Francisco Timothy P.L. Roberts, Ph.D., Associate Professor, University of Toronto The Primate Visual System John H. Kaas, Department of Psychology, Vanderbilt University Christine Collins, Department of Psychology, Vanderbilt University Neurosteroid Effects in the Central Nervous System Sheryl S. Smith, Ph.D., Department of Physiology, SUNY Health Science Center Modern Neurosurgery: Clinical Translation of Neuroscience Advances Dennis A. Turner, Department of Surgery, Division of Neurosurgery, Duke University Medical Center Sleep: Circuits and Functions Pierre-Hervé Luoou, Université Claude Bernard Lyon I, Lyon, France Methods in Insect Sensory Neuroscience Thomas A. Christensen, Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, AZ Motor Cortex in Voluntary Movements Alexa Riehle, INCM-CNRS, Marseille, France Eilon Vaadia, The Hebrew University, Jeruselum, Israel Neural Plasticity in Adult Somatic Sensory-Motor Systems Ford F. Ebner, Vanderbilit University, Nashville, TN Advances in Vagal Afferent Neurobiology Bradley J. Undem, Johns Hopkins Asthma Center, Baltimore, MD Daniel Weinreich, University of Maryland, Baltimore, MD The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology Josef T. Kittler, University College London Stephen J. Moss, University of Pennsylvania

© 2006 by Taylor & Francis Group, LLC

THE DYNAMIC SYNAPSE MOLECULAR METHODS IN IONOTROPIC RECEPTOR BI0LOGY Edited by

Josef T. Kittler University College London

Stephen J. Moss University of Pennsylvania

Boca Raton London New York

CRC is an imprint of the Taylor & Francis Group, an informa business

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Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2006 by Taylor & F rancis Group, LLC CRC Press is an imprint of Taylor & F rancis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-1891-2 (Hardcover) International Standard Book Number-13: 978-0-8493-1891-7 (Hardcover) Library of Congress Card Number 2005025723 This b ook co ntains in formation o btained f rom aut hentic a nd h ighly r egarded sources. Re printed ma terial i s quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been mad e t o pu blish r eliable d ata an d in formation, but t he au thor an d th e pu blisher cannot as sume responsibility for the validity of all materials or for the consequences of their use. No pa rt of this boo k ma y be reprinted, r eproduced, tr ansmitted, or utilized in any fo rm by an y el ectronic, mechanical, or other means, no w known or he reafter in vented, in cluding ph otocopying, mi crofilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, I nc. (CCC) 222 R osewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a v ariety o f users. F or organizations th at ha ve b een gr anted a p hotocopy license b y t he C CC, a se parate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data The dynamic synapse : molecular methods in ionotropic receptor biology / [edited by] Josef T. Kittler and Stephen J. Moss. p. ; cm. -- (Frontiers in neuroscience) Includes bibliographical references and index. ISBN 0-8493-1891-2 1. Synapses. 2. Neuroplasticity. 3. Neural transmission. 4. Neurotransmitters. [DNLM: 1. Synapses--physiology. 2. Carrier Proteins--physiology. 3. Neuronal Plasticity--physiology. 4. receptors, Amino Acid--physiology. 5. Synaptic Transmission--physiology. WL 102.8 D997 2006] I. Kittler, Josef T. II. Moss, Stephen J., 1962- III. Title. IV. Series: Frontiers in neuroscience (Boca Raton, Fla.) QP364.D96 2006 612.8--dc22

2005025723

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group is the Academic Division of Informa plc.

© 2006 by Taylor & Francis Group, LLC

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Preface Nerve cells in the brain communicate with each other at synapses, specialized sites of cell-cell contact formed between a pre-synaptic nerve terminal and a post-synaptic neuron. The properties of these synaptic connections determines, in part, how information in the brain is processed and changes in the strength of these connections (synaptic plasticity) is belie ved to be the molecular basis of information storage in the brain. At synapses, information is passed in the form of neurotransmitters that are released from the pre-synaptic terminal and dif fuse across the synaptic cleft to activate post-synaptic neurotransmitter g ated ion channels (also called ionotropic receptors). Activation of ionotropic receptors elicits the electroph ysiological responses essential for f ast synaptic transmission and changes in the acti vity and distribution of these receptors play a critical role in re gulating the strength of synapses. In this v olume in the CRC Press Methods and Ne w Frontiers in Neur oscience series, we focus on tools and technologies to study the acti vity and functional regulation of these important receptors and introduce the application of cutting-edge approaches to the study of synapse and receptor biology . The recognition that synapses can be studied in man y w ays and using a lar ge number of tools and approaches, from molecular biology and protein biochemistry to imaging, electrophysiology and in vivo studies, stimulated the production of this book, which aims to provide a resource covering many of the methods that have become most relevant to those interested in studying the synapse. The first t o chapters provide broad overviews of the excitatory and inhibitory synapse with Chapter 1 describing the v arious methods that ha ve been important for establishing the critical role of AMPA receptor traf ficking in the plasticity o excitatory synapses, and Chapter 2 focusing on the plasticity of inhibitory synapses. Chapter 3, Chapter 4 and Chapter 5 focus on approaches for studying the biochemical properties of the receptors and other proteins of the post-synaptic domain. Chapter 3 describes recent advances in the use of proteomics approaches for studying the complement of proteins in the post-synaptic density , whereas Chapters 4 and 5 focus on tools for studying post-translational modification o synaptic proteins by phosphorylation and palmito ylation, respectively. In Chapter 6 through Chapter 9, various state-of-the-art methods for studying the membrane trafficking and sur ace localization of receptors are described, including biochemical (Chapter 6), optical (Chapter 6, Chapter 7 and Chapter 8) and electrophysiological (Chapter 9) approaches. Chapter 10 through Chapter 14 cover established and newly emerging approaches for interfering with protein acti vity inside cells from neuronal transfection and transduction to the genetic manipulation of neurons by homologous recombination of ES cells. Chapter 10 focuses on the use of RNAi technologies for knocking do wn e xpression le vels of tar get proteins of interest,

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whereas Chapter 11 through Chapter 13 focus on v arious approaches for transfecting neural tissue, from the introduction of cDN As into primary neuronal cultures to transfecting neurons in brain slices and nerve cells in vivo. In particular, Chapter 12 and Chapter 13 describe ele gant advances in the use of viral v ectors to transfect neurons in vivo . In addition, Chapter 13 and Chapter 14 focus on methods for achieving long-term alterations in the genetic complement of neurons either using the emer ging po wer of lenti viral v ector systems for the ef fective delivery and expression of cDNAs and shRNAis to neurons in vivo (Chapter 13), or by homologous recombination and “knockin” approaches (Chapter 14). Finally, the last chapter explores the applicability of the “post-genomic” resources that are now increasingly available to biologist and neuroscientists for the study of receptor and synapse biology . Man y of the approaches described here are rele vant well beyond studies of neurotransmitter receptors and are thus applicable to studies of other important molecular and cellular components of the nerv ous system. We therefore hope that this book will be useful to both ne wcomers and e xperienced synaptic physiologists, in addition to the wider neuroscience community . We are particularly grateful to the participating authors for their hard w ork and excellent chapter contrib utions. We are also v ery grateful to Lorena ArancibiaCarcamo, whose help with the formatting of chapters pro ved invaluable. We also thank Pavel Osten, Jeremy Henley, Helene Marie, Sabine Levi and Lorena ArancibiaCarcamo for their contrib utions to the front co ver art. We also thank the staf f of CRC Press, in particular Barbara Norwitz, Robert Sims and Jill Jur gensen, for their help, patience and encouragement. Josef T. Kittler, Ph.D. London Stephen J. Moss, Ph.D . Philadelphia

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The Editors Josef T. Kittler, Ph.D., is a MRC fellow and Principal Investigator in the Department of Physiology, University College London. Dr. Kittler received a B.Sc. in Biochemistry from the Uni versity of Bath, U.K., and carried out predoctoral research in the Department of Ph ysiology and Pharmacology , Wake F orest Uni versity, WinstonSalem, North Carolina, U.S.A. Dr . Kittler carried out doctoral studies in Molecular Neuroscience in the MRC Laboratory for Molecular Cell Biology and postdoctoral studies in the MRC Laboratory for Molecular Cell Biology and in the Departments of Physiology and Pharmacology, University College London, U.K. Dr . Kittler has published over 30 papers and book chapters in the field of molecular and cellula neuroscience with a major focus on neuronal cell biology and the re gulation of inhibitory synapses by membrane traf ficking and recepto -associated proteins. Stephen J. Moss, Ph.D., is Professor of Neuroscience in the Department of Neuroscience at the Uni versity of Pennsylv ania. Dr . Moss recei ved a B.Sc. in Biochemistry from the University of Bath, U.K., and carried out his doctoral studies in Neurobiology at the Medical Research Council (MRC) Laboratory for Molecular Biology, Cambridge, U.K. Dr. Moss carried out postdoctoral studies on ion channel phosphorylation at the Ho ward Hughes Medical Institute, Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Following his postdoctoral w ork, Dr. Moss w as appointed as a lecturer and group leader in the Department of Pharmacology and MRC Laboratory for Molecular Cell Biology, University College London, U.K. In 2000, Dr . Moss w as appointed Professor of Molecular Pharmacology and Cell Biology in the Pharmacology Department at University College London. In 2003 Dr. Moss moved to the Department of Neuroscience at the Uni versity of Pennsylv ania. Dr. Moss has published o ver 100 publications on the re gulation of neurotransmitter receptor acti vity and traf ficking In addition, he has presented man y invited lectures and has been in volved in the organization of several symposia and international meetings in the areas of synapse function and ion channel re gulation.

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Contributors I. Lorena Arancibia-Cárcamo Department of Pharmacology University College London London, U.K. Yehezkel Ben-Ari Institut de Neurobiologie de la Méditerranée (INMED) Institut National de la Santé et de la Recherche Médicale (INSERM) Marseille, France David S. Br edt Department of Ph ysiology University of California at San Francisco San Francisco, CA, U.S.A. Tanjew Dittgen Department of Molecular Neurobiology Max Planck Institute for Medical Research Heidelberg, Germany Benjamin P. Fairfax John Radcliffe Hospital Headley Way, Headington Oxford, U.K. Brian D. Fernholz Department of Biochemistry NYU School of Medicine New York, NY, U.S.A.

Masaki Fukata Laboratory of Genomics and Proteomics National Institute for Longe vity Sciences Obu, Aichi, Japan Yuko Fukata Laboratory of Genomics and Proteomics National Institute for Longe vity Sciences Obu, Aichi, Japan Jean-Luc Gaiarsa Institut de Neurobiologie de la Méditerranée (INMED) Institut National de la Santé et de la Recherche Médicale (INSERM) Marseille, France Jonathan G. Hanley MRC Centre for Synaptic Plasticity Department of Anatomy School of Medical Sciences University of Bristol Bristol, U.K. Jeremy M. Henley MRC Centre for Synaptic Plasticity Department of Anatomy School of Medical Sciences University of Bristol Bristol, U.K.

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John T. R. Isaac Developmental Synaptic Plasticity Unit, NINDS Porter Neuroscience Research Center Bethesda, MD, U.S.A. Tija C. J acob Department of Pharmacology University College London London, U.K. Bryen A. Jordan Department of Biochemistry NYU School of Medicine New York, NY, U.S.A. Jasmina N. J ovanovic Department of Pharmacology School of Pharmac y University of London London, U.K. Josef T. Kittler Department of Ph ysiology University College London London, U.K. Hey-Kyoung Lee Department of Biology Neuroscience and Cognitive Sciences (NACS) Program University of Maryland College Park, MD, U.S.A. Sabine Lévi INSERM U.497 Ecole Normale Supérieure Paris, France Pawel Licznerski Department of Molecular Neurobiology Max Planck Institute for Medical Research Heidelberg, Germany

Robert C. Malenka Nancy Pritzker Laboratory Department of Psychiatry and Behavioral Sciences Stanford University School of Medicine Palo Alto, CA, U.S.A. Hélène Marie European Brain Research Institute Fondazioni Rita Levi-Montalcini Rome, Italy Stephen J. Moss Department of Neuroscience School of Medicine University of Pennsylvania Philadelphia, PA, U.S.A. Thomas A. Neubert Department of Biochemistry NYU School of Medicine New York, NY, U.S.A. Peter L. Oli ver MRC Functional Genetics Unit Department of Human Anatomy & Genetics University of Oxford Oxford, U.K. Pavel Osten Department of Molecular Neurobiology Max Planck Institute for Medical Research Heidelberg, Germany Pavel V. Perestenko MRC Anatomical Neuropharmacology Unit Oxford, U.K. Sophie Restituito Department of Biochemistry NYU School of Medicine New York, NY, U.S.A.

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Trevor G. Smart Department of Pharmacology University College London London, U.K.

Antoine Triller INSERM U.497 Ecole Normale Supérieure Paris, France

Philip Thomas Department of Pharmacology University College London London, U.K.

Edward B. Ziff Department of Biochemistry NYU School of Medicine New York, NY, U.S.A.

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Table of Contents Chapter 1 Methods for Unco vering the Mechanisms of AMPA Receptor Traffickin .............1 Sophie Restituito and Edwar d B. Zif f Chapter 2 Long-Term Plasticity at Inhibitory Synapses: A Phenomenon That Has Been Ov erlooked..............................................................................................23 Jean-Luc Gaiarsa and Yehezkel Ben-Ari Chapter 3 New Tricks for an Old Dog: Proteomics of the PSD .............................................37 Bryen A. Jordan, Brian D. F ernholz, Thomas A. Neubert, and Edward B. Zif f Chapter 4 Phosphorylation Site-Specific Antibodies as Research Tools in Studies of Native GABAA Receptors ......................................................................57 Jasmina N. Jovanovic Chapter 5 Protein Palmitoylation by DHHC Protein F amily ..................................................83 Yuko Fukata, David S. Br edt, and Masaki Fukata Chapter 6 Studying the Localization, Surf ace Stability and Endoc ytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation Approaches ........................................................................................91 I. Lorena Arancibia-Cárcamo, Benjamin P. Fairfax, Stephen J. Moss, and J osef T. Kittler Chapter 7 Visualization of AMPAR Trafficking and Sur ace Expression.............................119 Pavel V. Perestenko and Jeremy M. Henley Chapter 8 Neurotransmitter Dynamics ...................................................................................143 Sabine Lévi and Antoine Triller

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Chapter 9 Receptor Dynamics at the Cell Surf ace Studied Using Functional Tagging .......155 Philip Thomas and Trevor G. Smart Chapter 10 RNAi and Applications in Neurobiology ..............................................................177 Tija C. Jacob Chapter 11 Transfecting and Transducing Neurons with Synthetic Nucleic Acids and Biologically Active Macromolecules ....................................................................205 Josef T. Kittler, Jonathan G. Hanley, and John T. R. Isaac Chapter 12 Acute In Vivo Expression of Recombinant Proteins in Rat Brain Using Sindbis Virus ...............................................................................................241 Hélène Marie and Robert C. Malenka Chapter 13 Lentivirus-Based Genetic Manipulations in Neurons In Vivo ..............................249 Pavel Osten, Tanjew Dittgen, and Pawel Licznerski Chapter 14 AMPA Receptor Phosphorylation in Synaptic Plasticity: Insights from Knockin Mice ................................................................................ 261 Hey-Kyoung Lee Chapter 15 Genomic and Post-Genomic Tools for Studying Synapse Biology .....................279 Josef T. Kittler and P eter L. Oliver

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1

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking Sophie Restituito and Edward B. Ziff

CONTENTS 1.1 1.2

Introduction ......................................................................................................1 Approaches Used in the Study of AMPA Receptor Traffickin .....................3 1.2.1 Protein Interaction Assays ...................................................................3 1.2.2 Cell Biology .........................................................................................7 1.2.2.1 Microscopy ...........................................................................7 1.2.2.2 Cell Biology Assays ...........................................................11 1.2.3 Biochemistry ......................................................................................12 1.2.3.1 Biochemical Assays ............................................................12 1.2.3.2 Phosphorylation ..................................................................12 1.2.4 Electrophysiology...............................................................................13 1.2.4.1 Blocking Peptide.................................................................13 1.2.4.2 Rectification Ind x..............................................................13 1.2.4.3 Agonists and Toxins ...........................................................14 1.2.5 Pharmacology.....................................................................................15 1.2.6 Genetic Approach...............................................................................15 1.2.6.1 Murine Models.................................................................... 15 1.2.6.2 Knockout Mice ...................................................................15 1.2.6.3 C. Elegans ...........................................................................16 1.3 Synthesis, Summary and Speculation ............................................................16 References................................................................................................................17

1.1 INTRODUCTION Information storage in the brain involves alterations in the strength of communication between neurons. This requires activity-dependent, long-lasting changes in synaptic transmission. Long-term potentiation (LTP) is a long lasting use-dependent increase 1

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2

The Dynamic Synapse

in the ef ficien y of e xcitatory synaptic transmission that has been suggested to underlie certain forms of learning and memory [1]. The induction of L TP requires Ca2+ entry through the N-meth yl-D-aspartate receptor (NMD AR). Ho wever, the region within the synapse whose regulation results in LTP is still controversial. Some groups suggest a pre-synaptic modification that results in an increase in the amoun of glutamate released, whereas others suggest a post-synaptic modification, such a an increase in the number of receptors or a change in receptor properties [2]. Interestingly, the description of the silent synapse, synapses that contain NMD AR only b ut could acquire α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) as a result of synaptic acti vity, support a post-synaptic mechanism [3–5]. Glutamate receptors mediate most excitatory synaptic transmission in the brain. The ionotropic glutamate receptors are clustered with associated do wnstream signalling molecules that are found in the post-synaptic density (PSD), and the synaptic clustering of these receptors is critical for rapid and ef ficient synaptic transmission This comple x of receptors with signalling molecules can also under go dynamic changes. In particular , changing the number of glutamate receptors at the synaptic membrane could constitute a critical mechanism for rapidly altering synaptic strength. Two major classes of ionotropic glutamate receptors exist, the NMDAR and the AMPAR. A third class, the kainate receptors, will not be discussed here as it has been reviewed elsewhere [6]. Both NMD AR and AMPAR are highly concentrated at e xcitatory synapses link ed to the PSD b ut the y interact with dif ferent sets of scaffolding proteins. In addition, whereas NMD AR are v ery stably localized at the PSD, AMPAR cycle rapidly to and from the synaptic membrane. This difference in trafficking beh vior of NMD AR versus AMPAR may reflect their use of di ferent mechanisms for anchoring to the PSD. The conversion of a silent synapse to a synapse with AMPAR, a change that creates a functional synapse, has been proposed as one of the main mechanisms for LTP induction. F or these reasons, studies of the traf ficking of AMPAR and their cycling in and out of the synapse ha ve provided a large step in understanding L TP. Synaptic organization, assembly and traf ficking h ve been studied e xtensively for other receptors, such as the acetylcholine receptor at the neuromuscular junction [7]. AMPAR trafficking has been one of the most xtensively studied properties of the excitatory synapse because of its implication in synaptic plasticity . The complexity of the or ganization of the brain mak es assaying receptor properties without disrupting the system dif ficult. A complete picture of synaptic re gulation comes from combining a wide range of methodological approaches, such as electrophysiology, cell biology, biochemistry and genetics. The development of new methods or the adaptation of methods used in other systems has allo wed a better understanding of the traf ficking of AMPAR. Mechanisms of AMPAR traf fickin have been reviewed elsewhere [3,8,9], and this chapter will present an o verview of the different approaches that have been used to study the trafficking of AMPAR and its implication for synaptic plasticity , focusing on the contrib ution and the impor tance of each method.

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Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking

3

1.2 APPROACHES USED IN THE STUDY OF AMPA RECEPTOR TRAFFICKING AMPARs are hetero-oligomeric proteins composed of subunits GluR1–4 [10]. Each receptor complex contains four sub units [11]. In an adult hippocampus, tw o heteromeric receptor comple xes predominate: GluR1/2 and GluR2/3 [12]. All subunits have an extracellular domain, three transmembrane domains, a membrane re-entrant hairpin that forms the pore and a c ytoplasmic C-terminal tail. The tail can either be short or long. GluR1, GluR4 and one of the splice v ariants of GluR2 ha ve long, highly homologous cytoplasmic tails containing around 80 amino acids. In contrast, the predominant forms of GluR2 and GluR3 and one GluR4 splice v ariant ha ve short, homologous c ytoplasmic tails of around 50 amino acids [8]. Through their cytoplasmic tails, each sub unit interacts with specific ytoplasmic proteins. Man y of these interacting proteins ha ve single or multiple PDZ domains, which mediate protein-protein interactions with the e xtreme C-termini of the tar get proteins. Two classes of PDZ domains have been defined based on their binding specificities. Cla I PDZ domains ha ve a histidine at the αB1 position, b ut in class II, this residue is hydrophobic [13]. GluR1 has a class I PDZ lig and whereas GluR2, GluR3 and GluR4c have class II PDZ lig ands. Channels containing exclusively GluR1, GluR3 and GluR4 are Ca 2+ permeable, whereas inclusion of GluR2 makes a channel Ca2+ impermeable. This specific chang in permeability imparted by GluR2 is the result of a post-transcriptional modificatio introduced by RN A editing of the GluR2 mRN A residues at the Q/R site in the channel pore. Glutamine Q607 is replaced by an arginine in 99% of post-natal GluR2 [14]. Interestingly , this property of GluR2 dominates the permeability of the AMPAR. Indeed, its presence in a comple x confers Ca 2+ impermeability to the AMPAR [15]. The existence of different AMPAR heterotetramers will then generate channels with different electrophysiologic and trafficking properties

1.2.1 PROTEIN INTERACTION ASSAYS The use of protein interaction assays, such as the yeast tw o-hybrid screen, the coimmunoprecipitation assay and the pull do wn assay , has allo wed a great breakthrough in the characterization of partners ofAMPAR involved in receptor anchoring, trafficking and egulation ( Table 1.1). Indeed, these approaches h ave all owed the characterization of protein comple xes involving multiple partners, highlighting the complex regulation of AMPAR trafficking. The yeast two-hybrid screen is a potent technique to identify protein interactors in vivo [16,17]. This well-known technique consists of fusing the protein of interest to a DNA-binding domain and e xpressing it in a yeast host cell carrying a reporter gene under the control of the transcriptional acti vity of the DN A-binding domain. The fusion protein cannot acti vate transcription on its o wn, b ut it can be used as “bait” to screen a library of cDN A clones encoding proteins that are fused to an activation domain. The cDNA clones within the library that encode a fusion protein capable of interaction with the “bait” are identified by their ability to act vate expression of the reporter gene.

Two-Hybrid Screen

Co IP/ Biochemistry

Interaction with GluR2 [20,21,23] Interaction with liprin- and LAR [31]

Interaction with GluR2 [20,21,23] Interaction regulated by phosphorylation [53,54]

PICK1

Interaction with GluR2 [24] Interaction with GluR2 [25–27]

Interaction with GluR2 [19,24] Interaction with GluR2, SNAP [27] and PICK1 [19] Interaction with GluR1 [18] Interaction with GluR1 [30] Interaction with GluR1 [29] Interaction with AMPAR [65]

NSF

SAP97 RIL 4.1N STG

Interaction with GluR1 [30] Interaction with GluR1 [29]

© 2006 by Taylor & Francis Group, LLC

Mutation of interacting motif [28]

Microscopy Subcellular localization and colocalization with AMPAR by confocal microscopy [20,23] Subcellular localization and EM [21,22] Localization, EM of GRIP [32] Colocalization with liprin [31] Regulation of GluR2 surf ace accumulation [28] Subcellular localization and colocalization with GluR2 [24] Colocalization with GluR2 [27]

Physiology

Murine Models

Regulation of GluR2 trafficking [58

Regulation of GluR2 trafficking [26,55–57

Colocalization with AMPAR [29] Subcellular localization and colocalization with GluR4 [65]

Regulation of AMPAR trafficking [65

Cloning [65]

The Dynamic Synapse

ABP/GRIP

Mutagenesis

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4

TABLE 1.1 Summary of the Various Approaches Used to Characterize Proteins Interacting with AMPARs

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5

This interaction is usually confirmed by biochemical methods, such as co immunoprecipitation. This standard technique is based on the formation of a comple x between an antibody specific to a protein of interest, the protein of interest and al the proteins that interact with it. The complex is then isolated using beads to which the antibody is link ed [18]. A pull-do wn assay can also be used to confirm a interaction between two proteins. This method is an in vitro method very similar to co-immunoprecipitation, e xcept that a tagged bait protein is used instead of an antibody [19]. The major proteins interacting with the C-terminal domains of AMPAR subunits were identified using a yeast t o-hybrid screen. Thus, Dong et al. identified a n vel protein, GRIP (glutamate receptor -interacting protein), interacting with the C-ter minal tails of GluR2 and GluR3 [20] (Table 1.1). GRIP contains seven PDZ domains, is enriched in the brain and co-localizes with AMPAR at e xcitatory synapses. The interaction is between PDZ4 and 5 of GRIP and the last seven amino acids of GluR2 and GluR3, b ut not GluR1. Subsequently , Srivastava et al., using the same assay , identified another partner of GluR2, ABP (AMPA receptor-binding protein, Table 1.1) [21], which is closely related to GRIP . ABP has splice v ariants with either six or seven PDZ domains and also interacts with the C-terminal tails of GluR2 and GluR3 but not GluR4, GluR1 or NMDA-NR2A [21,22]. The interaction is between the C-terminal motif, VKI of GluR2 and PDZ5 of ABP. ABP and GRIP e xhibit 64 to 93% amino acid homology in the six PDZ domains.They have a similar structural organization, including tw o clusters of three PDZ domains, with each cluster followed by linker regions. These studies suggest that GRIP andABP could be involved in the ta rgeting of AMPAR to the synapse ( Figure 1.1). A palmit oylated form of ABP, pABP, w as also identified ( able 1.1) [23]. This splice v ariant is present in spines, whereas non-palmitoylated ABP and GRIP are found in the dendritic shaft. The authors propose that pABP andABP provide alternative anchorages for AMPAR at the synapse or intracellular membranes during receptor trafficking (Figure 1.1[3b and Figure 1.1[8b]). Xia et al. identified a n vel protein that also interacts with the C-terminal tail of GluR2, PICK1 (protein interacting with C kinase, Table 1.1) [24]. Through its PDZ domain, PICK1 interacts with the last seven amino acids of GluR2, GluR3 and GluR4, b ut not with GluR1. PICK1 is thought to function in the trafficking phase of AMPAR transport (Figure 1.1[3b] and Figure 1.1[5b]). The yeast two-hybrid approach has also identified r gulatory proteins associated with AMPARs. Several groups showed that NSF (N-ethylmaleimide sensitive fusion protein) interacts with the C-terminal tail of GluR2 and interacts weakly with GluR3 but not with GluR1 or GluR4 (Table 1.1) [25–27]. NSF is a homomeric ATPase and a component of the protein machinery responsible for v arious membrane fusion events. It interacts with α and β SNAP and disassembles the SN ARE comple x, driven by ATP h ydrolysis [19,28]. Osten et al. and Hanle y et al. suggest that the NSF/SNAP comple x could serv e as a chaperone in dissociating PICK1-AMP AR complexes involved in AMPAR cycling (Figure 1.1[3b] and Figure 1.1[6b]) [19,28]. Several proteins ha ve thus been sho wn to interact with the C-terminal domain of GluR2. To dissect the function and the implication of each protein in this complex, Osten et al. used mutagenesis of the C-terminal tail of GluR2 (T able 1.1) [28]. By removing the binding site for ABP/GRIP, Osten et al. demonstrated that the

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4b

5b

4a 3a

3b 8b 2a

6b

7b

ER golgi

9b 1

2b

GluR1/2

actin

RIL

4.1

10b

GluR2/3

SAP97

activity dependent

PKC PICK1 NSF SNAP

constitutive

GluR1/2+GluR2/3 ABP/GRIP

PSD95

NMDAR

Stg

NMDAR synaptic activity

FIGURE 1.1 (Color figure fol ows page 176.) Schematic representation of AMPAR traffick ing. Pathway (a) represents GluR1 traf ficking and path ay (b) represents GluR2 traf ficking AMPAR complexes exit from the ER/Golgi associated with star gazin (1). GluR1-containing AMPAR traf fic through the secretory path ay (2a) and are inserted at e xtrasynaptic sites through an acti vity-dependent mechanism (3a). Star gazin binds PSD95, which localizes the complex at the synapse where the receptor can bind other scaf folding proteins such as RIL and 4.1N (4a). Stargazin can then be released. GluR2-containing AMPAR traffic through th secretory pathway (2b) and are inserted at synaptic sites through a constituti ve mechanism (3b). PICK1 f acilitates the transport of the receptor possibly both to and from the plasma membrane but is remo ved from the AMPAR by the NSF/SN AP when the receptor reaches the plasma membrane. NSF/SNAP/ PICK1 forms a transient complex with GluR2-containing AMPAR (not shown on this schematic).AMPAR are stabilized at the membrane by scaffolding proteins. Phosphorylation by PKC pre vents interaction between ABP/GRIP and GluR2 (4b) and favors the PICK1/GluR2 interaction (5b). The AMPAR are then internalized follo wing a coupling with PICK1, and AMPAR/PICK1 complex are destabilized by NSF/SNAP when the receptor reaches its destination (6b). GluR2-containing AMPAR can then interact with an intracellular pool of ABP/GRIP, possibly in the endosome (7b), and get rec ycled to the membrane through interaction with PICK1 (8b) or tar geted to lysosomes (9b) and de graded (10b).

plasma-membrane tar geting of the receptor w as not dependent on PDZ-mediated binding but that the PDZ binding motif w as required for surf ace accumulation of GluR2-containing receptor at the synapse. They proposed that the binding of a PDZ protein, ABP or GRIP, but not PICK1, mediated accumulation of the AMPAR at the synapse by regulating the receptor’s local endocytic synaptic turnover (Figure 1.1).

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In a tw o-hybrid screen using the C-terminal domain of GluR1 as bait, Shen et al. sho wed an interaction between the membrane proximal re gion of C-terminal domains of GluR1 and protein 4.1N, a protein critical for the or ganization and maintenance of the spectrin-actin cytoskeleton (Table 1.1) [29]. The authors suggest that protein 4.1N might play a role in the anchoring of AMPAR to the actin c ytoskeleton and stabilizing the sur face expression of the receptors ( Figure 1.1). However, Schulz et al. found that the C-terminal tail of GluR1 is highly toxic to yeast [30]. To circumvent this condition, the y used the C-terminal tail of GluR2 fused to the last 10 amino acids of GluR1 as a bait in a two-hybrid screen and found an interaction between GluR1 and RIL (re version-induced LIM protein, Table 1.1) [30]. RIL belongs to a f amily of proteins that has been proposed to function as adaptors linking proteins to the actin c ytoskeleton. RIL can bind α-actinin via a PDZ domain and GluR1 by a LIM domain (Figure 1.1). Co-immunoprecipitation or pull-down assays from transfected cells expressing the proteins of interest or from brain lysate extracts were used to further characterize all of these interactions (Table 1.1) [21–24,29,30]. Leonard et al. chose a co-immunoprecipitation approach using various extraction conditions to identify partners of the GluR1 sub unit (Table 1.1) [18]. They characterized an interaction between SAP97, a member of the SAP f amily (synapseassociated proteins), and GluR1. The SAP97 f amily of proteins clusters NMD ARs at the postsynaptic membrane, and SAP97 could play a similar role for the AMPAR (Figure 1.1). These assays have also allowed the identification of la ger complexes involving multiple proteins. Using a tw o-hybrid screen, Wyszynski et al. identified a n vel partner for GRIP , liprin- α (Table 1.1) [31]. Liprin had first been identified by i interaction with a receptor tyrosine phosphatase, LAR. Liprin- α interacts through its C-terminal domain, with the PDZ6 of GRIP . The authors were able to coimmunoprecipitate a comple x containing GRIP, liprin- α, GluR2/3 and LAR. Their data implicate liprin and LAR in the surf ace expression or the synaptic tar geting of AMPAR. Similarly , Hanle y et al. were able to sho w, using pull-do wn and coimmunoprecipitation approaches, that GluR2, NSF, PICK1 and SNAP form a complex (Table 1.1) [19]. They showed that the NSF/GluR2 interaction stabilizes the AMPAR at the surf ace by NSF’ s ability to disrupt the PICK1/GluR2 comple x, preventing PICK1 dependent-endoc ytosis of the AMPAR (Figure 1.1[3b], Figure 1.1[8b], Figure 1.1[5b] and Figure 1.1[6b]).Together, these approaches have allowed the identification of la ge protein comple xes involved in the synaptic localisation, stabilization or re gulation of AMPAR at the synapse.

1.2.2 CELL BIOLOGY 1.2.2.1 Microscopy While biochemical assays allo wed the identification of components of AMPAR associated complexes, a further critical step w as to study the function of these proteins in more physiological systems. The use of classical techniques such as light and electron microscopy allowed the visualization of the proteins in tissues or culture.

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Electron microscopy, a classical technique that uses a beam of highly ener getic electrons to examine objects on a very fine scale, all ws the localization of a protein at an ultra-structural le vel. Using immunoperoxidase electron microscop y, Dong et al. showed that GRIP1 is present in the dendrite and dendritic spine, close to the PSD, b ut is also found in the endoplasmic reticulum (ER) and Golgi ( Table 1.1) [22]. These various localizations suggest a function of this protein at different points in the trafficking path ay and could implicate this protein in theAMPAR trafficking The results were confirmed using immunogold electron microsco y, which allo ws a higher resolution (T able 1.1) [21,22]. Similarly , Wyszynski et al. sho wed a preand post-synaptic localization of liprin- α and GRIP, raising potential new functions for these tw o proteins (T able 1.1) [31,32]. Using post embedding immunogold electron microscopy, Takumi et al. performed a very precise study of the relationship between AMPAR and NM DAR density at specific synapses Table 1.2) [33]. They were able to detect synapses without an y AMPAR staining, consistent with the existence of silent, AMPAR-deficient synapses. They also sho wed that the number of AMPAR in a synapse is proportional to the PSD area, whereas NMD AR number increases v ery little with synapse size. They sho wed a v ery lar ge v ariability in AMPAR number and heterogeneity in synapse size, with which it can be correlated and suggest that the ratio between AMPAR and NMDAR depends critically on PSD diameter. Thus, electron microscop y studies ha ve allowed the visualization of the ultra-structure of the PSD. They also gi ve an idea of the precise localizations of receptors at an ultra-structural le vel. Electron microscop y can be used for more comple x studies. Kiro v et al. took advantage of this technique to study changes in the number of spines during synaptic activity using hippocampal slices in culture. Using serial electron microscop y analysis, the y sho wed that a blockade of acti vity increases spine number [34]. They suggest that neurons can re gulate spine number depending on the le vel of activity. Confocal microscopy, a well-known technique that uses spatial filtering to elim inate out-of-focus light, has been widely used for a better characterization of protein subcellular localization, for the co-localization of se veral proteins and for definin possible functions of these proteins. F or example, by confocal microscop y, Xia et al. showed a colocalization of PICK1, GluR2 and protein kinase C (PKC) in hippocampal neurons in culture (T able 1.1) [24]. The use of recombinant tagged receptors and the development of overexpression techniques that may be emplo yed together with confocal microscop y studies ha ve allowed a more complex investigation of the AMPAR trafficking in neuronal models Specially, tagging with green fluorescent protein (GFP) has pr vided a major insight into the dynamics of AMPAR trafficking in l ving cells. Shi et al. o verexpressed Nterminally GFP-tagged GluR1 sub units in hippocampal neurons in culture or in organotypic slices using a Sindbis virus system and e xamined the beha vior of the subunit after tetanic stimulation (T able 1.2) [35]. The change in distrib ution w as monitored by time-lapse, tw o-photon laser scanning microscop y. GluR1-GFP w as mainly intracellular in the dendritic shaft. Interestingly , redistribution to the spine was observed after tetanic stimulation. However, GFP does not distinguish surf ace from intracellular AMPAR. To circumvent this problem, Passafaro et al. have used a thrombin surf ace cleavage assay

Biochemistry

GluR1

Activity-dependant insertion [35,36] Local synthesis [38]

Phosphorylation [52]

GluR2

Constitutive insertion at synapse [36] Trafficking of GluR2 in live neuron [37] Local synthesis [38] Movement of single receptor [40–42] EM relationship AMPAR and NMDAR [33]

Trafficking of GluR2 through secretory pathway [48,49] Phosphorylation [53,54]

AMPARs

AMPAR movement in/out surface [45–47]

Physiology/ Peptide Block/Drug

Physiology/ Rectification

Regulation of GluR1 trafficking by phosphorylation [52] Regulation of GluR2 trafficking by NSF [26,55–57] Regulation of GluR2 trafficking by ABP/GRIP [58]

Activitydependent insertion [59,60] Constitutive insertion at synapse [60,61]

Pharmacology

Animal Models Function of GluR1 [67]

Incorporation of GluR2 at synapse [61]

Function of GluR2 [68–71]

Trafficking of AMPAR [64]

Function of GluR2 and GluR3 [72] Function of GluR [73]

9

© 2006 by Taylor & Francis Group, LLC

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TABLE 1.2 Summary of the Various Approaches Used to Study Trafficking of AMPARs

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to study AMPAR surface delivery (Table 1.2) [36]. AMPAR subunits tagged at the N-terminal domain with a hemagglutinin (HA) tag followed by a thrombin cleavage site were transfected into hippocampal neurons and the localization of the sub units was assessed by confocal microscopy. They were able to show that GluR1 and GluR2 have distinctive roles in AMPAR surface accumulation and synaptic plasticity ( Figure 1.1[2a] and Figure 1.1[2b]). HA-GluR1 has a much slower time course of surface reinsertion than HA-GluR2. Activity promotes GluR1 reinsertion b ut not GluR2 (Figure 1.1[3a] and Figure 1.1[3b]). Ho wever, GluR1 acts dominantly o ver GluR2, and GluR1 properties will predominate when it is in a comple x with GluR2. Using a synaptic marker, the authors were able to sho w that most HA-GluR1 accumulates during the first f w minutes follo wing reinsertion in a non-synaptic area of neuron surface, whereas ne wly inserted HA-GluR2 accumulates directly at the synapse. They concluded that GluR2 e xocytosis is rapid and constituti ve and GluR1 is slo w and inducible. They also concluded that GluR1 determines the rate and site of exocytosis and GluR2 controls the recycling and endocytosis. For the first time, thi approach has allowed the study of reinsertion of AMPAR; however, it presents some limitations in temporal resolution. Ashby et al. used a modified GFP to assess the m vement of AMPAR at the cell surface (Table 1.2) [37]. The pH-sensitive GFP (ecliptic pHluorin) is nonfluo rescent at pH less than 6.0 and its brightness increases up to pH 8.5. Placed on the N-terminal domain of GluR2, the protein will be fluorescent at the cell sur ace and not inside the cell, where the en vironment is mainly acidic. Using this mutant GFP , surface GluR2 was then assessed by live cell confocal microscopy. The authors were able to sho w that synaptic and e xtrasynaptic AMPAR act dif ferently follo wing NMDAR acti vation. They could sho w a rapid internalization of e xtrasynaptic AMPAR that preceded the internalization of synaptic AMPAR. Recently, Ju et al. took adv antage of properties of no vel dyes to study the local synthesis of AMPAR (Table 1.2) [38]. Local synthesis has been broadly studied and reviewed else where [39]. The biarsenical dyes bind to short peptide sequences containing four c ysteine residues. They are nonfluorescent until th y bind to the tetracysteine motif and then become green (FlAsH-EDT 2) or red (ReAsH-EDT 2). The authors introduced the tetrac ysteine motif in the C-terminal tails of GluR1 and GluR2, HA/thrombin N-terminally tagged (GluR1c y4 and GluR2c y4). Using this technique, they could compare the dendritic traf ficking of pre- xisting and recently synthesized AMPAR subunits. They were able to sho w that a chronic blockade of activity, which enhances synaptic strength, causes an increase in the amount of both pre-existing and recently synthesized GluR1c y4 but not GluR2cy4. However, highK+ depolarization that w ould mimic L TP increased both recently synthesized GluR1cy4 and GluR2cy4. They concluded that AMPAR subunit mRNAs are targeted to proximal and distal dendrites where the sub units can be locally translated, processed and inserted at or near synaptic sites. This novel approach could be used to study other aspects of AMPAR trafficking Borgdorff et al. de veloped a v ery original approach based on confocal microscopy to study the mo vement of single endogenous receptors at the cell surf ace (Table 1.2) [40]. They used 0.5 µm late x beads coated with antibodies ag ainst the N-terminal domain of GluR2 to study the lateral mobility of nati ve GluR2-containing

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AMPAR [40]. Each bead was held in contact with the surface of a neurite with laser tweezers for 5 to 10 sec to allo w binding to GluR2. The bead’ s trajectory w as followed by video microscop y for 200 sec with a spatial resolution of 5 to 10 nm. They found that the GluR2-containingAMPAR exists in two states, one characterized by a rapid dif fusion movement and the other a confined, sl w moving state. They showed that surrounding the synapse w as a region of low receptor diffusion. Basal neuronal activity or Ca2+ regulate the movements of AMPAR. However, using caged Ca2+, which allo ws a local increase of Ca 2+, they could sho w that local Ca 2+ influ triggers lateral GluR2 mo vement. The same group adapted this technique to study AMPAR movement within the synaptic membrane ( Table 1.2) [41,42]. They used Cy3-coupled antibodies or quantum dot coupled antibodies to label the receptor and to follow its mo vement inside the synapse. Quantum dots are fluorescent semicon ductor nanocrystals that are encapsulated in phospholipids, allo wing their use as probes in biological imaging [43,44]. The authors were able to sho w that AMPAR are mobile between synaptic and extrasynaptic locations (Figure 1.1) [41]. Changes in neuronal activity modified AMPAR but not NMDAR localization [42]. Thus, complementary approaches have been employed to understand the movement of AMPAR at the synapse. All suggest a v ery high mobility of the receptor at the surf ace, even if the receptor is part of a lar ge complex of proteins. They also showed that this mo vement seems to be dependent on the sub unit composition of the heterotetramer and that the insertion and exit of the receptor from the cell surface takes place at specific sites on the plasma membrane 1.2.2.2 Cell Biology Assays Other approaches, combining microscop y and biochemistry , ha ve been used to understand the mechanisms that re gulate the mo vement of AMPAR in and out of the synapse (Table 1.2) [45–47]. Ehlers used an antibody-feeding assay to study this aspect of trafficking [45]. Hippocampal neurons in culture were stained, while lving, with an antibody ag ainst GluR1 or GluR2 and internalized receptors were detected after acidic treatment that remo ves all the receptors remaining at the cell surf ace. This technique allowed the authors to determine the rate of endocytosis. Co-staining with specific mar ers of the endoc ytosis and de gradation pathways allowed Ehlers to identify the pathways [45]. Ehlers and Lin et al. used also a biotinylation approach to complement the antibody-feeding approach [45,46]. Biotin is a small molecule that can be conjug ated to a protein of interest. It can then interact with strepta vidin and the comple x can be isolated and detected. This approach is more quantitati ve and more sensitive than using immunofluorescent staining. Using these approaches several distinct pathw ays for AMPAR endoc ytosis ha ve been described. AMPAR can be endoc ytosed via NMD A-dependent or independent pathw ays [45,47] or an insulin-dependent pathway [46,47]. AMPAR endocytosed through a NMDA-dependent pathw ay go to the rec ycling endosome and are reinserted into the plasma membrane (Figure 1.1[7b] and Figure 1.1[8b]). This pathway involved Ca 2+ flu es and protein dephosphorylation [45,47], whereas AMPAR endoc ytosed through AMPA-dependent internalization are de graded [45] (Figure 1.1, Figure 1.1[9b] and Figure 1.1[10b]).

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1.2.3 BIOCHEMISTRY 1.2.3.1 Biochemical Assays Greger et al. chose a biochemical approach to study the traf ficking of AMPAR through the secretory path way (Table 1.2) [48,49]. Taking advantage of subcellular and sucrose gradient fractionations, the y identified t o forms of GluR2, one that is mature and present in the synaptic fraction and a second that is immature and enriched in the ER [49]. Using de glycosylation methods and pulse chase e xperiments, they were able to sho w that the majority of GluR2 is immature, retained in the ER and not associated with GluR1 ( Figure 1.1). By mutagenesis, they identifie several regions in the C-terminal tail of GluR2 in volved in the ER retention. Most significantl , they also sho wed that R607, at the Q/R editing site, also controls ER retention [49]. Using h ydrodynamic methods and nati ve P AGE gels that mak e possible the molecular weight characterization of comple xes, the y sho wed that tetramerisation but not dimerization is controlled by the re-entrant pore loop containing the Q/R editing site [48]. They proposed that the edited R-sub units remain mainly dimeric and ER-retained unless comple xed with unedited Q sub units, whereas the unedited Q-subunits can tetramerize and traffic to the cell surace (Figure 1.1). Thus, the Q/R site affects subunit stoichiometry and, in turn, channel function. This editing site affects the functional properties ofAMPAR at two levels: one during assembly, by restricting the number of GluR2 incorporated into tetramers, and the other during ion conduction by controlling major AMPAR transmission properties, in particular Ca2+ permeability [48]. Thus, biochemical and cell biologic approaches have allowed a dissection at a molecular le vel of the traf ficking of the AMPAR. 1.2.3.2 Phosphorylation Phosphorylation is one of the major mechanisms of re gulation of AMPAR function. Its importance has also been highlighted in L TP and long-term depression (L TD) by se veral groups [3,8,50,51]. A classic approach to study phosphorylation is to label the cells with 32P orthophosphate and immunoprecipitate the protein of interest, as done by Roche et al., to identify the phosphorylation sites of the GluR1 sub unit [52]. Two-dimensional phosphopeptide mapping after trypsin digestion and treatment with drugs to stimulate the various kinases are also commonly used to identify possible phosphorylation sites of a protein [52]. Potential phosphorylation sites can then be confirmed by mutagenesis [52]. Using these approaches, Roche et al. shwed that GluR1 w as phosphorylated at S845 by protein kinase A (PKA) and S831 by PKC [52]. They showed a potentiation of the peak amplitude of whole cell glutamategated current after stimulation by purified PKA. This effect disappeared after mutation of S845. Using a phosphorylation assay , Matsuda et al. sho wed that phosphorylation of S880 of the GluR2 sub unit by PKC reduced the af finity of GluR2 fo GRIP, and thus could be important in controlling the stabilization of the receptor at the synapse [53]. Hayashi et al. chose a different approach to study the phosphylation of the GluR2 sub unit [54]. They generated a phospho-specific antibody a ainst a sequence surrounding T876, which is very similar to the tar geting sequence for Src family of tyrosine kinases, and sho wed that GluR2 w as tyrosine phosphorylated.

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They also showed that the Lyn, Src and Fyn kinases could phosphorylate the GluR2 C-terminal tail. Tyrosine phosphorylation of GluR2 could regulate AMPAR traffick ing to the cell surf ace. This study suggests that GluR2 is phosphorylated on T876 in vivo and in vitro by the Src f amily of tyrosine kinases. The authors suggest that the ef fect on GluR2 traf ficking ould be through a destabilization of the GluR2/GRIP interaction.

1.2.4 ELECTROPHYSIOLOGY 1.2.4.1 Blocking Peptide Another approach to understand the mechanism of AMPAR traf ficking and it physiological implication w as to block the interaction of a sub unit with its protein partners in vivo in hippocampal slices to e xamine the ef fects on basal synaptic transmission and synaptic plasticity . Nishimune et al. first characterized th GluR2/NSF interaction by two-hybrid screen and by biochemical approaches (Table 1.1) [26]. They then characterized the p hysiological function by infusing a peptide that blocks the GluR2/NSF interaction, or a specific antibody of NS , into a patch pipette and recorded evoked excitatory post-synaptic current (EPSC). They described a reduction of the EPSC amplitude and suggested that a rapid NSF-dependent insertion exists of GluR2-containing AMPAR at the post-synaptic membrane (Figure 1.1[3b]). Using the same blocking peptide, Noel et al. further characterized the role of this re gulation in AMPA mEPSC and sho wed a reduction in mEPSC frequenc y (Table 1.1) [55]. They also sho wed a reduction of AMPAR surf ace le vels upon expression of this peptide in cultured hippocampal neurons. Luthi et al. and Luscher et al. analyzed the effects on plasticity using, respectively, the NSF/GluR2 blocking peptide, or agents known to block various steps of exocytosis, and endocytosis (Table 1.1) [56,57]. Their experiments suggest that AMPAR cycle in and out of the synapse and that certain forms of plasticity such as LTD could utilize this mechanism for its expression (Figure 1.1[3b]). Similarly , Da w et al. block ed the interaction of PICK1/ABP/GRIP1 and GluR2 with a peptide and sho wed an increase in basal AMPAR-mediated transmission and a block of the generation of L TD (Table 1.1) [58]. The authors proposed a model in which the proteins interacting with the Cterminal domain of GluR2 are involved in the expression of LTD. Thus, this approach complemented microscopy data obtained at the same time and presented previously. 1.2.4.2 Rectification Index The current-voltage (IV) relationship of AMPAR is determined largely by the GluR2 subunit. AMPAR with GluR2 sho w a linear IV relation whereas AMPAR without GluR2 conduct little outward current at +40 mV. Taking advantage of this property, Hayashi et al. studied the importance of GluR1 in synaptic act ivity (Table 1.2) [59]. The recombinant AMPAR lacked GluR2. Thus, the incorporation of this recombinant receptor into a synapse w ould increase rectification of synaptic responses. The overexpression of GluR1 in or ganotypic hippocampal slices did not ha ve any effect on the amplitude or rectification of synaptic transmission in the absence of act vity. However, expression of GluR1 and CaMKII resulted in an increase of rectification

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suggesting an insertion of homomeric GluR1-GFP into the synapse ( Figure 1.1 and Figure 1.1[3a]). Interestingly , mutation of the PDZ binding site present on the Cterminal domain of GluR1 could block the CaMKII ef fect and L TP, suggesting a requirement for an interacting protein.Thus, the authors suggested that incorporation of GluR1-containing AMPAR into the synapse is a major mechanism underlying plasticity. Taking advantage of the same IV relationship properties, Shi et al. studied the molecular determinant on AMPAR that re gulates their synaptic deli very (Table 1.2) [60]. Unlike GluR1, the expression of homomeric GluR2 was sufficient for it incorporation at the synapse. To determine the requirements for GluR2 traf fickin in synaptic transmission, Shi et al. mutated GluR2-R to Q at the pore apex, allowing a distinct “electrophysiological signature.” These receptors showed a marked inward rectification in voked AMPAR-mediated synaptic transmission. With this tool, the authors were able to sho w that no activity was required for the continuous synaptic replacement of GluR2, unlike GluR1 (Figure 1.1 and Figure 1.1[3b]). The incorporation of this subunit was accompanied by a remo val of a fraction of the pre viously existing receptor. They also showed that GluR2 was not delivered to silent synapses, whereas GluR1 was able to do so when coexpressed with an active form of CaMKII. Thus, the y suggested tw o distinct synaptic deli very processes controlled by the subunit composition of the receptor . The first one requires a su unit with a long Ctail, such as GluR1, and participates in plasticity (Figure 1.1[3a]), and the second requires subunits with short C-tails, such as GluR2, and replaces synaptic receptors independent of synaptic acti vity (Figure 1.1[3b]). Ho wever, GluR1 properties are dominant when expressed in a heteromer. Thus, GluR1 + GluR2 require activity for their synaptic delivery and GluR2 + GluR3 comple xes are formed and deli vered to synapses continuously. 1.2.4.3 Agonists and Toxins Using specific bloc ers and taking adv antage of the dif ference in the inw ardly rectifying IV relationship observ ed when GluR2 is present or not in AMPAR, Liu et al. studied the composition of AMPAR at the parallel fibre stellate cell synaps during acti vity (T able 1.2) [61]. The authors used spermine, which is kno wn to confer voltage-dependent block of AMPAR lacking GluR2, Joro spider toxin (JST), a sub unit specific bloc er of GluR2-lacking AMPAR, and pentobarbital, which selectively inhibits GluR2-containing AMPAR at a specific concentration, to sh w that stellate cells e xpress a population of GluR2-containing AMPAR but predominantly at e xtrasynaptic sites. Using dif ferent frequencies of stimulation, the y were able to show that the level of GluR2-containing AMPAR at the parallel fibre stellat cell synapse undergo dynamic changes controlled by local Ca 2+ influx. In the basa state, synaptic AMPAR are mainly Ca 2+ permeable and so lack the edited GluR2. During high frequenc y stimulation, the increased Ca 2+ entry through synaptic AMPAR triggers the insertion of GluR2-containing AMPAR into the synapse and the removal of GluR2-lacking AMPAR. This results in a reduction of EPSC amplitude at +60 mV and a change in Ca 2+ permeability.

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1.2.5 PHARMACOLOGY The pharmacology of AMPAR has been e xtensively studied and agonists or antagonists of AMPAR or NMD AR have been broadly used to analyze the functions of these receptors [62,63]. Here, we described an e xample of the use of AMPAR pharmacology in the study of the traf ficking of this recepto . Using antagonists of NMDAR or AMPAR (APV and CNQX, respecti vely), Liao et al. sho wed that the level of AMPAR and NMD AR acti vity could re gulate the proportion of putati ve silent synapses (Table 1.2) [64]. The authors suggested that most excitatory synapses physically contain NMD AR but not AMPAR. Thus, the modulation of the endogenous activity of AMPAR and NMDAR regulates the number of silent synapses.

1.2.6 GENETIC APPROACH 1.2.6.1 Murine Models Use of mutant or genetically modified mice represents another means for iden tifying novel interacting components or to better understand receptor functions. Thus, Chen et al. took adv antage of an ataxic and epileptic mutant mouse, stargazer, to identify a ne w pathw ay re gulating AMPAR trafficking Table 1.1) [65]. The stargazer mouse (stg/stg) exhibit seizures and cerebellar ataxia and lack of functional AMPAR on granule cells [66]. The defective protein, stargazin (stg), exhibits a low homology with the sub unit of Ca 2+ channels. Chen et al. sho wed that stg/stg mice present no spontaneous acti vity, have lower GluR4 staining in cerebellar culture and no GluR2/3 staining in granule cells. They were able to co-immunoprecipitate stg with GluR4 and GluR2/3. Through its PDZ domain, stg can also interact with PSD95 and SAP97. The authors suggested that stg has two distinct roles. First, stg can re gulate the deli very of AMPAR to the cell surface (Figure 1.1). This does not require interaction of the C-terminal stg PDZbinding domain with a PDZ protein. But stg can also mediate the synaptic targeting of the receptor (Figure 1.1[2a], Figure 1.1[2b], Figure 1.1[3a] and Figure 1.1[3b]). This effect requires a PDZ interaction. 1.2.6.2 Knockout Mice Several groups ha ve used knock out approaches to understand the importance of GluR1 [67], GluR2 [68–71] and GluR3 sub units (Table 1.2) [72]. Mice lacking GluR1 exhibit AMPA-mediated neurotransmission but fail to generate LTP. They present a selecti ve loss of e xtrasynaptic AMPAR. However, spatial learning is normal [67]. Using mice lacking the GluR2 sub unit, Jia et al. showed an increase in Ca2+ permeability and an enhancement of LTP. They suggested that GluR2 was not required for L TP but seems to play a ne gative regulatory role [70]. Later , Sans et al. showed aberrant formation of GluR1/3 heteromers and GluR1 and GluR3 homomers in mice lacking the GluR2 sub unit, suggesting that GluR2 plays an important role in controlling the assembly of AMPAR [71]. Brusa et al. and Feldmeyer et al. used mice not able to edit GluR2 to sho w the importance of the edited GluR2 subunit in the AMPAR function [68,69]. Mice with unedited GluR2 present

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a higher Ca2+ permeability and develop seizures. The generation of a double-knockout mouse lacking GluR2 and GluR3 confirmed the importance of GluR1 in th generation of LTP and the critical involvement of GluR2 and GluR3 in maintaining basal synaptic transmission [72]. Thus, modification at the l vel of the whole animal can help an understanding of the function of a receptor in a more inte grated system. Ho wever, mice are v ery complex animals to handle; modifications ta e time. In addition, the e xistence of several genes implicated in the same pathway can lead to compensatory effects when the knockout is restricted to one of them. These compensatory effects can mask the function of a protein. 1.2.6.3 C. Elegans Simpler systems such as Caenorhabditis elegans have also been used to characterize the function of AMPAR in the whole animal. Genetic modifications are easier an quicker to assess in such systems. Thus, random mutations can allo w the identification of a n w pathw ay. In addition, less redundanc y is observ ed in simpler systems. Finally , the identification of a gene conser ed from w orm to mammal highlights the primordial function of such a gene. Thus, Hart et al. identified GLR 1, a receptor 40% identical to an AMPAR subunit, during a screen for a mutation that disrupts ASH-mediated nose-touch sensitivity [73]. The authors suggested that GLR-1 acts in synaptic tar gets of the ASH neurons.

1.3 SYNTHESIS, SUMMARY AND SPECULATION The use of the v arious methods discussed in this chapter has allo wed a better understanding of the traf ficking of AMPAR. AMPAR comple xes e xit from the ER/Golgi associated with stargazin (Figure 1.1; Table 1.1) [65]. GluR1-containing AMPAR traf fic through the secretory path ay (Figure 1.1[2a]) [48,49] and are inserted at e xtrasynaptic sites through an acti vity-dependent mechanism (Figure 1.1[3a]; Table 1.2) [35,36]. Sta rgazin binds PSD95, which localizes the compl ex at the synapse where the receptor can bind other scaf folding proteins such as RIL and 4.1N (Figure 1.1[4a]; Table 1.1) [29,30]. GluR2-containing AMPAR traf fi through the secretory pathw ay (Figure 1.1[2b]) and are inserted at synaptic sites through a constitutive mechanism (Figure 1.1[3b]; Table 1.2) [59,60]. PICK1 [24] facilitates the transport of the receptor , possibly both to and from the plasma membrane, but it is removed from the AMPAR by the NSF/SNAP when the receptor reaches the plasma membrane. NSF/SNAP/PICK1 forms a transient comple x with GluR2-containing AMPAR (Table 1.2) [19,25–27,55–57]. AMPAR are stabilized at the membrane by scaf folding proteins [20–22,58]. Phosphorylation by PKC prevents interaction between ABP/GRIP and GluR2 (Figure 1.1[4b]; Table 1.2) [53] and favors PICK1/GluR2 interaction (Figure 1.1[5b]; Table 1.2). The AMPAR are then internalized follo wing a coupling with PICK1, and AMPAR/PICK1 complexes are destabilized by NSF/SN AP when the receptor reaches its destination (Figure 1.1[6b]; Table 1.1) [19,27]. GluR2-containing AMPAR can then interact with an intracellular pool of ABP/GRIP, possibly in the endosome (Figure 1.1[7b];

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Table 1.2) [23] and get re cycled to the membrane through interaction with PICK1 (Figure 1.1[8b]; Table 1.2) or get targeted to lysosomes (Figure 1.1[9b]; Table 1.2) and degraded (Figure 1.1[10b]; Table 1.2) [45–47]. Research on the trafficking of AMPAR has led to pioneering studies in the fiel of the dynamics of synaptic proteins because of the importance of AMPAR traffick ing in synaptic plasticity . Ho wever, similar techniques and approaches ha ve been used to understand the dynamics of other neuronal proteins, such as other channels or receptors that determine and control properties and specificity of a neuron. The next phase will involve development of new and more sophisticated techniques and approaches that will allow linking properties of a protein to its functions in the whole animal.

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Long-Term Plasticity at Inhibitory Synapses: A Phenomenon That Has Been Overlooked Jean-Luc Gaiarsa and Yehezkel Ben-Ari

CONTENTS 2.1 2.2

Introduction ....................................................................................................23 Long-Term Changes in the Strength of Inhibitory Synapses .......................24 2.2.1 Minimal Requirements and Characteristics .......................................24 2.2.2 Expression ..........................................................................................29 2.2.3 Functional Relevance .........................................................................31 2.3 Conclusion......................................................................................................32 References................................................................................................................33

2.1 INTRODUCTION Synaptic plasticity describes the ability of individual synapses to alter their strength of transmission in response to dif ferent stimuli or en vironmental cues. Persistent activity-dependent changes are often referred to as long-term potentiation (LTP) and long-term depression (LTD), and represent respecti vely an increase and a decrease in the effica y of synaptic transmission. Initially studied as a model for learning and memory, L TP and L TD are also thought to play a crucial role in the netw ork hyperexcitability observ ed in pathological conditions and in the establishment of appropriate synaptic connections. The most extensive characterization of the cellular mechanisms in volved in the induction and maintenance of long-term plasticity has been undertaken at glutamatergic synapses. However, considering the ubiquitous distribution of inhibitory synapses and their role in shaping indi vidual and population acti vity, acti vity-dependent changes in the strength of inhibitory synapses w ould have important consequences on the development and the proper functioning of neuronal networks and, ultimately, on cognitive processes. Thus, information on long-term plasticity at inhibitory synapses is required to fully understand ho w acti vity-dependent changes in synaptic 23

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effica y contribute to brain de velopment and function in concert with plasticity at glutamatergic synapses. Surprisingly, plasticity at inhibitory synapses has only recently been reported. In this chapter, we summarize our current understandings of the cellular and molecular processes underlying inhibitory synaptic plasticity and the possible functions that this plasticity might serv e in the de veloping and adult nerv ous system.

2.2 LONG-TERM CHANGES IN THE STRENGTH OF INHIBITORY SYNAPSES 2.2.1 MINIMAL REQUIREMENTS

AND

CHARACTERISTICS

In the past decade, long-term changes in the strength of inhibitory synapses ha ve been reported in a large number of developing and adult structures, including Mauthner cells of the goldfish [34], the hippocampus [9,22,24,40,41,49,54,56], the cort x [21,30], the cerebellum [23], the deep cerebellar nucleus [43], the lateral superior olive [36], lateral amygdal [2] and the brain stem [4,17] (Figure 2.1 and Figure 2.2). In all these structures, attention had be focused to demonstrate that GAB Aergic and glycinergic synapses themselv es under go long-term changes in synaptic ef fica y. Long-term plasticity was observed on pharmacologically isolated inhibitory postsynaptic potentials (IPSPs) or currents (IPSCs) or on unitary IPSCs e voked by direct stimulation of the interneurons. To date, most studies on plasticity at GAB Aergic synapses ha ve focused on GABAA-receptor mediated postsynaptic potentials (GAB AA-PSPs). However, activation of either pre- or post-synaptic GABAB receptors during the conditioning protocol appears to have a key role in the induction of long-term plasticity at both GABAAergic and glycinergic synapses (Figure 2.1a and Figure 2.2c) [4,22,31,35,49,53], although in one case activation of GABAB receptors has been reported to pre vent the induction of GABAergic synaptic plasticity [25,26]. In most studies, plasticity at inhibitory synapses can be induced by high- or lowfrequency stimulations [15]. Although such conditioning protocol pro vides a good tool to study the mechanisms by which plasticity are triggered and e xpressed, such patterns of acti vity are rather unlik ely to occur in vivo . Thus, the rele vance of the conditioning protocol to physiological or pathological conditions must be taken into account to gain insight into the role that this plasticity might serve in the developing and adult nerv ous system. Compelling e vidence shows that conditioning protocols relevant to ph ysiological or pathological conditions can also induce plasticity at GABAergic and glycinergic synapses. The best example was provided by Korn and colleagues, who sho wed that auditory stimulations in the goldfish trigger TP of glycinergic synapses in vivo [47] that shares similar properties with L TP induced by tetanic stimulation [34,46]. More recently , Cherubini and colleagues ha ve reported that spontaneously occurring netw ork oscillations, which constitute a hallmark of de veloping netw orks, can trigger L TPGABA-A in the de veloping rat hippocampus [24]. In the hippocampus and corte x, plasticity of inhibitory synapses can also be triggered by post-synaptic firing of the ta get cells [5,38] or by coincidence of back-propag ating action potentials with pre-synaptic stimulation of inhibitory

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terminals [21,56]. Such conditioning protocols are related to the sort of acti vity that pyramidal neurons might e xperience in vivo . When stimulation of presynaptic inhibitory terminals is required, L TP and L TD is only expressed by the conditioned fibers. In the adult rat hippocampus, TDGABA-A is a highly localized phenomenon that spreads less than 20 µm a way from the conditioned fibers along the apical dendrite [9]. In this particular xample, the local re gulation of GABAergic synaptic strength is due to the restricted spread of the retrograde signal required for LTDGABA-A induction to synapses impinging on a small portion of the CA1 p yramidal cell dendrite. The interneurons e xhibited a great heterogeneity . One of the most striking dif ferences is found on the restricted and specific lamina distribution of their axonal terminals on dendritic and perisomatic re gions of their target cells [14]. This heterogeneity provides the possibility that the different interneuronal populations may selecti vely influence the e fica y of af ferent inputs and the emergence and maintenance of netw ork oscillations. Thus, depending on the type of interneurons, local changes in the ef fica y of inhibitory synapses will ha ve different consequences on the input-output relationship of the tar get neurons. Both homosynaptic and heterosynaptic plasticity at inhibitory synapses ha ve been reported ( Figure 2.1 and Figure 2.2 ). In the neonatal rat hippocampus, the induction of L TPGABA-A requires a membrane depolarization, pro vided by the activation of GABAA receptors during the conditioning protocol [18,41]. This depolarization is strong enough to allo w activation of voltage-dependent calcium channels (VDCCs), which will in turn trigger a cascade of e vents leading to homosynaptic LTPGABA-A (Figure 2.1a). In the adult and neonatal rat hippocampus, tetanic stimulation triggers heterosynaptic LTDGABA-A. In both cases, LTDGABA-A is triggered postsynaptically via the acti vation of glutamater gic receptors during the conditioning protocol. That is, glutamate released during repetiti ve stimulation activates group I metabotropic glutamate receptors [9] on adult CA1 p yramidal cells (Figure 2.2a) or N-methyl-D-aspartate (NMDA) receptors on neonatal CA3 p yramidal cells [6,42] (Figure 2.1a) or adult CA1 pyramidal cells (Figure 2.2b) [40,55]. Activation of these receptors will, in turn, trigger LTDGABA-A. Such heterosynaptic plasticity will locally affect dendritic integration of synaptic input, for instance, by decreasing or increasing the inhibitory shunt of neighboring glutamatergic afferents. Moreover, several studies have reported that a conditioning protocol can trigger L TP at e xcitatory synapses and, concomitantly, LTD at inhibitory synapses [40,55]. This opposite change in excitatory and inhibitory synaptic strength can change the ability of the e xcitatory synaptic potential to dischar ge an action potential [40,55], thereby changing the excitability of neuronal netw orks. As with glutamatergic synaptic plasticity, a rise in intracellular Ca 2+ concentration is important in shaping the strength of inhibitory synapses, although the source and location of this calcium rise and the consequences on inhibitory synaptic strength will vary depending on the structure considered and the conditioning protocol used (Figure 2.1 and Figure 2.2). In the neonatal rat hippocampus, the induction of LTPGABA-A requires a calcium influx through postsynaptic VDCCs (Figure 2.1a) [5,18], while in the corte x (Figure 2.1c) [31] and cerebellum (Figure 2.2d) [20], activation of postsynaptic calcium stores is required. An influx of calcium throug VDCCs triggers L TD in the corte x [38] and deep cerebellar nuclei (Figure 2.1b)

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−

+

Mg 2+

VDCCs

NMDA

GABAA

Pyr. cell

Cl−

Ca2+

Ca2+

Depolarization RyR-Ca2+ stores (a)

Mg2+ GABAA

VDCCs Pyr. cell

Ca2+

NMDA

Ca2+

Cl−

LTDGABA−A

LTDGABA−A (b)

AP

α1 Pyr. cell

GABAA

GABA B PLC

Ca2+ stores

VDCCs

+ IP3 Ca2+

LTPGABA-A (c)

Cl−

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FIGURE 2.1 Long-term changes in the strength of inhibitory synapses in the de veloping brain. (a) In the neonatal rat hippocampus, high-frequency stimulation leads to LTD and LTP of GABAergic synapses. Both forms of synaptic plasticity require a membrane depolarization provided by activation of GABAA receptors during the stimulation. This depolarization leads to a postsynaptic influx of calcium through oltage-dependent calcium channels (VDCCs) and the induction of LTP. The depolarization also removes the Mg2+ block of NMDA channels, leading to an influx of calcium through these channels. This calcium increase, amplified b the acti vation of ryanodine (RyR)-sensiti ve calcium stores, triggers L TD. Both forms of plasticity are e xpressed as a modification in the probability of GA A release through yet unknown mechanisms. (b) In the de veloping neocortex, high-frequency stimulation triggers heterosynaptic LTD through the activation of NMDA receptors. LTD of GABAergic synaptic transmission can also be triggers by post-synaptic firing of the ta get cells. The mechanisms underlying the expression of this LTD are presently unknown. (c) In the developing neocortex, tetanic stimulation triggers LTP. The induction of LTP requires the activation of postsynaptic GABAB receptors. Activation of these receptors f avors the release of calcium from postsynaptic IP3-sensitive stores induced by α1 adrenoreceptors.

[44]. An influx of calcium through postsynaptic NM A channels also triggers LTDGABA-A in the neonatal and adult rat hippocampus (Figure 2.1a and Figure 2.2b) [6,40,55] as well as in the neocorte x (Figure 2.1b) [32]. Interestingly , the initial depolarization required for the remo val of the Mg 2+ block of NMD A channels depends on the de velopmental stage. This depolarization is pro vided by acti vation of postsynaptic GABAA receptors in the neonates (Figure 2.1a) and by α-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors (Figure 2.2b) in the adult. Release of calcium by intracellular calcium stores could also trigger LTPGABA-A in the neocortex (Figure 2.1c) [31] and hippocampus (Figure 2.2c) [22]. In these peculiar e xamples, GABA released during the conditioning protocol activates postsynaptic metabotropic GAB AB receptors, which then causes or f avors intracellular release of calcium. In most cases, the calcium rise that triggers plasticity of inhibitory synapses occurs in the post-synaptic tar get cells, as post-synaptic loading with a calcium chelator prevents its induction. Ho wever, the calcium rise could also occur in the presynaptic terminal [7,16] or in neighboring astrocytes (Figure 2.2c) [22]. Thus, in the adult hippocampus, repetiti ve firing of a single interneuron triggers a calcium dependent LTPGABA-A on pyramidal cells. The calcium rise is triggered on neighbor ing astroc ytes follo wing the acti vation of GAB AB receptors by GAB A released during the conditioning protocol [22]. This calcium rise then causes the release of a retrograde messenger acting on GAB Aergic terminals [22]. Interestingly, the same conditioning protocol can lead to both L TP and L TD, depending on the experimental conditions (Figure 2.1a). The source or the magnitude of the postsynaptic Ca 2+ rise has been proposed to determine the polarity of the plasticity. In the neonatal rat hippocampus, high-frequenc y stimulation can lead to either LTPGABA-A or LTDGABA-A, depending on whether or not NMDA receptors were activated during the conditioning protocol [41]. Gi ven that both forms of plasticity require a postsynaptic rise in calcium [41], these observ ations suggest that the polarity of synaptic changes might be determined by the source of calcium influx i.e., an influx through VDCCs triggers L TPGABA-A [5], whereas an influx throug

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The Dynamic Synapse

Heterosynaptic LTDGABA-A

Heterosynaptic LTDGABA-A Schaﬀer col.

Schaﬀer col. Mg2+ NMDA

AMPA

DAG PLC

mGluR

CB1

DAG-L

Depol.

Ca2+

? -

2-AG

GABAA

GABAA Pyr. cell

Cl−

Pyr. cell

Calcineurin

(a)

−

P

Cl−

(b) Rebound potentiation

LTPGABA-A +

Climbing ﬁber

Astrocyte Ca2+ GABAB

GABAA

GABAA Pyr. cell

Cl−

Purkinje cell

VDCCs P Cl−

+

CaMKII

(c)

AMPA

Ca2+

Depolarization

IP3 Ca2+ stores

(d)

FIGURE 2.2 Long-term changes in the strength of inhibitory synapses in the adult brain. (a) In the adult rat hippocampus, repetiti ve activation of Schaffer collaterals triggers heterosynaptic LTD that is induced via the acti vation of postsynaptic group I metabotropic glutamate receptors (mGluR). Activation of mGluR leads to PLC activation, formation of diacyl-glyceral (DAG) that is converted by DAG lipase (DAG-L) into 2-arachidonoyl glycerol (2-AG). 2-AG acting on presynaptic CB1 receptors decreases GAB A release. (b) In the adult rat hippocampus, high-frequenc y stimulation of Schaf fer collaterals triggers heterosynaptic L TD that is induced via the activation of AMPA and NMDA receptors. The calcium influx through NM A receptors likely leads to a dephosphorylation of post-synaptic GAB AA receptors. (c) In the adult rat hippocampus, repetitive pre-synaptic firing of GA Aergic interneurons triggers LTP that is induced by the acti vation of GAB AB receptors on neighboring astroc ytes. Activation of GABAB receptors leads to a calcium rise that causes the release of retrograde messenger acting on GABAergic terminals. (d) In the cerebellum, direct depolarization of Purkinje cells or stimulation of climbing fibers triggers a long lasting potentiation of GA Aergic synapses, termed rebound potentiation. The induction of this potentiation requires a post-synaptic rise in calcium, amplified by the act vation of IP3-sensiti ve stores and leading to the acti vation of the calcium/calmodulin-dependent kinase II (CaMKII).

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NMDA channels triggers LTDGABA-A (Figure 2.1a) [6]. Alternatively, the magnitude of the calcium rise could determine the polarity of synaptic changes, as documented in the deep cerebellar nucleus. In this structure, the number of spikes and the related amount of Ca 2+ that enters the post-synaptic neurons during the post-inhibitory rebound depolarization seems to determine whether L TPGABA-A or L TDGABA-A is induced with lar ge numbers of spik e leading to LTPGABA-A [1].

2.2.2 EXPRESSION Long-term changes in the strength of synaptic ef fica y can be accounted for by at least four none xclusive mechanisms: modifications in the number or properties o receptors at functional synapses; changes in the re versal potential of post-synaptic responses; modifications in the probability of transmitter release; and modificatio in the number of functional synapses though either pre- or post-synaptic changes. Probably because fewer studies have been performed to date, the locus of expression of plasticity at inhibitory synapses appears less controversial than at their excitatory counterparts. If the amount of GAB A released during synaptic stimulation reaches the concentration that saturates postsynaptic GAB AA receptors, the total number or properties of receptors at functional synapses will be an important limiting f actor. In the adult dentate gyrus, a direct relationship between synaptic GABAA receptor number and quantal size at potentiated GAB Aergic synapses has been reported in an e xperimental model of temporal lobe epilepsy [45]. In this model, insertion of ne w GABAA receptors underlies the increase in amplitude of unitary IPSCs. Other studies ha ve reported that the pathw ay through which this Ca 2+ rise is translated into long-term changes of synaptic ef fica y in volves changes in the properties of post-synaptic GABAA receptors through activation of protein kinases or phosphatases [29]. Thus, in the adult hippocampus, the e xpression of LTDGABA-A involves a do wn-regulation of GABAA receptors by the calcium-sensitive phosphatase, calcineurin (Figure 2.2b) [40,55]. In the cerebellum, the e xpression of L TPGABA-A required the acti vation of post-synaptic calcium-calmodulin-dependent kinase II (CaMKII, Figure 2.2d) [23,26]. In the lateral superior olive, both post-synaptic CaMKII and protein kinases A and C participate in the L TD of inhibitory synapses [37]. Changes in the strength of inhibitory inputs can also result from modificatio in the re versal potential of GAB Aergic synaptic responses, as documented in the hippocampus [56]. In this structure, coincident pre- and post-synaptic spiking leads to a persistent decrease in GAB Aergic synaptic strength associated with a depolar izing shift of the reversal potential of GABAA receptor-mediated synaptic potentials (EGABA). Similarly, pairing e xogenously applied GAB A with postsynaptic depolar ization leads to a long-lasting transformation of h yperpolarizing GAB Aergic responses into depolarizing responses [12]. GAB AA receptors are permeable to chloride and the intracellular concentration is regulated by different chloride cotransporters [50]. In their study, Woodin et al. have shown that coincident pre- and postsynaptic spiking decreases the cation/chloride cotransporter KCC2 activity, resulting in the shift of EGABA to more positive values [56]. Long-lasting change in EGABA have

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been also reported in epileptic tissue [10,27], supporting the contrib ution of such phenomenon in the emer gence and maintenance of pathological netw ork activity. However, recent studies have reported that saturation of GABAA receptors does not occur at all sites of GAB A release [19]. In this case, change in the probability of GAB A release or in the number of functional release sites might underlie the expression of synaptic plasticity . Compelling e vidence has accumulated in support of this h ypothesis. Thus, long-term changes in the strength of inhibitory synapses are often associated with changes in the f ailure probability of unitary inhibitory postsynaptic currents (IPSCs) [22,24,46]; changes in the coef ficient of ariation of evoked IPSCs [6,16,46]; changes in the paired pulse ratio of e voked IPSCs [9,24]; and changes in the frequenc y, b ut not amplitude, of miniature IPSCs [5,22] or asynchronous quantal IPSCs e voked in the presence of strontium [6]. A modification in the number of functional release sites has been directl demonstrated in the goldfish [8]. Thus, in dual recordings of presynaptic glycinergic interneurons and postsynaptic Mauthner cells, 25% of the interneurons produced no detectable postsynaptic response. Morphological examination revealed that the number of synaptic contacts made by these “silent” interneurons is similar to that of “functional” interneurons. After a tetanic stimulation of the VIIIth nerve, that produces LTPgly , the “silent” interneurons become functional. Similarly, in the neonatal rat hippocampus, postsynaptic application of a conditioning protocol leading to LTPGABA-A leads to the appearance of functional GAB Aergic synapses in previously “silent” CA3 p yramidal neurons [18]. It remains to be determined whether the modifications in the number of functional release sites is due to all-or none modi fications in the number of postsynaptic receptors or to presynaptic switching on i transmitter release. If plasticity is e xpressed at the postsynaptic le vel while induction requires a postsynaptic rise in calcium, the information should be transmitted back from the postsynaptic cell to the presynaptic inhibitory terminal. Such synaptic feedback provided by retrograde messengers has been demonstrated in the rat hippocampus. Chevaleyre and Castillo [9] recorded from adult hippocampal slices, and found that high frequenc y stimulation induces an NMD A-independent L TDGABA-A. This LT-GABA-A is triggered postsynaptically via acti vation of group I metabotropic glutamatergic receptors (mGluRs), b ut is e xpressed presynaptically (Figure 2A). Thus activation of postsynaptic group I mGluRs leads to the release of endocannabinoids, which then causes a persistent reduction of GABA release. The implication of a trans-synaptic messenger in the induction of L TPGABA-A in the rat hippocampus has also been demonstrated in an ele gant study by Kang and collaborators [22] (Figure 2.2c ). In this stud y, repetit ive firing of hippocampal interneurons leads t the acti vation of GAB AB receptors on astroc ytes, and a subsequent increase in intracellular calcium concentration. This calcium rise causes the release of a messenger from astroc ytes, probably glutamate, which in turn triggers an increase in the probability of GABA release. Similar feedback regulation of inhibitory synaptic transmission has been reported in the rat cerebellum, although this control occurs in a short (seconds to minutes) time scale. Thus, short depolarization of Purkinje cells leads to a transient (tens of seconds) decrease follo wed by a short-term (10 to 15 minutes) increase in the frequency of miniature inhibitory post-synaptic currents

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(mIPSCs). Both phenomena, termed respecti vely depolarization-induced suppression of inhibition (DSI) [39] and depolarization-induced potentiation of inhibition (DPI) [13], require a post-synaptic rise in calcium concentration and are mediated presynaptically. In DSI, the post-synaptic calcium rise initiates endocannabinoid synthesis that activates presynaptic CB1 receptors, resulting in inhibition of GAB A release for tens of seconds [48]. In DPI, the post-synaptic rise in calcium induces the release of glutamate from Purkinje cells [13]. Glutamate acti vates pre-synaptic NMDA receptors, leading to calcium influx into the pre-synaptic terminal that, wit the activation of ryanodine-sensitive calcium stores, induces a short-lasting increase of GABA release. In all studies, modified synaptic strength seems to be maintained by a mechanis independent of neuronal activity. In contrast, in the developing rat visual cortex, the maintenance of L TPGABA-A requires firing of pre-synaptic inhibitory terminals an pre-synaptic calcium influx throug VDCCs (Figure 2.1c) [33]. Thus, if stimulation of the test pathw ay w as stopped after L TPGABA-A induction, potentiated responses returned to a baseline level. Because this plasticity could underlie experience-dependent refinement of visual inputs early in life, this obser ation suggests that this refinement might not persist unless strengthened synapses are act vated by visual stimulation.

2.2.3 FUNCTIONAL RELEVANCE Most of our interests on long term changes in synaptic ef fica y stem from the possible functions that LTP and LTD might serve in the developing and adult nervous system. In the adult nerv ous system, the role of plasticity at inhibitory synapses w as previously thought to re gulate the occurrence of plasticity at e xcitatory synapses. However, plasticity of inhibitory synapses, in parallel with plasticity at e xcitatory synapses, can also affect the probability of the target neurons to fire action potentials and will contribute to hyperexcitability of neuronal networks observed in pathological conditions. The balance between excitation and inhibition in neuronal networks can critically influence the l vel and type of spontaneous synaptic acti vity within the netw ork, and changes in synaptic strength could lead to profound netw ork modifications. or instance, in the adult rat hippocampus, L TDGABA-A induced by tetanic stimulation re vealed latent e xcitatory synaptic connections between hippocampal pyramidal cells. Such phenomenon lik ely contributes to the emer gence or maintenance of epileptiform acti vity [54]. Plasticity at inhibitory synapses alone, without changes in glutamater gic synaptic strength, could also ha ve beha vioral consequences. This w as directly addressed in the goldfish with the plasticity a glycinergic synapses onto Mauthner cells. Activation of the Mauthner cell system by sound is kno wn to contrib ute to an escape reaction that orients the fish way from the predator [59]. In an ele gant study performed in vivo , sound stimulation sufficient to produce TP of glyciner gic synapses, b ut inef ficient to modify th excitatory inputs onto Mauthner cells, w as reported to decrease the probability to escape in the conditioned goldfish [57,58], whereas the basic properties of the escap refl x were not modified

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Plasticity at inhibitory synapses could also play a crucial role in the de veloping brain. Thus, both L TP and L TD of inhibitory synapses ha ve been described in different developing brain regions, including the lateral superior olive [36], the cortex [30] and the hippocampus [41]. In all these structures, plasticity is induced during a restricted period of de velopment that closely matches the period of functional synaptic maturation. In the auditory system, the tonotopic organization of glycinergic projections is achieved through synapses elimination [51], a process involving activity-dependent mechanisms [52]. The structural refinement of axonal arbors eme ges gradually and is preceded by a functional elimination and strengthening of GABA/glycine connections [28]. The period during which L TD is induced in this structure coincides with the period of functional elimination of inhibitory synapses [36]. To strengthen the link between activity-dependent maturation of inhibitory synapses and long-term plasticity showing that the activity involved in the development of inhibitory synapses is able to produce modifications in inhibitory synaptic strengt is necessary. The presence of spontaneous pattered synaptic acti vity is a hallmark of the de veloping network [3]. In the neonatal hippocampus, this netw ork activity, termed giant depolarizing potentials (GDPs), consists of b ursts of action potentials associated with post-synaptic influx of calcium through VDCCs. Thus, the minimal requirements to induce LTPGABA-A in the neonatal rat hippocampus are present during GDPs. This activity could therefore represent the ph ysiological pattern of acti vity leading to the functional maturation of inhibitory synapses through L TP/LTD-like mechanisms [18]. In agreement with this h ypothesis, pharmacological blockade of the GDPs pre vents the functional maturation of GAB Aergic synapses [11], and application of a conditioning protocol that mimics, at least in part, the post-synaptic consequences of GDPs triggers L TPGABA-A during a narro w postnatal time windo w [18]. Moreo ver, the same protocol also leads to the appearance of functional GABAergic synapses on previously silent cells, thus mimicking the functional maturation of GAB Aergic synapses occurring in vivo [5,18]. Finally, pairing e voked GABAergic synaptic responses with GDPs leads to L TPGABA-A in the neonatal rat hippocampus [24]. A key issue that might unco ver the link between acti vity-dependent functional maturation and synaptic plasticity is to sho w that the same cellular mechanisms are involved in both phenomena. In this context, neurotrophins and related Trk receptorcoupled protein tyrosine kinases (PTKs) ha ve been implicated in synapse de velopment and plasticity, and could likely represent the signal linking long-term plasticity and acti vity-dependent maturation of inhibitory synapses. In agreement with this hypothesis, recent results suggest that TrkB receptors participate in the induction of LTDGly in the developing auditory brain stem [35] and L TPGABA-A in the developing rat hippocampus (Gaiarsa and Gubellini, unpublished results).

2.3 CONCLUSION The data reviewed here indicate that inhibitory synapses undergo calcium-dependent, long-term changes in synaptic ef fica y. This plasticity can be triggered by conditioning protocol rele vant to ph ysiological and pathological conditions, pointing to

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a possible contribution in the de veloping and adult brain. Moreo ver, although most studies have been performed in vitro, a study has reported that similar process occurs in vivo , and demonstrates behavioral consequences following the induction of plasticity at glyciner gic synapses. Thus, we must consider plasticity at inhibitory synapses to fully understand the consequences of acti vity-dependent plasticity on the development and function of the neuronal netw ork. However, one should consider the heterogeneity of GAB Aergic interneurons [14]. In the adult hippocampus, different interneurons impinge precisely on specifie areas and differentially control the excitability of their target cells. Persistent strength modifications of di ferent types of interneurons will likely produce different changes on integrative functions and in future studies, inserting the heterogeneity of inter neuronal types in this general scheme will be important. For instance, heterosynaptic plasticity that results from interactions with glutamatergic synapses will be expressed by dendritic synapses where glutamatergic inputs impinge on target cells. Therefore, to completely understand the o verall ef fect of long-term plasticity at inhibitory synapses on the acti vity generated by a neuronal netw ork, the morphological identification of the interneurons underlying this plasticity will be required

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The Dynamic Synapse 12. Collin, C. et al., Long-term synaptic transformation of hippocampal CA1 g ammaaminobutyric acid synapses and the effect of anandamide, Proc. Natl. Acad. Sci. USA, 92, 10167–10171, 1995. 13. Duguid, I.C. and Smart,T.G., Retrograde activation of presynaptic NMD A receptors enhances GABA release at cerebellar interneuron-Purkinje cell synapses, Nat. Neurosci., 7, 525–533, 2004. 14. Freund, T.F. and Buzsáki, G., Interneurons of the hippocampus, Hippocampus, 6, 347–470, 1996. 15. Gaïarsa, J.L. et al., Long-term plasticity at GAB Aergic and glyciner gic synapses: Mechanisms and functional significance, Trends Neurosci., 25, 564, 2002. 16. Glaum, S.R. and Brooks, P .A., Tetanus-induced sustained potentiation of monosynaptic inhibitory transmission in the rat medulla: Evidence for a presynaptic locus, J. Neurophysiol., 76, 30–38, 1996. 17. Grabauskas, G. and Bradley, R.M., Potentiation of GABAergic synaptic transmission in the rostral nucleus of the solitary tract, Neuroscience, 94, 1173–1182, 1999. 18. Gubellini, P. et al., Activity- and age-dependent GABAergic synaptic plasticity in the developing rat hippocampus, Eur. J. Neurosci., 14, 1937–1946, 2001. 19. Hájos, N. et al., Cell type- and synapse-specific ariability in synaptic GAB AA receptor occupancy, Eur. J. Neurosci., 12, 818, 2000. 20. Hashimoto, T. et al., Release of Ca 2+ is the crucial step for the potentiation of IPSCs in the cultured cerbellar Purkinje cells of the rat, J. Physiol. Lond ., 497, 611–627, 1997. 21. Holmgren, C.D. and Zilberter , Y., Coincident spiking acti vity induced long-term changes in inhibition of neocortical p yramidal cells, J. Neurosci., 21, 8270–8277, 2001. 22. Kang, J. et al., Astrocyte-mediated potentiation of inhibitory synaptic transmission, Nat. Neurosci., 1, 683–692, 1998. 23. Kano, M. et al., Ca (2+)-induced rebound potentiation of g amma-aminobutyric acidmediated currents requires acti vation of Ca 2+/calmodulin-dependent kinase II, Proc. Natl. Acad. Sci. USA , 93, 13351–13356, 1996. 24. Kasyanov, A.M. et al., GABA-mediated giant depolarizing potentials as coincidence detectors for enhancing synaptic effica y in the developing hippocampus, Proc. Natl. Acad. Sci. USA , 101, 3967–3972, 2004. 25. Kawaguchi, S.-Y. and Hirano, T., Suppression of inhibitory synaptic potentiation by presynaptic acti vity through postsynaptic GAB AB receptors in a Purkinje neuron, Neuron, 27, 339–347, 2000. 26. Kawaguchi, S.-Y. and Hirano, T., Signaling cascade regulatig long-term potentiation of GABAA receptor responsiveness in cerebellar Purjinje neurons, J. Neurosci., 22, 3969–3976, 2002. 27. Khalilov, I. et al., In vitro formation of a secondary epileptogenic mirror focus by interhippocampal propagation of seizures, Nat. Neurosci., 6, 1079–1085, 2003. 28. Kim, G. and Kandler , K., Elimination and strengthening of glyciner gic/GABAergic connections during tonotopic map formation, Nat. Neurosci., 6, 282–290, 2003. 29. Kittler, J.T. and Moss, S.J., Modulation of GAB AA receptor activity by phosphorylation and receptor traf ficking: Implications for the e fica y of synaptic inhibition, Curr. Opin. Neurobiol., 13, 341–347, 2003. 30. Komatsu, Y., Age-dependent long-term potentiation of inhibitory synaptic transmission in rat visual corte x, J. Neurosci., 14, 6488–6499, 1994.

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31. Komatsu, Y., GABAB receptors, monoamine receptors, and postsynaptic inositol trisphosphate-induced Ca 2+ release are involved in the induction of long-term potentiation at visual cortical inhibitory synapses, J. Neurosci., 16, 6342–6352, 1996. 32. Komatsu, Y. and Iw akiri, M., Long-term modification of inhibitory synaptic trans mission in de veloping visual corte x, Neuroreport, 4, 907–910, 1993. 33. Komatsu, Y. and Yoshimura, Y., Activity-dependent maintenance of long-term potentiation at visual cortical inhibitory synapses, J. Neurosci., 20, 7539–7546, 2000. 34. Korn, H. et al., Long-term potentiation of inhibitory circuits and synapses in the central nervous system, Proc. Natl. Acad. Sci. USA , 89, 440–443, 1992. 35. Kotak, V.C. et al., GABAB and Trk receptor signaling mediates long-lasting inhibitory synaptic depression, J. Neurophysiol., 86, 536–540, 2001. 36. Kotak, V.C. and Sanes, D.H., Long-lasting inhibitory synaptic depression is age- and calcium-dependent, J. Neurosci., 20, 5820–5826, 2000. 37. Kotak, V.C. and Sanes, D.H., Postsynaptic kinase signaling underlies inhibitory synaptic plasticity in the lateral superior oli ve, J. Neurobiol., 53, 36–43, 2002. 38. Kurotani, T. et al., Postsynaptic firing produces long-term depression at inhibitor synapses of rat visual corte x, Neurosci. Lett., 337, 1–4, 2003. 39. Llano, I. et al., Calcium entry increases the sensiti vity of cerebellar Purkinje cells to applied GABA and decreases inhibitory synaptic currents, Neuron, 6, 565–574, 1991. 40. Lu, Y.M. et al., Calcineurin-mediated L TD of GAB Aergic inhibition underlies the increased e xcitability of CA1 neurons associated with L TP, Neuron, 26, 197–205, 2000. 41. McLean, H.A. et al., Bidirectional plasticity e xpressed by GAB Aergic synapses in the neonatal rat hippocampus, J. Physiol. Lond., 496, 471–477, 1996. 42. McLean, H.A. et al., Spontaneous release of GAB A activates GABAB receptors and controls netw ork acti vity in the neonatal rat hippocampus, J. Neurophysiol., 76, 1036–1046, 1996. 43. Morishita, W. and Sastry, B.R., Long-term depression of IPSPs in rat deep cerebellar nuclei, Neuroreport, 4, 719–722, 1993. 44. Morishita, W. and Sastry , B.R., Postsynaptic mechanisms underlying long-term depression of GAB Aergic transmission in neurons of the deep cerebellar nuclei, J. Neurophysiol., 76, 59–68, 1996. 45. Nusser, Z. et al., Increased number of synaptic GAB A(A) receptors underlies potentiation at hippocampal inhibitory synapses, Nature, 395, 172–177, 1998. 46. Oda, Y. et al., Long-term potentiation of glycinergic inhibitory synaptic transmission, J. Neurophysiol., 74, 1056–1074, 1995. 47. Oda, Y. et al., Inhibitory long-term potentiation underlies auditory conditioning of goldfish escape beh viour, Nature, 394, 182–185, 1998. 48. Ohno-Shosaku, T. et al., Endogenous cannabinoids mediated retrograde signals form depolarized postsynaptic neurons to presynaptic terminals, Neuron, 29, 729–738, 2001. 49. Patenaude, C. et al., GAB AB receptor- and metabotropic glutamate receptor -dependent cooperative long-term potentiation of rat hippocampal GAB AA synaptic transmission, J. Physiol., 553, 155–167, 2003. 50. Payne, J.A. et al., Cation-chloride co-transporters in neuronal communication, development and trauma, Trends Neurosci., 26, 199–206, 2003. 51. Sanes, D.H. and Si verls, V., The de velopment and specificity of inhibitory axona arborizations in the lateral superior oli ve, J. Neurobiol., 22, 837–854, 1991. 52. Sanes, D.H. and Tackacs, C., Actvity-dependent refinement of inhibitory connections Eur. J. Neurosci., 5, 570–574, 1993.

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The Dynamic Synapse 53. Shew, T. et al., Mechanisms in volved in tetanus-induced potentiation of f ast IPSCs in rat hippocampal CA1 neurons, J. Neurophysiol., 83, 3388–3401, 2000. 54. Stelzer, A. et al., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy , Nature, 326, 698–701, 1987. 55. Wang, J.H. and Stelzer ,A., Shared calcium signalling pathw ays in the induction of long-term potentiation and synaptic disinhibition in CA1 p yramidal cell dendrites, J. Neurophysiol., 75, 1687–1702, 1996. 56. Woodin, M.A. et al., Coincident pre- and postsynaptic acti vity modifies GA Aergic synapses by postsynaptic changes in Cl-transporter acti vity, Neuron, 39, 807–820, 2003. 57. Yang, X.D. and Faber, D.S., Initial synaptic effica y influences induction and xpression of long-term changes in transmission,Proc. Natl. Acad. Sci. USA, 88, 4299–4303, 1991. 58. Yang, X.D. et al., Long-term potentiation of electronic coupling at mix ed synapses, Nature, 348, 542–545, 1990. 59. Zotelli, S.J., Correlation of the startle refl x and Mauthner cell auditory response in unrestrained goldfish, J. Exp. Biol., 66, 243–254, 1997.

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New Tricks for an Old Dog: Proteomics of the PSD Bryen A. Jordan, Brian D. Fernholz, Thomas A. Neubert, and Edward B. Ziff

CONTENTS 3.1

Introduction ....................................................................................................37 3.1.1 Why Proteomics? ...............................................................................38 3.2 The Brain and the PSD ..................................................................................39 3.2.1 Proteomics of the PSD .......................................................................40 3.3 Methods..........................................................................................................41 3.3.1 2DE-LC-MS/MS ................................................................................41 3.3.2 1DE-LC-MS/MS ................................................................................42 3.3.3 2DE-DIGE..........................................................................................43 3.3.4 Relative Quantification by Stable Isotope Labeling and Mass Spectrometry .............................................................................43 3.3.5 Immobilized Metal Affinity Chromatograp y (IMAC).....................44 3.3.6 Affinity Purificati ...........................................................................45 3.4 A Comparison of PSD Proteomics Studies ...................................................46 3.5 Challenges ......................................................................................................47 3.6 Conclusions ....................................................................................................49 References................................................................................................................50

3.1 INTRODUCTION Most questions in modern cell biology ha ve been approached using reductionist methods, i.e., by studying one gene, one protein or one specific protein modificati at a time. This reductionism has been necessary, given the complexity of biological systems and lack of tools for de veloping more inte grative methodologies. It is, nonetheless, responsible for most of our knowledge of biological systems. However, a more thorough understanding of comple x systems will require the simultaneous observation of their many characteristics. Proteomic methodologies, especially those that are mass spectrometry based, ha ve enabled the lar ge-scale study of protein

37

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modifications, protein a undance and protein interactors. Only by using such integrative approaches will we de velop rigorous models that more accurately reflec actual cellular processes. Proteomics is the study of the set or subset of all proteins xepressed in an organism, tissue or cell culture. While proteomics is by no means a no vel idea, recent adv ances in mass spectrometry and the determination of the complete genomic sequences of several organisms greatly enhance its usefulness [1–3]. Only by mass spectrometry can one ef ficiently identify the ind vidual protein components deri ved from protein complexes, and only lar ge-scale genomic data allo ws amino acid sequences to be reliably assigned to peptide fragments identified by mass spectrometr . Most proteomics-based studies today seek to answer four basic questions:Which proteins were found? What is the relati ve abundance of the proteins found? What modifications were found on the proteins? Which proteins physically associate with one another? One type of proteomics e xperiment, protein profiling, i volves the identification of the proteins present in a compl x, cell culture, tissue or or ganism. However, unlik e genomes, proteomes are dynamic with changes that reflect thei current functional state. Thus, analyzing changes in the abundance and modification of proteins in a comple x could be considered a functional study (functional proteomics). While several proteomic-based methods exist, most are based on a standard sequence of experiments: a protein complex is obtained by biochemical prefractionation or affinity purification; the sample is subjected to enzymatic digestion (usual trypsin); the resulting peptides can be labeled for future analysis; and the peptides are analyzed by mass spectrometry to determine their identity and characteristics. At the core of most modern proteomic studies lies mass spectrometry (MS) [2,3]. A mass spectrometer is, in essence, a detector that measures the mass-to-charge ratio (m/z) of ionized particles and detects the relati ve number of ions at each m/z ratio. In general, peptide fragments generated by enzymatic clea vage (typically trypsin) are ionized most commonly by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) and injected into a mass spectrometer. In most cases, peptides with specific m/z ratios can be selected by quadrupole e xclusion or time of flight ( OF) selection. These peptides can then be fragmented by cleavage at peptide bonds via collision with a g aseous matrix. The masses of the fragments can then be determined by a second mass analysis (often by TOF or ion trap MS) to determine the amino acid sequence of peptides. Prefractionation of samples by nanofl w highperformance liquid chromatograph y (HPLC) and increases in sensiti vity and accuracy of mass spectrometers allo ws for the identification of up to 1000 or mor peptides per sample. In this w ay, complex mixtures can be resolv ed and the protein compositions elucidated.

3.1.1 WHY PROTEOMICS? A recent analysis of the finalized human genome predicts as f w as 20,000 to 25,000 genes [4]. This number represents a significant d wnward revision of the already surprisingly lo w number of 30,000 genes predicted upon the completion of the human genome [5]. This number is only somewhat higher than the 14,000 genes of Drosophila, barely abo ve the 19,500 found in the nematode C. elegans and below

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the 27,000 found in the tin y plant Arabidopsis. However, pre-, co- and post-translational modifications result in a la ge number of dif ferent protein products from single genes. Pre-translational modifications occur as a result of splicing or editin events of transcribed messages, resulting in se veral unique mRN As from a single gene. Co-translational modifications can also result in changes to primary sequences Modifications of traditional amino acids and special tR As can result in the incor poration of nontraditional amino acids such as selenoc ysteine or p yrrolysine. The largest di versifying e vent results from post-translational modifications (PTMs) Aside from the more prominently studied phosphorylation, lipidation and ubiquitination, over 100 kno wn modifications are found on amino acids [6]. While many could be a result of post-e xtraction chemistry, several exhibit functional rele vance (nitrosylation, sulfation, methylation, amidation, acetylation and oxidation) [7]. Protease-mediated protein cleavage can also significantly alter protein function. H w a single gene could result in hundreds of dif ferent protein states is easy to imagine. Regulation of protein ab undance is an important e vent in biological systems. Thirty years ago, researchers suggested that humans and other greater primates differ not primarily due to the structural changes in proteins resulting from their roughly 1% difference in genomic DN A but in quantitati ve differences in gene e xpression [8]. Recently Enard et al. ha ve shown that whereas the relative extent of changes in gene expression between humans, chimpanzees and macaques is similar for blood and liver, a pronounced difference in brain mRNA expression levels exists [9]. Large differences were also observed in brain protein intensity patterns by two-dimensional electrophoresis between chimpanzee and humans, something not observ ed between different mouse species. These results suggest the brain proteome has diverged more rapidly than other or gans. One could speculate that gi ven the similarities between human and chimpanzee genomes (98.7%), the changes in ab undance, and not function, of brain proteins contrib ute substantially to our cogniti ve differences.

3.2 THE BRAIN AND THE PSD The brain presents a particular challenge for proteomics as neurons are thought to contain more different proteins than an y other type of cell [10]. Moreo ver, the vast diversity of cell types in the brain introduces additional comple xity to the study of this organ at the tissue le vel. However, proteomics has been successfully applied in studying region-specific neurons [11] or particular subcellular r gions such as the post-synaptic density (PSD). The PSD is of particular importance in neuron function and is well suited for proteomic studies. Methodologies to detect changes in protein abundance and composition of the PSD can help elucidate the mechanism of longterm changes in synaptic plasticity. Therefore, the fact that a variety of studies have recently characterized the protein composition of the PSD is not surprising [12–15]. The PSD is a protein complex critically important for synaptic function. Its name stems from original observ ations of a densely stained re gion in direct apposition to nerve terminals in neuromuscular junctions and at contact points between cortical neurons [16,17]. The PSD is directly opposite synaptic acti ve zones and therefore poised to recei ve and transmit the chemical signals from a pre-synaptic cell. Early work e xploring the molecular composition of the PSD identified multiple protein

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responsible for a di verse set of cellular functions. The proteins identified suggeste that the PSD could participate in v arious cellular e vents, including signal amplifica tion, c ytoskeletal anchorage, re gulation of biochemical signaling and clustering of ionotropic as well as metabotropic receptors [18–21]. Ionotropic receptors at synapses, such as the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and Nmethyl-D-aspartate (NMD A) glutamate-g ated channels, transform pre-synaptically released chemical messages into electrical and biochemical signals. Proteins of se veral secondary messenger systems, including G-proteins, kinases and phosphatases, play roles in signal amplification and modulation of synaptic transmission Cytoskeletal proteins, including actin and its modulators, are major components of this matrix and underscore its structural role and potential for reor ganization based on rapid actin dynamics. This characteristic is of particular importance gi ven that changes in the size and composition of the PSD are associated with stable changes in synaptic strength [22,23]. Ph ysiologically relevant increases in synaptic strength are collectively known as long-term potentiation (LTP). LTP is often used as a model for learning and memory [24,25] and attrib uted in part to changes in the ab undance of key factors in the PSD. Activity-dependent changes in a v ariety of kno wn PSD components ha ve been demonstrated by biochemical means [26]. Changes in dendritic spine morphology have been correlated to hippocampal L TP, as observ ed by tw o-photon microscopy [27–29]. Depolarization of rat hippocampal neurons leads to a rapid and transient thickening of the post-synaptic density [22] caused in part by the rapid association of CaMKII. This result is consistent with the importance of CaMKII in the formation of LTP [30,31]. By EM and immunoreactivity, several groups observed that shorter, stockier dendritic spines contain larger PSDs and a greater amount of AMPA receptors when compared to small PSDs in the hippocampus [32–34]. Ho wever, the mechanisms that re gulate protein levels in the PSD are unclear .

3.2.1 PROTEOMICS

OF THE

PSD

Some of the earliest proteomic studies were aimed at analyzing the protein composition of PSDs. These and later studies ha ve greatly benefited from the ability t purify the PSD to a relati vely high de gree. Fiszer and De Robertis [35] found that the nonionic deter gent Triton X-100 could solubilize the adjoining membrane in crude synaptosomal preparations while leaving the “subsynaptic structure” intact as assessed by electron microscop y (EM). Later modifications by Cohen et al. an Carlin et al. [36,37] involving additional sucrose gradient centrifugation steps yielded PSDs devoid of pre-synaptic contamination as assayed by EM and enzymatic assays. Purified PSDs resembled conc ve circular structures, which were virtually identical to those observed in vivo . This purification yields PSDs with little or no membran contamination, an ef fect of the KCl and deter gent used during the e xtraction. The most significant contamination obser ed was that from mitochondrial resident proteins as determined by ATPase activity and cytochrome C oxidase activity, although this contamination w as determined to be v ery small. This protocol has been the foundation for the majority of studies on the compositional nature of the PSD.

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Early proteomic-based methods to identify PSD constituents in volved staining one- and two-dimensional Western blots of purified PSD protein with antibodies fo known proteins [36–38]. One interesting method involved creating antibodies against PSD proteins by injecting purified and enzymatically digested PSDs into rabbit [39]. Sera collected from these rabbits were later used to screen e xpression clones and the expressed proteins were identified. This method resulted in over 200 cDNAs collected. Modern day approaches ha ve relied on one- or tw o-dimensional gel electrophoresis to prefractionate samples, follo wed by HPLC to resolv e peptides deri ved from the enzymatic digestion of protein mixtures prior to mass spectrometric analysis. After trypsin digestion of the protein, the peptides are often separated by HPLC and analyzed by quadrupole time-of-flight (Q OF) mass spectrometry. These techniques have resulted in a wealth of knowledge about many readily purifiable fraction [2,40]. Recent proteomic studies ha ve investigated the composition of the PSD by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis (2DE) followed by mass spectrometry [12–15,41,42] and ha ve resulted in the identification of hundreds of proteins in the PSD The growing number of proteins identified by these n vel mass spectrometry– based methods redefines what we kn w about synaptic function. The hundreds of identified PSD constituents represent a dverse set of signaling components including proteins involved in cell polarity, protein translation and de gradation, kinases/phosphatases, G-proteins and direct re gulators of these groups and a lar ge number of proteins of unidentified function. This information suggests that the PSD possesses a capacity for biochemical signal transduction that could greatly e xceed that of its electrical signal processing. Due to the application of no vel methodologies in proteomics, we can hope to more fully understand the function of the PSD.

3.3 METHODS The following subsections illustrates some of the proteomic-based methods that were used to study the post-synaptic density as well as other protein comple xes.

3.3.1 2DE-LC-MS/MS The studies by Yoshimura et al. [15] and Li et al. [13] relied in part on tw odimensional electrophoresis (2DE) to pre-fractionate PSD samples prior to mass spectrometry. Protein spots resolved by 2DE were then subjected to tryptic digestion followed by mass spectrometric analysis. 2DE has been used for se veral decades, but recent adv ances that o vercome pH drifts by crosslinking ampholytes to the acrylamide gels have greatly increased protein resolution and, more importantly, the reproducibility of results. This method, which adds resolution by separating proteins via their isoelectric points, of fers advantages over traditional one-dimensional gels; however, it has significant dr wbacks. Hydrophobic proteins tend to precipitate out of solution at their isoelectric points, due to the lack of net char ge that helps solubilize proteins [43]. This fact is problematic especially for the PSD, a very densely packed and poorly soluble protein comple x containing many principal components that are

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Paralemmin Aralar 1 Neurofilament L Actin C-AMP PKI Protein Gα

P32-Rack

(a)

(b)

FIGURE 3.1 20 µg of PSDs were solubilized by either (a) 4% CHAPS, 8 M urea or (b) 2% ASB-14, 1% Tx-100, 7 M urea, 2 M thiourea, 10 mM Tris. Both samples contained 50 mM DTT. Both samples were rehydrated onto a 3.0 to 10.0 pH 7-cm strip (Amersham) and focused for 5 hr on a Multiphor apparatus (3500V max). Notice thatASB is able to solibilize additional PSD proteins for 2DE. Identified protein spots are sh wn as e xamples.

hydrophobic or multipass transmembrane proteins. Thus that neither of these studies identified important transmembrane receptors such as AMPA or NMD A receptors is not surprising. We have noticed poor resolution of PSD proteins in 2D gels using traditional rehydration buffers such as 8M urea and 2% CHAPS. Ho wever, the use of no vel zwitterionic deter gents, such as ASB-14 or increased urea and CHAPS concentrations, can impro ve protein resolution of the PSD in 2D gels. Figure 3.1 shows increased number — and better resolved — protein spots using a combination of 2% ASB-14, 1% Tx-100 and 7 M urea, as well as 9.4 M urea and 4.5% CHAPS. 2DE presents additional problems in that higher molecular weight proteins, v ery basic or very acidic proteins do not enter the second dimension, or often appear as smears. These problems greatly decrease the set of proteins a vailable for identification, which is most li ely why Yoshimura et al. [15] and Li et al. [13] found significantly f wer proteins at the PSD by 2DE (250 visualized and analyzed spots corresponded to about 90 unique proteins) than studies using traditional one-dimensional SDS-PAGE.

3.3.2 1DE-LC-MS/MS Peng et al. [14] and Jordan et al. [12] used simple one-dimensional electrophoresis (1DE) to pre-fractionate PSDs. In both cases, the entire length of the in-gel sample was cut into 10 to 30 bands and digested with trypsin. The extracted peptides were further resolv ed by HPLC in a nanocolumn using re verse-phase chromatograph y infused directly into a mass spectrometer. This method allowed the identification o more than three times the number of proteins than by 2DE and with 40-fold less material (20 to 80 µg compared to 3 mg used for 2DE). These results point to a

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clear advantage of one-dimensional SDS-P AGE coupled to LC-MS/MS o ver 2DE for profiling proteomics of the PSD. Yoshimura et al. [15] used tw o-dimensional liquid chromatography coupled to tandem mass spectrometry. Tryptic peptides were resolved via two rounds of HPLC, an anion-e xchange column and subsequently via a reverse phase column. This technique w as applied to a lar ge quantity of PSDs (6 mg) and resulted in a lar ge number of identified proteins

3.3.3 2DE-DIGE While proteome profiling is an important and necessary step for a complete unde standing of the function of a protein comple x, proteomics can also be applied to study global proteome function. Unlike genomes, proteomes are dynamic and many proteomic studies focus on e xamining changes in proteome composition under various conditions. Changes in the protein composition of the PSD underlie changes in synaptic strength, and can ultimately influence learning and memor . Thus, the PSD is particularly suited for such studies. Methods such as immunoblotting with a variety of antibodies to known PSD components have been used to study changes in the protein composition of the PSD upon increased or decreased synaptic acti vation [26]. Ho wever, the adv ent of no vel technologies such as ICA T or 2D fluores cence dif ferential in-gel electrophoresis (2D-DIGE) mak es possible the study of these changes on a global le vel. Both of these methods rely on the incorporation of tags onto a population of proteins to differentiate it from a second population. While 2DE has the drawbacks mentioned above for profiling, its impr ved resolution over one-dimensional SDS-PAGE allows for monitoring changes of kno wn or unkno wn proteins. In this method, one set of purified PSDs (for xample, control neurons) is labeled with a c ysteine-reactive fluorescent dye (FITC). A second set of PSDs (for example, from glutamate-treated neurons) is labeled with a second fluorescent dy (Rhodamine), which is both weight and pI-matched for the first dye. In this ay, the location of spots on 2DE gels will not be altered with respect to each other due to differences in their respective fluorescent labels. The combined samples are then subjected to 2DE and visualized by fluorescence imagers to detect the a undance of proteins in specific spots. Equal l vels of protein from both samples should result in yellow spots, whereas all changes w ould be measured as shifts to ward green or red. While this technique is ideal for observing global changes in PSD composition, it w ould represent only those proteins resolv able by 2DE and requires e xpensive reagents and instrumentation. Moreover, as seen by Li et al. [13] and in Figure 3.1, single spots can contain man y dif ferent proteins. The co-migration of proteins in 2DE w ould yield ambiguous results. In these cases, a narro w pH range or longer isoelectric focusing strips could help resolv e these proteins.

3.3.4 RELATIVE QUANTIFICATION BY STABLE ISOTOPE LABELING AND MASS SPECTROMETRY A second and often f avored technique for e xamining relati ve changes in protein abundance is isotope-coded af finity tagging (IC T) [44]. Proteins are labeled with (usually cysteine) reactive tags that contain both biotin af finity tags and a specif

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isotopically encoded linker. Two or more sets of proteins can be labeled with dif ferent isotopically labeled tags, mixed and digested with trypsin. Labeled peptides can then be purified by a finity chromatograp y using the biotin tag and then subjected to tandem MS. Modern mass spectrometry instruments can resolv e the small mass difference between dif ferently isotopically labeled peptides. Thus, peaks dif fering in mass by the mass of the isotope tags can be measured and dif ferences in the relative abundance between the two peptide populations can be assessed.The original chemistry involved sulfhydryl-based chemistry; however, concerns over the relative scarcity of cysteines resulted in tags reacti ve to a v ariety of groups. ICAT has been successfully applied in a v ariety of biological systems, such as identification o matrix metalloproteinase substrates [45], changes in acti vated liver carcinoma cells [46] and erythroid dif ferentiation [47]. A related method called stable isotope labeling by amino acids in cell culture (SILAC) [48] metabolically labels all proteins through the incorporation of stable isotopically labeled amino acids. The relative abundance of experimentally induced changes in protein e xpression can then be assessed in a similar manner to ICA T, i.e., by measuring the relati ve changes in intensities between peaks that dif fer by the mass differences of the isotopic forms of the amino acids. Alternatively, whole organisms can be fed radiolabeled materials for in vivo incorporation of tags. Rats fed for 44 days with 15N-containing alg al cells were found to contain more than 90% 15N enrichment in liver and plasma. These rats were subsequently treated with cycloheximide and global changes in protein ab undance were assessed [49].

3.3.5 IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC) Analysis of PTMs using standard profiling techniques can yield substantial func tional information about protein comple xes. While current bioinformatics and mass spectrometry can identify only a fraction of the more than 100 kno wn PTMs, significant ad ances ha ve been made in the identification of some of the mor prominent PTMs such as phosphorylation. Phosphorylated peptides can be enriched using immobilized metal affinity chromatograp y (IMAC) [50]. Typically, Fe3+ columns are used to trap negatively charged ions, such as phosphorylated peptides. Ions trapped are then eluted and analyzed by mass spectrometry . These methods ha ve resulted in the identification of s veral “phosphoproteomes” [51] as well as changes in the phosphorylated state of a set of proteins in response to stimulation, such as adrenergic stimulation of cardiac myoc ytes [52] and endothelin treatment of lung fibroblasts [53]. Alternative methods e xploit the f act that phosphate moieties are labile at high pH and replace these groups with biotin tags for subsequent purificatio using avidin [54]. Jaffe et al. used a gallium-based metal affinity column to identif phosphorylated proteins in PSDs [55] and identified s veral novel phosphorylation sites on scaf folding proteins such as PSD-95. Collins et al. recently identifie phosphorylated proteins in synaptic preparations by enrichment of phosphopeptides using similar methods [56]. This group observed 289 unambiguously assigned phosphorylation sites in 79 proteins, including no vel phosphorylation sites of PSD-95 and delta and alpha catenin. Surprisingly absent from both studies were wellestablished phosphorylation sites for highly ab undant PSD components such as

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NMDA or AMPA. Ho wever, this study demonstrates that significant amounts o important functional data can be obtained; 92% of all identified phosphorylatio sites were unique.

3.3.6 AFFINITY PURIFICATION The identification of protein “interactosomes” via a finity purification and ma spectrometry has re vealed additional layers to the comple xity of protein function. Immunoprecipitation has been used for decades to confirm the presence of singl proteins by Edman AA sequencing. Mass spectrometry–based identification of co precipitating protein complexes is an important method to obtain lar ge quantities of functional data for proteins. This method has resulted in v aluable information on NMDA receptor complexes [57], the TNF/NFkB signaling pathway [58] and inward rectifying potassium channels comple xes [59]. Ho wever, immunoprecipitation of PSD-resident protein complexes presents significant technical challenges. PSDs ar purified, in la ge part, because of their insensitivity to detergent-based protein extraction (0.5% Tx-100). The purification of PSDs results in a highly compact an insoluble pellet that is only fully solubilized in the presence of 1 to 2% sodium dodecyl sulphate (SDS). These conditions are incompatible with preserving noncovalent protein interactions, including the antibody/antigen interactions necessary for immunopurification. The question is thus how to extract proteins from the PSD using gentle enough conditions to preserv e protein comple xes. Several groups ha ve used 1% Deoxycholic acid (DOC) at a pH of 9.0 follo wed by dialysis ag ainst a weakly solubilizing buffer (usually 0.1% Triton X-100, pH 7.5 and 150 mM NaCl). DOC is a naturally occurring bile acid that is used endogenously to emulsify f ats for absorption in the intestine. Ho wever, it can lead to protein denaturation, leading to the loss of protein binding sites and must thus be dialyzed out for optimal immunopurification. Other methods include solubilization of PSDs in radioimmunopre cipitation assay (RIPA) or equivalent buffers containing 0.5% DOC as well as 0.1% SDS. While these represent relatively harsh solubilizing buffers, proteins have been effectively co-immunoprecipitated in these buffers. Husi et al. identified 77 protein that interact with NMD A receptors [57]. NMD A receptor comple xes (NRCs) from RIPA- or DOC-solubilized synaptosomes were purified with a arose beads coated with NMDA receptor antibodies. The results re vealed man y pre viously identifie NMDA receptor interacting proteins as well as unkno wn members. Ho wever, proteins identified were obtained from the entire SDS-based eluate from the colum and thus include a great number of unspecifically bound proteins. G ven the substantial number of nonspecific interactions resulting from such assays, sveral groups have developed tandem purification protocols to further purify captured compl xes [60]. In tandem af finity purification AP), proteins are tagged with tw o af finit tags, often separated by specific protease substrate sequences. Solubilized complxes are captured using affinity matrices, gently rem ved from the column using specifi proteases, and subsequently purified a ain using a second af finity matrix. These techniques substantially reduce the amount of nonspecific background and hve been used to identify man y macromolecular comple xes. Regardless of the method, that “interactosomes” likely represent a heterogeneous population of protein comple xes

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present in the initial tissue used is important. Thus, that all proteins identified i these studies are components of single protein comple xes is unlik ely.

3.4 A COMPARISON OF PSD PROTEOMICS STUDIES The recent profiling studies of the PSD all w a unique opportunity to compare proteomics-based techniques and results. We have obtained the sets of PSD proteins identified by all four groups and h ve compared them using the BLASTCLUST program to perform pairwise comparisons and cluster proteins into homology groups. BLASTCLUST returns clusters of proteins that e xhibit a gi ven homology, which we set at 80%. This setting usually clusters orthologs and other ambiguities arising from database redundancies and errors; Table 3.1 illustrates the dif ferences found. The PSD preparation methods varied slightly in all cases. Moreover, in some studies, only forebrains were used, which could significantly alter the results The studies from Peng et al. [14] and Li et al. [13] include synaptosomal membrane fractions because no second sucrose step centrifug ation was performed on Triton X-100 extracted synaptosomes; thus, these studies could include additional proteins found in lipid rafts (for e xample, the set of proteins found by Peng et al. [14] includes flotillin, a lipid raft mar er). Different methodologies were also used between the four groups, including ICA T [44], SDS-PAGE or 2DE-PAGE coupled to liquid chromatograph y and tandem mass spectrometry (MS/MS) or tw o-dimensional liquid chromatography (2D-LC) coupled to MS/MS. While these differences are sure to impact the outcome, of the at least 911 unique proteins found by all four groups, only 39 — less than 5% — were found by all four groups. The number of

TABLE 3.1 Comparison of PSD Proteins Found between Four Different Groups Research Proteins Method Used (Mgs of protein analyzed) Proteins Identified Unique to this Group Found Group (% of total # of unique proteins) Jordan, BA 452 SDS-PAGE + LC-MS/MS (0.025mgs) 219 (48%) Li, KW 175 2DE + LC-MS/MS (3 mgs) + ICAT (0.1 mgs) 54 (31%) Yoshimura, Y 480 2DE + LC-MS/MS (0.2 mgs) + 2D LCMS/MS (6 mgs) 214 (45%) Peng, J 351 SDS-P AGE + LC-MS/MS (0.080mgs) 127 (36%) Number % of TOTAL Total # of unique proteins (Proteins that are <80% homologous) 911 Number of Proteins found by all four groups 39 4% Number of Proteins found by at least three groups 117 13% Number of Proteins found by at least tw o groups 259 28% Note: For each group, all proteins found by all methods were consolidated into one protein list. The list of accession numbers or gi numbers, whiche ver were given, were submitted as a batch genbank query and a F ASTA-searchable database w as created from the protein’ s downloaded. In some cases not all proteins were returned, usually due to modifications in the NCBI database resulting fro corrections or general upk eep, thus the numbers reported here may be dif ferent than the numbers reported by the groups. When databases from all four groups were constructed, the y were subjected to BLASTCLUST clustering softw are set at an 80% threshold with no restriction for length (thus fragments could match with 100%).

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proteins found by at least three groups is not much lar ger at 117 (about 13%). The number of proteins unique to each set v aries from 30 to 50%.

3.5 CHALLENGES An additional significant technical challenge for functional proteomics of the PS is its lo w abundance relative to starting material. Most purification protocols yiel about 100 to 150 µg of PSDs per gram of brain tissue. ICAT or 2D-DIGE is typically performed using quantities in this order of magnitude. However, obtaining sufficien quantities of experimentally treated PSDs for these types of experiments is difficult Whereas broad-based pharmacologic treatment of li ve animals could solv e this problem, standard LTP- or LTD-inducing protocols are not available for large quantities of tissue. Ehlers [26] has successfully isolated PSDs from high-density cortical cultures to study changes in abundance of key PSD components by Western blotting in response to increased synaptic transmission. Ho wever, the mid-zeptomole sensitivity of immunoblotting is several orders of magnitude higher than the corresponding mid-femtomole sensitivity of mass spectrometry. Thus, at this stage, functional proteomics of neural cultures presents significant technical challenges. We have had some success in treating and isolating PSDs from synaptoneurosome preparations [61]. Whole brains dounce homogenized in an isotonic sucrose solution and passed through increasingly fine ylon filters can generate a crude synaptosomal fraction These can be then incubated in the presence of an ATP regeneration system (5 mM Mg-ATP, 80 mM creatine phosphate and 0.5 mg/ml creatine phosphokinase) to yield functionally active isolated synapses. Synaptosomes isolated in this w ay have been used to assay a v ariety of synaptic functions including AMPA receptor traf fickin [62]. We have observed that K +-induced depolarization, NMDA or glutamate treatment of synaptoneurosomes results in changes in GluR1 and GluR2 associated with PSDs (Figure 3.2). This method could easily be scaled to produce enough PSD for ICAT, 2D-DIGE or other comparative analysis. An exciting new study demonstrates that isolated PSDs can be inserted into giant liposomes, resulting in preparations which exhibit physiological currents [63]. Because mass spectrometry–based protein identification is ery sensiti ve and able to detect lo w-level contaminants, sample preparation is critical for the rele vance of identified proteins. All profiling studie of the PSD to date identified o vious contaminants such as histones, DN A polymerase and glial fibrillary acidic protein (G AP). Most if not all PSD purificatio protocols rely on the original studies from the lab of Siek evitz [36,37]. These methods yield a highly pure PSD fraction as observ ed by EM, although some mitochondrial contamination w as detected using enzymatic assays. Ho wever, from the profiling assays, these samples are clearly contaminated with presynaptic, mito chondrial and nuclear components. Dosemeci et al. ha ve shown that a significant portion of CaMKI α present in the PSD fraction is of nonsynaptic origin, and state that man y Triton-insoluble protein comple xes have densities similar to that of the PSD [64]. EM analysis of the PSD fraction shows that it is contaminated with spectrin and neurofilament fibe as well as nonsynaptic CaMKII clusters [65]. Vinade et al. used magnetic beads coated with antibodies to PSD-95 to further purify the PSD fraction and reduce

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N M D A K+ (1 ) K+ (2 ) K+ (3 )

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G lu

48

114 WB-GluR2 PSD 114 WB-GluR2 Synaptosomes

PSD

Raft

+Glutamate PSD Raft

114 WB-GluR1

64 50

WB-Flotilin

FIGURE 3.2 Synaptoneurosomes were treated with 1 mM Glutamate for 30 min (Glu), 40 µM NMDA for 5 min (NMD A), 60 mM KCl for 45 min (K + (1)), 60 mM KCl for 5 min + (2)), or identical to (K + (2)) followed by 10 min with artificial cerebrospinal fluid aCSF + but repeated four times, (K (3)). Note that GluR2 associated with PSDs is increased by these treatments. No changes are observ ed in the synaptosome fraction suggesting changes in the distribution of the GluR2 within synaptoneurosomes. PSDs and lipid rafts isolated from glutamate-treated (1 mM glutamate for 30 min) synaptoneurosomes shows the disappearance of GluR1 from PSDs b ut not from raft fractions. Flotillin immunostaining demonstrates the purity of the fractions.

nonsynaptic contaminants [65]. These experiments resulted in a fraction with substantially reduced amounts of the GFAP and neurofilaments, frequent contaminant of the traditionally purified PSD fraction. This method represents a significant ste forward in purifying the PSD and, if combined with mass spectrometry , could yield a much higher quality PSD protein composition, with the ca veat that only PSD-95 containing PSDs will be isolated. The general consensus in the field of proteomics is that protein lists are gettin larger and more meaningless. While the general scope of proteomics is to e xplore biology on a global scale, key proteins or post-translational modifications identifi in a protein complex will invariably lead to the development of newer, more refine models of its function. We belie ve that proper v erification of data resulting fro large-scale proteomics based studies is critical. Considering the controls necessary to v erify most biological w ork, which often studies a single gene or protein, dismissing these simply because the scale of the e xperiment has changed w ould be unwise. Ho wever, the v erification of la ge datasets is, for ob vious reasons, v ery difficult to achi ve. Biological v erification should be preceded by bioinformati verification. Proteomics today is possible due to the a undance of genomic data. It often deals with staggeringly lar ge amounts of data, which are interpreted by a

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variety of softw are packages. The statistical analysis of mass spectrometric data is a critical aspect for correct protein identification. Therefore, great care should be taken to develop a rigorous statistical package to minimize the introduction of f alse positive protein identifications based on random matches. The identification of proteins relies on the comparison of potential amino aci sequences determined by the mass spectrometer (actually , peptide fragmentation mass patterns) to those produced by virtual tryptic digestions of the entire genome of an or ganism (and often multiple or ganisms). This attribute is due to a generally unsolved problem regarding direct de novo sequencing from MS data. Most software designed for protein identification by mass spectrometry ta es into consideration the error range of the mass analyzer , the quality of the data and the size of the database to return a probability the mass spectrum matches a peptide in a database. However the increasingly lar ge number of queries (mass spectra) used for searches and larger and larger databases searched (especially when including multiple organisms or multiple post-translational modifications) generates a significant probabili that certain peptides might match purely by random chance. F ollowing the lead of Fenyo and Bea vis [66], we generated e xpectation v alues for our protein matches that take into account the number of MS/MS spectra (queries) we used in our search; few software packages return a probability of randomly matching a database entry (p-value) for identified proteins. We calculated a mathematical E-value, i.e., the number of times the protein w ould be e xpected to match a tar get randomly in one database search, and set an E-value threshold at less than 1 (proteins that w ould not match randomly even once). Importantly, we also determined an empirical E-value cutoff by searching our entire set against a randomized NCBI database using similar parameters. We believe developing rigorous statistical methods to verify such results is important. Statistical verification should be foll wed by biochemical v erification. Tagging identified proteins with GFP presents a relat vely simple method for confirming, t some degree, the presence of identified proteins in the compl xes studied. This has been performed for the centrosome [67] and for the PSD [12].We cloned 17 proteins and found that at least 16 are at least somewhat enriched in the PSD, with a majority highly enriched. This would ensure that future functional assays are established on solid ground.

3.6 CONCLUSIONS Proteomics is a rapidly e volving field with technologies capable of generatin enormous amounts of data. As e xpected, solid biological interpretation of these datasets is lagging w oefully behind. This f act is, in lar ge part, due to the lack of methodologies to statistically and biologically v erify the data created by proteomic studies. Our study of post-synaptic densities initially identified more than 400 proteins. A significant challenge as to collapse this dataset by removing redundancies based on database sequencing errors, cross-species orthologs, protein fragments and a myriad of other inconsistencies between different available databases. Because so much of protein identification relies on the databases used during mass spectro scopy data searches, ha ving effective software to help na vigate the often-confusing

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bioinformatic world is important. Once we had eliminated these redundancies from the list, we were left with around 750 proteins. Only after applying the statistical analysis described abo ve did we arri ve at our final number of 452 proteins Most proteomic-based studies ha ve been viewed as launching pads that lead to reductionist-based biological methods. In this respect, proteomics can be enormously successful in that it could catalyze decades of future research. Ho wever, this would undermine its principal goal, which is to obtain an o verview of protein comple x function. In neuroscience, we already be gin to see problems in vie wing synaptic plasticity by reductionist methods. A general fear among neuroscientists that study synaptic function is that the o verexpression of almost an y synaptically enriched protein can alter spine size or synaptic strength. Therefore, we believe the strength of proteomics lies in its ability to characterize the multiple characteristics of complex biological pathways. Initiatives by Grant et al. to characterize synaptic comple xes represent exciting studies that could help elucidate synaptic function [68,69]. Their work includes the establishment of protein netw ork maps based both on original research as well as work published in the scientific literature. Such approaches coul ultimately effectively integrate the biological study of synapses with applied mathematics and ph ysics and current theories of comple xity.

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34. Nusser, Z., Lujan, R., Laube, G., Roberts, J.D., Molnar , E. and Somogyi, P ., Cell type and pathway dependence of synaptic AMPA receptor number and v ariability in the hippocampus, Neuron, 21(3), 545–559, 1998. 35. Fiszer, S. and De Robertis, E.D., Action of Triton X-100 on ultrastucture and membrane-bound enzymes of isolated nerv e endings from rat brain, Brain Res. , 5(1), 31–44, 1967. 36. Cohen, R.S., Blomberg, F., Berzins, K. and Siekevitz, P., The structure of postsynaptic densities isolated from dog cerebral corte x. I. Overall morphology and protein composition, J. Cell Biol., 74(1), 181–203, 1977. 37. Carlin, R.K., Grab, D.J., Cohen, R.S. and Siekevitz, P., Isolation and characterization of postsynaptic densities from v arious brain re gions: Enrichment of dif ferent types of postsynaptic densities, J. Cell Biol., 86(3), 831–845, 1980. 38. Walsh, M.J. and K uruc, N., The postsynaptic density: Constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing, J. Neurochem., 59(2), 667–678, 1992. 39. Langnaese, K., Seidenbecher , C., Wex, H., Seidel, B., Hartung, K., Appeltauer, U., Garner, A., Voss, B., Mueller , B., Garner , C.C. and Gundelfinge , E.D., Protein components of a rat brain synaptic junctional protein preparation, Brain Res. Mol. Brain Res., 42(1), 118–122, 1996. 40. Taylor, S.W., Fahy, E. and Ghosh, S.S., Global organellar proteomics, Trends Biotech., 21(2), 82–88, 2003. 41. Satoh, K., Takeuchi, M., Oda, Y., Deguchi-Tawarada, M., Sakamoto, Y., Matsubara, K., Nag asu, T. and Takai, Y., Identification of act vity-regulated proteins in the postsynaptic density fraction, Genes Cells, 7(2), 187–197, 2002. 42. Walikonis, R.S., Jensen, O.N., Mann, M., Pro vance, D.W ., Jr ., Mercer , J.A. and Kennedy, M.B., Identification of proteins in the postsynaptic density fraction by mas spectrometry, J. Neurosci., 20(11), 4069–4080, 2000. 43. Santoni, V., Molloy, M. and Rabilloud, T., Membrane proteins and proteomics: Un amour impossible? Electrophoresis, 21(6), 1054–1070, 2000. 44. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H. and Aebersold, R., Quantitative analysis of comple x protein mixtures using isotope-coded af finity tags, Nat. Biotech., 17(10), 994–999, 1999. 45. Tam, E.M., Morrison, C.J., Wu, Y.I., Stack, M.S. and Ov erall, C.M., Membrane protease proteomics: Isotope-coded af finity tag MS identification of undescrib MT1-matrix metalloproteinase substrates, Proc. Natl. Acad. Sci. USA , 101(18), 6917–6922, 2004. 46. Yan, W., Lee, H., Deutsch, E.W., Lazaro, C.A., Tang, W., Chen, E., Fausto, N., Katze, M.G. and Aebersold, R., A dataset of human li ver proteins identified by protei profiling via isotope-coded a finity tag (IC T) and tandem mass spectrometry , Mol. Cell. Proteomics, 3(10), 1039–1041, 2004. 47. Brand, M., Ranish, J.A., K ummer, N.T., Hamilton, J., Ig arashi, K., Francastel, C., Chi, T.H., Crabtree, G.R., Aebersold, R. and Groudine, M., Dynamic changes in transcription factor complexes during erythroid dif ferentiation revealed by quantitative proteomics, Nat. Struct. Mol. Biol ., 11(1), 73–80, 2004. 48. Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A. and Mann, M., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to e xpression proteomics, Mol. Cell. Pr oteomics, 1(5), 376–386, 2002.

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The Dynamic Synapse 49. Wu, C.C., MacCoss, M.J., Ho well, K.E., Matthe ws, D.E. and Yates, J.R., III, Metabolic labeling of mammalian or ganisms with stable isotopes for quantitati ve proteomic analysis, Anal. Chem., 76(17), 4951–4959, 2004. 50. Scanff, P., Yvon, M. and Pelissier , J.P., Immobilized Fe 3+ affinity chromatographi isolation of phosphopeptides, J. Chrom., 539(2), 425–432, 1991. 51. Salih, E., Phosphoproteomics by mass spectrometry and classical protein chemistry approaches, Mass Spectrom. Rev., 24(6): 828–46, 2005. 52. Chu, G., Egnaczyk, G.F ., Zhao, W., Jo, S.H., F an, G.C., Maggio, J.E., Xiao, R.P . and Kranias, E.G., Phosphoproteome analysis of cardiomyoc ytes subjected to betaadrenergic stimulation: Identification and characterization of a cardiac heat shoc protein p20, Circ. Res., 94(2), 184–193, 2004. 53. Stannard, C., Soskic, V. and Godo vac-Zimmermann, J., Rapid changes in the phosphoproteome show diverse cellular responses follo wing stimulation of human lung fibroblasts with endothelin-1, Biochemistry, 42(47), 13919–13928, 2003. 54. Oda, Y., Nagasu, T. and Chait, B.T., Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome, Nat. Biotech., 19(4), 379–382, 2001. 55. Jaffe, H., Vinade, L. and Dosemeci, A., Identification of n vel phosphorylation sites on postsynaptic density proteins, Biochem. Biophys. Res. Com ., 321(1), 210–218, 2004. 56. Collins, M.O., Yu, L., Coba, M.P ., Husi, H., Campuzano, I., Blackstock, W.P., Choudhary, J.S. and Grant, S.G., Proteomic analysis of in vivo phosphorylated synaptic proteins, J. Biol. Chem., 280(7): 5972–82, 2005. 57. Husi, H., Ward, M.A., Choudhary, J.S., Blackstock, W.P. and Grant, S.G., Proteomic analysis of NMD A receptor -adhesion protein signaling comple xes, Nat. Neurosci., 3(7), 661–669, 2000. 58. Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P.O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., Hopf, C., Huhse, B., Mangano, R., Michon, A.M., Schirle, M., Schlegl, J., Schwab, M., Stein, M.A., Bauer, A., Casari, G., Dre wes, G., Ga vin, A.C., Jackson, D.B., Joberty , G., Neubauer , G., Rick, J., Kuster, B. and Superti-Furga, G., A physical and functional map of the human TNFalpha/NF-kappa B signal transduction pathw ay, Nat. Cell Biol ., 6(2), 97–105, 2004. 59. Leonoudakis, D., Conti, L.R., Radek e, C.M., McGuire, L.M. and Vandenberg, C.A., A multiprotein traf ficking compl x composed of SAP97, CASK, Veli, and Mint1 is associated with inw ard rectifier Kir2 potassium channels, J. Biol. Chem ., 279(18), 19051–19063, 2004. 60. Rigaut, G., She vchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B., A generic protein purification method for protein compl x characterization and proteome exploration, Nat. Biotech., 17(10), 1030–1032, 1999. 61. Schwartz, R.D., Sk olnick, P., Hollingsw orth, E.B. and P aul, S.M., Barbiturate and picrotoxin-sensitive chloride efflux in rat cerebral cortical synaptoneurosomes, FEBS Lett., 175(1), 193–196, 1984. 62. Bernard, J., Massicotte, G. and Baudry , M., Potassium-induced depolarization of rat telencephalic synaptoneurosomes increases [3H]amino-3-h ydroxy-5-methylisoxazole-4-propionate receptor binding, J. Neurochem., 58(1), 387–389, 1992. 63. Wyneken, U., Marengo, J.J. and Orre go, F ., Electroph ysiology and plasticity in isolated postsynaptic densities, Brain Res. Br ain Res. Re v., 47(1-3), 54–70, 2004. 64. Dosemeci, A., Reese, T.S., Petersen, J. and Tao-Cheng, J.H., A novel particulate form of Ca(2+)/calmodulin-dependent [correction of Ca(2+)/CaMKII-dependent] protein kinase II in neurons, J. Neurosci., 20(9), 3076–3084, 2000.

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65. Vinade, L., Chang, M., Schlief, M.L., Petersen, J.D., Reese, T.S., Tao-Cheng, J.H. and Dosemeci, A., Affinity purification of PSD-95-containing postsynaptic co plexes, J. Neurochem., 87(5), 1255–1261, 2003. 66. Fenyo, D. and Bea vis, R.C., A method for assessing the statistical significance o mass spectrometry-based protein identifications using general scoring schemes,Anal. Chem., 75(4), 768–774, 2003. 67. Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A. and Mann, M., Proteomic characterization of the human centrosome by protein correlation profiling Nature, 426(6966), 570–574, 2003. 68. Grant, S.G. and Blackstock, W.P., Proteomics in neuroscience: From protein to network, J. Neurosci., 21(21), 8315–8318, 2001. 69. Grant, S.G., Systems biology in neuroscience: Bridging genes to cognition, Curr. Opin. Neurobiol., 13(5), 577–582, 2003.

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4

Phosphorylation SiteSpecific Antibodies as Research Tools in Studies of Native GABAA Receptors Jasmina N. Jovanovic

CONTENTS 4.1 4.2

Introduction ....................................................................................................58 Design, Production and Characterization of GAB AA Receptor Phospho-Site Specific Antibodies .................................................................63 4.2.1 Phosphopeptide Design and Preparation ...........................................64 4.2.2 Preparation of Phosphopeptide Antigens...........................................65 4.2.2.1 Phosphopeptide Coupling Protocol Based on Terminal Cysteine Residue .................................................65 4.2.2.2 Phosphopeptide Coupling Protocol Based on Terminal Lysine Residue ....................................................68 4.2.3 Rabbit Immunization Protocol ...........................................................68 4.2.4 Phosphorylation State-Specific Antisera Screening and Purificatio .........................................................................................69 4.2.4.1 First Screen: Dot-Immunoblotting of Synthetic Peptides ...............................................................................69 4.2.4.2 Second Screen: Immunoblotting of Dephospho/ Phospho Forms of the Holoprotein ....................................70 4.2.4.3 Third Screen: Expression of wt and Phospho-Site Mutants in Heterologous Cell Line Systems .....................71 4.2.4.4 Fourth Screen: Dephospho/Phospho Peptide Block ..........72 4.2.5 Affinity-Purification of Phosphorylation State-Specif Antibodies .75 4.2.5.1 Purification of Total IgGs from the Phospho-Specific Antiserum............................................................................75 4.2.5.2 Purification of Phospho-Specifi Antibodies Using Affinity Chromatograp y with Peptide Columns ..............76

57

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4.3 Concluding Remarks ......................................................................................78 4.4 Acknowledgments ..........................................................................................78 References................................................................................................................78

4.1 INTRODUCTION Type A γ-aminobutyric acid (GABAA) receptors represent a large and diverse family of ligand-gated CI– channels that mediate the majority of fast inhibitory neurotransmission in the brain [1] by causing transient reduction in the probability of action potential firing due to plasma membrane yperpolarization. Modifications of GA AA receptor function are belie ved to be critical for de velopment and coordination of neuronal activity underlying all physiological and behavioral processes, and the vast majority of neurons in the brain e xpress these receptors on their plasma membrane. Native, bicuculline-sensiti ve GAB AA receptors are hetero-pentamers of sub units with multiple isoforms classified as: α (1–6), β (1–3), γ (1–3), δ, ε, π and θ [2–4], with a common transmembrane topology comprising a lar ge N-terminal domain, four transmembrane domains (TMs), and a major intracellular domain between TMs 3 and 4 [5] (Figure 4.1b and Figure 4.1c). Some of the most common subunits, such as β3 [6] and γ2 [7] are essential for synaptic inhibition and or ganism survival. In contrast, indi vidual isoforms of the α subunit are highly specialized to maintain function of neuronal circuits associated with specific beh vioral phenotypes, including sedation, memory, anxiety, arousal and others [8,9]. GAB AA receptors are also the main sites of action for a v ariety of compounds with potent sedati ve, hypnotic, anxiolytic and anticonvulsant effects, such as benzodiazepines, barbiturates, neurosteroids and anaesthetics [10,11]. One of the key morphological features of GABAergic synapses allowing for the high effica y of stimulus/transmission coupling is the presence of GABAA receptors clustered at the post-synaptic membrane opposite of GAB A-releasing presynaptic terminals (Figure 4.1a). Targeting of GAB AA receptors to these sites is f acilitated, at least in part, by interactions with a v ariety of re gulatory proteins [12], whereas their stabilization at post-synaptic sites is partially dependent on multifunctional protein gephyrin [13,14]. Receptor removal from synaptic sites, largely via clathrindependent endocytosis, is mediated by dynamic association with the AP2 adaptin complex [15]. Intracellular trafficking of GA AA receptors is thus critical for shaping the response of the post-synaptic neuron to released GAB A and operates o ver the time scale of tens of minutes to hours [16]. In contrast, the acute and transient modulation of GABAA receptor function in a variety of neuronal cell types, occurring within seconds or minutes, is believed to involve a reversible modification of GA AA receptor subunits via direct phosphorylation. However, at the present time, the direct evidence for phosphorylation of native GABAA receptors is just beginning to emerge and it is f acilitated by the production of phosphorylation state-specific antibodie designed to bind to specific GA AA receptor subunits. The ef fects of acti vation of v arious signal transduction pathw ays ha ve been shown to range from potentiation to inhibition of GAB A-gated currents in different types of neurons [17]. For example, activation of protein kinase A (PKA) in cultured

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(a) α (1−6) β (1−3) γ (1−3) δ ε π θ

α

β

α

β δ

γ

(b)

N

C

P (c)

FIGURE 4.1 Structural hierarch y in or ganization of nati ve GABAA receptors. (a) GAB AA receptors are enriched at the post-synaptic membrane opposing the pre-synaptic GAB A releasing terminal where the y form clusters of functional receptor molecules. (b) The prototypical GABAA receptor is composed of a heteropentameric assembly of tw o α, two β and one γ subunit, isoforms of which are classified within separate groups on a basis of sequenc similarity. Currently, several classes of GABAA receptor subunits exist with multiple isoforms: α (1–6), β (1–3), γ (1–3), δ, ε, π and θ. (c) The common molecular structure of all GAB AA subunits contains a lar ge extracellular N-terminal domain, four transmembrane domains and a short e xtracellular C-terminal domain. The long intracellular loop between TMs 3 and 4 contains multiple sites for phosphorylation and binding of v arious protein kinases.

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superior cervical g anglia neurons, spinal cord neurons, cerebellar granule cells, hippocampal p yramidal cells and olf actory b ulb granule cells [18–22] has been demonstrated to correlate with a decrease in GAB A-activated currents. In contrast, activation of PKA in hippocampal dentate granule neurons, cerebellar Purkinje neurons and olf actory bulb granule cells has been correlated with an enhancement of GABA-activated currents [23,24]. Similar cell type-specific di ferences have been observed by activation of various G-protein coupled receptors (GPCR). For example, activation of D1 receptors by specific agonists, or injections of cAMP analogues were both found to cause a decrease in GAB A-activated currents in acutely dissociated neostriatal neurons. The role of PKA in this process w as confirmed by th application of specific PKA inhibitors [22]. Similar PKA-dependent inhibition o GABA-activated currents w as detected in olf actory bulb granule cells in response to D1 receptor acti vation by dopamine. In contrast, in olf actory bulb mitral cells, dopamine was shown to enhance GAB AA receptor function through the acti vation of D2 receptors and concomitant stimulation of Ca2+/phospholipid-dependent protein kinase (PKC) [25]. One possible e xplanation for the di versity of functional ef fects in dif ferent neuronal cell types is the functional compartmentalization of D1 and D2 receptors with dif ferent GABAA receptor subtypes. Establishing whether these differences are the direct consequence of changes in the phosphorylation state of different GAB AA receptor subtypes, or in volve alternati ve mechanisms such as activation of different signalling pathways, variations in levels of kinases and phosphatases and their anchoring molecules [26,27], or phosphorylation of other proteins associated with GAB Aergic synapses is important. Di verse functional ef fects on GABA-gated currents were also recorded in response to ele vated intracellular Ca 2+ levels and, again, these effects were found to depend on a neuronal cell-type studied [28,29]. Downstream of c ytosolic Ca 2+ increases, both Ca 2+/calmodulin-dependent 2+/calmodulin-dependent protein phosprotein kinases (CaM kinase II) and Ca phatases (protein phosphatase 2B/calcineurin) could underlie the re gulation of GABA-currents, gi ven that both classes of signalling molecules were sho wn to modulate GABAA receptor phosphorylation state, at least in vitro, by phosphorylating [30] or dephosphorylating [31] se veral sites in β and γ subunits. CaM kinase II and PKA were independently found to play important roles in the phenomenon known as “rebound potentiation ” of GAB Aergic currents in cerebellar Purkinje neurons, where glutamater gic stimulation leads to an increase in intracellular Ca 2+ and an enhancement of GAB Aergic currents [23,32]. In some neuronal cell types, activation of tyrosine kinases was correlated with functional modulation of GABAA receptors. The increase in the amplitude of mIPSCs in hippocampal neurons and brain slices in response to insulin w as proposed to be mediated by both tyrosine [33] and serine/threonine-specific kinases [31]. Similar enhancement of GA Aergic currents w as observ ed in cultured superior cervical g anglion (SCG) neurons and spinal cord neurons in response to c-src acti vation [34]. In Purkinje neurons, the activation of TrkB receptor by brain-deri ved neurotrophic f actor BDNF w as found to promote the ef fect of c-src tyrosine kinase acti vation, resulting in an increase in the amplitude of mIPSCs [35]. Notwithstanding the aforementioned e vidence for the role of phosphosphorylation/dephosphorylation in the modulation GAB AAR function, among the lar ge

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number of GABAA receptor subunit classes, members of only tw o of these classes, β and γ subunits, ha ve been demonstrated to comprise highly conserv ed serine, threomine and tyrosine residues localized within the intracellular loop between TMs 3 and 4 [17]. Early in vitro studies using intracellular domains of v arious β and γ subunits prepared as GST -fusion proteins, ha ve indicated that these residues are in vitro substrates for a variety of kinases including: PKA, PKC, CaM kinase II, cGMPdependent protein kinase (PKG) and the non-receptor tyrosine kinase, c-src [36]. Individual amino acid residues phosphorylated in vitro have been characterized by site directed mutagenesis. One remarkable result from these in vitro phosphorylation studies was the observation that multiple protein kinases appear equally capable of phosphorylating the same set of phosphorylation sites in the β and γ subunits of GABAA receptors (Figure 4.2). Some of the phosphorylation studies with GST-fusion proteins of β and γ subunit domains have been recapitulated within the conte xt of the full-length sequence of the receptor subunits expressed in heterologous expression systems (HEK293 cells, COS-7 cells), and correlated further with functional modulation of GAB AA receptors. Phosphorylation of β1, β2 and β3 subunits by PKA on conserved serine residues (Ser409/410 present in all three β isoforms, together with Ser408 present only in the β3 sub unit) w as correlated with a bidirectional modulation of GAB A-gated currents which was shown to depend on the type of β subunit expressed [37]. Thus, the functional outcome of β1 subunit phosphorylation on Ser409 appeared to be a decrease in GAB A-gated currents, whereas β3 subunit phosphorylation on Ser408 and Ser409 led to an enhancement in GABA-gated currents. Phosphorylation of both β and γ subunits of GAB AA receptors by PKC w as demonstrated to cause an inhibition in GAB A-evoked currents in heterologous systems [38], whereas phosphorylation of conserv ed tyrosine residues in the γ subunit by c-src resulted in an increase in GABA-gated currents [34].

γ

α β

γ2L: Ser327 P γ 2S: Ser327 P Ser348 P Thr350 P Tyr365 P

Ser343 P Ser348 P

β1: Ser384 P Ser 409 P

Thr350 P

β2: Ser410 P

β3: Ser383 P Ser 408 P Ser 409 P

Tyr367 P

Src

PKC

CaMKII

PKA

PKG

AKT

FIGURE 4.2 In vitr o phosphorylation of GAB AA receptor sub units by multiple protein kinases. Individual phosphorylated residues within the TM 3 and 4 intracellular loops of β1, β2, β3 and γ2 subunits, and corresponding protein kinases, are depicted schematically . Note that multiple protein kinases sho w the ability to phosphorylate in vitro the same phosphorylation sites in the GAB AA receptor subunits.

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Given the potent effects of direct phosphorylation of GAB AA receptors on their functional properties in heterologous systems [37,38], studying the phosphorylationdependent mechanisms re gulating GABAA receptors in neuronal systems and their modulation by physiologically relevant neuron-specific xtracellular and intracellular signals is imperati ve. Currently, the most adv anced understanding of signalling mechanisms regulating native GABAA receptors is based on biochemical and electrophysiological evidence for a direct association of PKC βII and receptor for activated C kinase (RA CK-1) with the β1 through 3 sub units. The binding of PKC to the β subunits has been localized to the sequence encompassing the major phosphorylation sites, Ser408/409(410) [39]. RA CK-1 was found to bind independently of PKC to the sequence in the β subunits localized immediately upstream of the PKC binding sequence [39]. RACK-1 binding stimulates PKC activity, leading to a further increase in phosphorylation state of β subunits on Ser408/409 [17]. In addition, PKA signalling pathways have also been implicated because A-kinase (PKA) anchoring proteins (AKAPs) mediate binding of PKA to some GABAA receptor subtypes [40]. Thus, direct phosphorylation of GAB AA receptors has emer ged as an essential mechanism for the dynamic modulation of the channel activity and might contribute to long-term changes in neuronal function, such as those e xperimentally observed as long-term potentiation/depression, and vie wed as cellular correlates of learning and memory [26]. The complex regulation of GAB AA receptor function in situ , in correlation with the phosphorylation of GAB AA receptors by multiple protein kinases, at least in vitro, raises man y important questions re garding the specificit of GABAA receptor regulation by phosphorylation, such as the mechanisms of spatial or temporal inte gration of signalling cascades as well as selection of signals. We have begun to uncover answers to some of these questions by characterizing in detail the molecular pathw ay that couples the acti vation of neurotrophin/T rk receptors to phosphorylation and functional modulation of GAB AA receptors in neurons [41]. Our experiments have demonstrated that a temporally bi-phasic mode of functional modulation of GAB Aergic currents by BDNF is achie ved by f acilitating dynamic changes in GAB AA receptor phosphorylation. These include acute and transient enhancement of β3 subunit phosphorylation at sites Ser408/409, followed by a rapid dephosphorylation to le vels significantly l wer than the phosphorylation state seen in untreated samples. These short-term biochemical and functional changes are dependent on the acti vities of PKC and protein phosphatase 2A (PP2A), and are mediated by transient recruitment of these signalling proteins to GAB AA receptors as judged by co-immunoprecipitation. In response to BDNF , an increase in the amount of GAB AA receptor–associated PKC, RA CK-1 and PP2A were detected within the first 10 minutes and as followed by dissociation of PKC and RA CK-1 at later time points (30 minutes in the presence of BDNF). However, PP2A remained associated with the receptor at later time points, coinciding with a rapid dephosphorylation of Ser408/409. Importantly, the extent of PKC binding to the receptor w as significantly decreased by phosphorylation of Ser408/409, whereas association o PP2A was enhanced in in vitro binding assays. Phosphorylation of sites Ser408/409 thus re gulates not only channel acti vity b ut also the af finity of arious signalling molecules in their direct association with GAB AA receptors [41].

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TABLE 4.1 Characterized Phosphorylation Sites in GABAA Receptor and Subunits P-Site β1 β2 β3

γ2S

γ2L

Ser384 Ser409 Ser410 Ser383 Ser408 Ser409 Ser327 Ser348 Thr350 Tyr365 Tyr367 Ser327 Ser343 Ser348 Thr350 Tyr365 Tyr367

In Vitro CaMKII PKA, PKG, PKC, CaMKII PKA, AKT, PKC, CaMKII CaMKII PKC, PKA PKC, PKA, CaMKII, PKG PKC CaMKII CaMKII Src Src PKC PKC CaMKII CaMKII Src Src

In Heterologous Systems

In Neuronal Systems

PKA, PKC AKT

AKT

PKC, PKA PKC, PKA PKC

PKC, PKA PKC, PKA

Src Src PKC PKC

Src Src

Src Src

Src Src

In summary, the e xperimental evidence for a direct phosphorylation of nati ve GABAA receptors is greatly facilitated by the development of phosphorylation statespecific antibodies for specific receptor s units (Table 4.1) [31,41]. Using these powerful research tools, we have obtained the first insights into the neuronal signal ling mechanisms operating at the le vel of GABAA receptors, pointing to a v ery fin mode of regulation determined by neuronal cell-type, specific subcellular compart mentalization and possibly e ven the history of signalling e vents.

4.2 DESIGN, PRODUCTION AND CHARACTERIZATION OF GABAA RECEPTOR PHOSPHO-SITE SPECIFIC ANTIBODIES Phosphorylation state-specific antibodies pr vide us with a remarkably sensitive and specific immunochemical tool to detect and quantify changes in the phosphorylatio state of particular amino acid residues within the full-length protein sequence in intact tissue and cell preparations. These features are of particular importance in instances where a high de gree of sequence similarity between v arious isoforms of a protein of interest, often co-e xpressed within the same cell type, complicate the use of more traditional methods to studyin situ protein phosphorylation, for example, by prelabeling of intracellular ATP pool with 32P-orthophosphate, or by alternati ve post-hoc approaches such as “back” phosphorylation. Unlike these methods, the use

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of phosphorylation state-specific antibodies i volves, in the majority of cases, relatively simple and ine xpensive immunoblotting procedures. In addition, the great advantage of phosphorylation state-specific antibodies in studies of in situ protein phosphorylation becomes apparent when these antibodies can additionally be reliably employed for use in immunoc ytochemical studies, pro viding the great benefi of being able to visualize the subcellular distrib ution of phosphorylated forms of a protein of interest. Following the early attempts to raise phosphorylation-dependent polyclonal antibodies by immunization with phosphorylated forms of intact proteins [42], an alternative approach utilizing phosphorylated and unphosphorylated forms of synthetic peptides w as developed in the laboratory of Professor P aul Greengard at the Rockefeller University in Ne w York, NY [43]. This method has become a general protocol for the production of phosphorylation site-specific antibodies for protein with characterized sites of phosphorylation and it is today commercially e xploited in a lar ge-scale production of such antibodies by commercial concerns.

4.2.1 PHOSPHOPEPTIDE DESIGN

AND

PREPARATION

The high quality of phosphorylation site-specific antibodies is necessitated to displa their specificity in t o important aspects: to recognize only the protein of interest, and to recognize either the phosphorylated or unphosphorylated form with respect to a gi ven phospho-site within this protein. Thus, one of the most critical steps in raising a ne w phosphorylation site-specific antibody is to determine the lengt (optimally up to 10 residues) and sequence of peptides that will sufficiently resembl the amino acid sequence surrounding a particular phosphorylated residue in a protein of interest but, at the same time, allow the sufficient xposure of this site as the main part of the epitope while reducing the exposure of other phosphorylation-independent residues within the epitope. The presence of char ged amino acid residues such as aspartate and glutamate, as well as ar ginine and lysine, are kno wn to impro ve the antigenicity of synthetic peptides and are often part of consensus sequences recognized by many protein kinases. In addition, the presence of tw o or more phosphor ylated residues within the same peptide has pro ven to significantly increase th antigenicity of synthetic peptides. This phenomenon became e vident during the production of phosphorylation site-specific antibodies for GA AA receptor β subunits. The tw o most ab undant β subunit isoforms ( β2 and β3) contain a highly conserved stretch of basic amino acid residues within the lar ge intracellular loop between TMs 3 and 4. This sequence includes a well-characterized phosphorylation site, Ser410, present in the β2 subunit, or Ser409, present in the β3 subunit, with one important dif ference between the tw o isoforms — namely , the β3 sub unit contains an additional phosphorylation site Ser408 within the same sequence F ( igure 4.3a). The presence of t wo phosphorylated serine residues significantly imp oved the antigenicity of this peptide resulting in the successful production of an antiserum specific for phospho β3 subunit after the first round of immunization Modifications of the nat ve amino acid sequence can be introduced at the N- or C-terminus of a synthetic peptide to enable the conjug ation to the carrier protein as well as direct coupling to the resin of a column for af finity purification of t

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antiserum. The coupling procedure is routinely based on the presence of an N- or C-terminal cysteine residue in the synthetic peptide, pro viding the sulfhydryl group m-maleimidodibenzoyl for coupling via m-maleimidodibenzo yl sulfosuccinimide ester bond. Thereby, the coupling reaction will occur at one end of the peptide, allowing the other end to be fully accessible as the epitope. Phosphorylated residues should ideally be positioned closer to that end of the peptide to be suf ficientl exposed to the immune response. This same feature is also important for the efficien purification of the phosphorylation site-specific pool of antibodies from the antis rum using af finity chromatograp y, where the coupling reaction to the resin will occur at one end of the peptide, lea ving the other end fully accessible to binding of specific antibodies. The alternative phospho-peptide coupling reactions are based on the presence of a tyrosyl residue at one of the ends of the peptide, thus requiring the use of bis-diazotized benzidine [44] as a coupling reagent, or the presence of εamino group of lysine residue, in volving the use of glutaraldeh yde as a coupling reagent [45]. Two other important determinants of peptide antigenicity are the distance of the phosphorylated residue from the free end of the peptide (N- or C-terminal) and the nature of the amino acid at the free end of the peptide; this should ideally contain a small side-chain, such as alanine [46]. Today, numerous companies pro vide services for the synthesis and purification of phosphorylated peptides.We routinely use the Protein Sequencing F acility at Rock efeller Uni versity. The methodologies employed by this facility have been described in detail elsewhere [46,47], and further information could be obtained online from http://pdtc.rockefeller.edu/syn/syn1.html.

4.2.2 PREPARATION

OF

PHOSPHOPEPTIDE ANTIGENS

For successful production of phosphorylation site-specific antibodies, phospho ylated peptide antigens are recommended to be short in sequence as pre viously mentioned. Due to their small size, these peptides are required to be coupled to large carrier proteins to invoke a sufficiently strong immune response. This also prevents their degradation by tissue peptidases and prolongs their half-life within the immunized animal. Here, tw o routinely used protocols for coupling of phosphopeptides to the carrier proteins are described. 4.2.2.1 Phosphopeptide Coupling Protocol Based on Terminal Cysteine Residue Cysteine-containing phosphopeptides are coupled to the carrier proteins using the heterobifunctional cross-linker sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Pierce). Sulfonated NHS-ester cross-link ers, supplied as sodium salts, are water soluble, non-cleavable and membrane impermeable. The maleimide group is the most selective for sulfhydryl group when the pH of the reaction is k ept between 6.5 and 7.5, and a stable thioether linkage formed in this reaction cannot be clea ved under ph ysiological conditions. The NHS group of sulfo-MBS is firs coupled to free amino groups (N-terminal and ε-amino group of lysine) in the carrier protein and then, after e xcess cross-link er is remo ved by a desalting column, the

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β1: Pro-Gly-Lys-Gly-Arg-Ile-Arg-Arg-Arg-Ala-Ser409-Gln-Leu β2: Gln-Lys-Lys-Ser-Arg-Leu-Arg-Arg-Arg-Ala-Ser410-Gln-Leu β3: His-Lys-Lys-Thr-His-Leu-Arg-Arg-Arg-Ser408-Ser409-Gln-Leu (a) st 1 screen

ng. pep 100

UCL39 pre-immune serum

50 25 5

100 50

UCL39 test bleed 1

25 5

100 50

UCL39 test bleed 2

25 5 +

−

−

−

deP-peptide

−

+

−

−

diP408/409-peptide

−

−

+

−

P409-peptide

−

−

− (b)

+

P408-peptide

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FIGURE 4.3 First screen: Characterization of anti-P-β3 antiserum (UCL39) using dot-immunoblotting assay. (a) High de gree of similarity in amino acid sequence encompassing the phosphorylation sites Ser409 in the β1 subunit, Ser410 in the β2 subunit and Ser408/Ser409 in the β3 subunit. (b) Increasing amounts (5, 25, 50 and 100 ng) of dephospho, di-phospho Ser408/409, and single-phospho Ser408 or Ser409 forms of the antigen peptide were spotted directly onto the PVDF membrane. Immunoblotting analysis using 1:100 dilution of the preimmune serum (upper panel), first test bleed (middle panel) and second test bleed (l wer panel) emplo yed an anti-rabbit secondary antibody coupled to alkaline phosphatase. The binding of antisera to dot-blotted peptides w as visualized by accumulation of a purple precipitate formed due to con version of NBT and BCIP substrates by the bound alkaline phosphatase.

cysteine-containing epitope-peptide is coupled via reaction with the maleimide group. The most common carrier proteins in use are: Limulus hemocyanin (LHC, Sigma), Keyhole limpet hemocyanin (KLH, Sigma), bo vine thyroglobulin (Sigma) and bovine serum albumin (BSA, Sigma). The protocol for coupling [46] is briefl summarized below. 1. Reconstitute 4 mg of the carrier protein in 2 ml of 75 mM Na-phosphate, pH 7.2/250 mM NaCl. Filter the protein solution through the 0.45 µm syringe filter (Millipore) 2. Dissolve sulfo-MBS immediately prior to use in the same buffer to obtain 2.5 mg/ml stock solution. 3. Add 125 µl of this solution to 1.25 ml of carrier protein solution and incubate for 60 min at room temperature. 4. Prepare one 10-ml Speedy desalting column (Pierce) by equilibrating it with about ~70 ml of the b uffer. 5. Load carrier protein/MBS solution onto the column and elute 0.5 ml fractions with 0.5 ml aliquots of the b uffer. 6. Determine the protein content by using Bradford assay or reading the absorbance of each fraction at λ280. Pool tw o or three fractions with the highest protein content to mak e a final olume to 1.5 ml. 7. Dissolve 2 mg of peptide in 200 µl of buffer and perform tw o important checks before continuing with the coupling procedure. First, check the pH of the peptide solution and adjust it to pH 7.0 if acidic (this often results from the HPLC-purification of the peptide). Second, check th presence of free sulfh ydryl group using Ellman’ s reagent, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, Pierce) and measuring the absorbance at λ412. 8. Add the peptide solution to the solution of acti vated carrier protein and incubate for 2 hours at room temperature. 9. Dilute the mixture to a final olume of 1.8 ml with H 2O and prepare the following aliquots: 0.6 ml aliquot for initial immunization of tw o rabbits (equal to 300 µg of phosphopeptide/rabbit), and four aliquots of 0.3 ml for subsequent boosts (equal to 150 µg of phosphopeptide/rabbit). Store aliquots at 20°C.

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4.2.2.2 Phosphopeptide Coupling Protocol Based on Terminal Lysine Residue Phosphopeptides containing either a free NH 2-terminal group or ε-NH2 group of a lysine residue can be coupled to the carrier protein using glutaraldeh yde as a crosslinking reagent. The coupling ef ficien y could v ary with the molecular weight of the peptide and weight-percent of the peptide, assuming that peptides are lik ely to be 75% pure by weight. Peptide-to-carrier protein ratio should optimally be 25:1. This protocol emplo ys bovine thyroglobulin (Sigma) as a carrier protein. The protocol for coupling is briefly summarized bel w. 1. Reconstitute 33 mg of th yroglobulin in 3 ml of 125 mM sodium b uffer, pH 7.5, and filter through 0.45-µm syringe filter (Millipore) into a sma glass vial containing a small magnetic stirring bar . 2. Dissolve peptide in 200 µl H2O to check the solubility and determine the pH with indicator paper . If necessary, adjust the pH to 7.5. 3. Place vial on magnetic stirrer in a cold room. 4. Dilute 25% glutaraldeh yde stock (Sigma) to 0.2%. Add 2.5 ml glutaraldehyde slowly while stirring o ver a 20 min period. Incubate further for total of 120 min. 5. Dissolve 10 mg of Na-boroh ydride (Sigma) in 800 µl of H 2O. Add 150 µl of this solution to the stirred vial to quench the coupling reaction. Note that the solution will tend to foam as H 2 gas is released. Stir for 30 min before dialysis. 6. Dialyse solution against 2 l of 10 mM PBS, pH 7.5.After dialysis, prepare the following aliquots: one 1 ml aliquot for the initial immunization of two rabbits and four aliquots of the remaining solution for subsequent boosts of tw o rabbits.

4.2.3 RABBIT IMMUNIZATION PROTOCOL Today, a large number of companies provide services for the immunization of rabbits and production of antisera. We routinely use services provided by Cocalico Biologicals Inc. of Philadelphia, PA (http://www.cocalicobiologicals.com). The procedure used is briefly summarized here. Prior to injection, pre-immune serum is collecte from each rabbit. The immunization procedure starts with the intradermal injection of the phosphopeptide-carrier protein solution (300 µg for initial immunization/rabbit) mixed with complete Freund’s adjuvant (Difco). This is followed by two booster injections (150 µg/rabbit) during the third and fourth week of the procedure. The first bleed is collected during the fifth week The third boost is gi ven during the sixth week and second bleed collected during the seventh week. Serum is then tested and further boosts depend on the le vel of the immune response. If the response is negative after four or more boosts, continuing the immunization procedure with an alternative immunogen containing the same phosphopeptide conjugated to a different carrier protein is recommended.The titer of antibodies should be monitored regularly

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and the final xsanguination of the rabbit should be preceded by a final boost wit the antigen.

4.2.4 PHOSPHORYLATION STATE-SPECIFIC ANTISERA SCREENING AND PURIFICATION Newly obtained antiserum is subjected to a series of assays in order to be fully characterized as a tool with which to monitor and quantify the phosphorylation state of a protein of interest. Here, the production and characterization of GABAA receptor anti-P-β3 antibody is described in detail. The amino acid sequence of a phosphopeptide used for immunization is depicted in the Figure 4.3a. In addition to testing for specificity of this antibody t ward the phosphorylated form of the β3 subunit, we have also tested whether this antibody binds preferentially to the diphospho form of the β3 subunit, containing both P-Ser408 ands P-Ser409 residues, in comparison with single phospho forms of this sub unit, containing either P-Ser408 or P-Ser409. This additional level of specificity as important to determine at this stage to ensure that the P-β3 antibody specifically binds to the phosphorylated β3 subunit, the only isoform of β subunits containing two phosphorylated residues, without cross-reacting with the β1 and β2 subunits. 4.2.4.1 First Screen: Dot-Immunoblotting of Synthetic Peptides The initial screen of a newly obtained antiserum is routinely carried out using a dotimmunoblotting assay, which emplo ys both dephospho and phospho forms of the peptide used as antigen. The result of this screen usually indicates the specificity o the antiserum toward the phosphorylated peptide, as well as the titre of the phosphospecific pool of antibodies within the antiserum. The dot-immunoblotting assay [46] used for the initial characterization of the GAB AA receptor anti-P-β3 antiserum is described below. The primary screen of the GAB AA receptor anti-P- β3 antiserum (UCL39) w as performed using increasing amounts of dephospho-, P-Ser408-, P-Ser409- and PSer408/409 peptides (Figure 4.3b). Peptides were dissolv ed in 50 mM HEPES, pH 7.6, and increasing amounts (5, 25, 50 and 100 ng) spotted directly onto the polyvinylidiene difluoride (PVDF) membrane (Immobilon , Millipore). The binding of peptides w as confirmed by probing the membranes with Ponceau S protein stain Immunoblotting was carried out in 50 mM Tris, pH 7.5/200 mM NaCl/0.05% (v/v) Tween-20 (Sigma). To reduce the nonspecific binding, membranes were first incubat in the same b uffer containing 2 mg/ml BSA for 30 min. This step was followed by a 90 min incubation with the follo wing test bleeds (all at the dilution of 1:100): preimmune serum, first and second test bleed. Membranes were ashed four times and incubated further in the presence of an anti-rabbit secondary antibody coupled to alkaline phosphatase (Promega) for 60 min, at a dilution of 1:1000. Membranes were washed four times with TBS/T, followed by two washes with TBS and a single w ash with alkaline phosphatase buffer (100 mM Tris, pH 9.5/100 mM NaCl/10 mM MgCl2). Two color substrates specific for alkaline phosphatases — nitro blue tetrazolium (NB, 35 µl/5 ml of buffer, Promega) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, 17.5

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µl/5 ml of b uffer, Promega) — were added and the purple precipitate w as developed to visualize the binding of antibodies to dot-blotted peptides. The results of this assay are presented in Figure 4.3b. This initial screen pro vides us with important information as to whether and how to proceed with the immunization protocol. Often the antiserum contains a mixture of Immunoglobulin G ( IgG) with the reacti vity toward both phospho- and dephospho-peptide, and the ratio of different IgG pools can change during the course of immunizations and boosts. Ho wever, e ven if the result of dot-immunoblotting assay indicates the presence of high-titer antibodies, partially or completely specifi for the phosphorylated form of the peptide, secondary tests must be carried out to confirm the immunoreact vity toward the same phosphorylation sites within the fulllength sequence of a protein of interest. 4.2.4.2 Second Screen: Immunoblotting of Dephospho/Phospho Forms of the Holoprotein Secondary screening of a ne wly obtained antiserum requires the a vailability of sufficient purified amounts of the protein of interest. wever, if the nati ve holoprotein is not easily available, its recombinant form expressed either as a holoprotein or as a lar ge fragment of the protein can be used. Most commonly , these proteins are purified from E. coli as GST -tagged or His-tagged fusion-proteins. F or this screen, purified protein is in vitro phosphorylated at specific phosphorylation site to a high stoichiometry (phospho). Control, dephosphoprotein is incubated under the same conditions b ut in the absence of the kinase acti vity (mock-phospho). Increasing amounts of both mock-phospho and phospho proteins (0.1 to 1 µg) are then directly spotted onto the PVDF membrane and dot-immunoblotting is per formed. Alternately, proteins can be resolv ed by SDS-P AGE and transferred onto the nitrocellulose membrane (Schleicher and Schuell). Immunoblotting assay is carried out as described in the pre vious section. The secondary screen carried out to confirm the specificity of G AA receptor anti-P-β3 antiserum (UCL 39) emplo yed GST-fusion proteins containing the TM3TM4 intracellular loop of the β3 subunit (GST-β3il), due to the limited a vailability of the holo- β3 subunit expressed as a GST -fusion protein. GST-β3il protein was in vitro phosphorylated at Ser408/409 by purified PKC to a stoichiometry of 0.85 mo P/mol protein (phospho). Mock-phospho protein w as incubated under the same conditions in the absence of the kinase. Increasing amounts of both mock-phosphoand phospho-GST fusion proteins (100 and 200 ng) were resolved using SDS-PAGE and transferred onto the nitrocellulose membrane. Immunoblotting assays using 1:100 dilution of UCL39 were carried out as described in the pre vious section. The primary antibody binding w as detected using [ 125I]-labeled anti-rabbit secondary antibody (Amersham) and Phosphoimager scanning (Molecular Dynamics, Figure 4.4). The secondary screen of UCL39 confirmed the exclusive binding of this antiserum to the phosphorylated form of the GAB AA receptor β3 subunit. For any further analysis of a ne wly raised phosphorylation state-specific antise rum, isolating a pool of IgGs that specifically binds to the phosphorylated epitop of the protein of interest is important. The methods routinely used to purify a

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2nd screen 66

46

30 +

−

−

−

mockP-GSTβ3 100ng

−

+

−

−

mockP-GSTβ3 200ng

−

−

+

−

P-GSTβ3 100ng

−

−

−

+

P-GSTβ3 200ng

FIGURE 4.4 Second screen: Characterization of anti-P-β3 antiserum (UCL39) using in vitro phosphorylated GST -β3il fusion protein. GST -β3il protein w as in vitr o phosphorylated at Ser408/409 by purified PKC and increasing amounts (100 and 200 ng) of both mock-phospho GSTβ3 (lanes 1 and 2), and phospho-GST β3 fusion proteins (lanes 3 and 4) were resolv ed using SDS-P AGE. Immunoblotting assays using 1:100 dilution of UCL39. The primary antibody binding w as detected using [ 125I]-labeled anti-rabbit secondary antibody and Phosphoimager scanning.

phosphorylation site-specific antibody using a described in a later section.

finity chromatograp y will be

4.2.4.3 Third Screen: Expression of wt and Phospho-Site Mutants in Heterologous Cell Line Systems GABAA receptor β subunits e xhibit a high de gree of similarity in the sequence surrounding the conserv ed phosphorylation sites Ser408 and Ser409 (or Ser410 in the β2 subunit). The strategy used to raise a phosphorylation site-specific antibod , which w ould be both phospho-site specific and su unit-specific in this case, as based on the presence of tw o phosphorylated residues, Ser408 and Ser409, e xclusively in the β3 sub unit. Once the primary and secondary screens confirmed th specificity of antiserum UCL39 t ward the phospho forms of the β3 sub unit, the third screen w as designed to test whether the af finity-purified UCL39 antiseru referred to as anti-P- β3 antibody, specifically recognized the β3 subunit only when both Ser408/409 residues were phosphorylated.This screen employed the expression of recombinant β3 subunit containing the wt sequence including both Ser408 and Ser409 residues and mutated β3 subunits containing the following serine to alanine substitutions: S408-A, S409-A and S408/409-A. These constructs were tranfected into the COS-7 cell-line and the e xpression levels were tested after 48 hours. Prior to preparation of SDS lysates (2% final concentration), cell cultures xpressing each of the constructs were incubated in the absence or presence of forsk olin to activate endogenous PKA and stimulate phosphorylation of these sites. Protein samples were

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resolved by SDS-P AGE, follo wed by transfer onto the nitrocellulose membrane. Immunoblotting assays using 0.5 µg/ml of anti-P- β3 antibody and 1 µg/ml of β3specific, phosphorylation state-independent antibody were carried out in parallel, a described in an earlier section. Immunoblotting with anti-P-β3 Ab revealed a specifi band of about ~58 kDa present only in lysates from COS-7 cells transfected with the wt β3 subunit under control conditions, which was strongly enhanced by forskolin (Figure 4.5, lane 3 (–) (+) Forsk, respectively). The total level of transfected wt β3, as detected by β3-specific antibod , remained constant (Figure 4.5, lane 3). The antiP-β3 immunoreactive band w as absent in lysates from mock transfected controls, cells transfected with α1 and γ2 subunits only, or cells transfected with either the single-phospho-site (β3S408A or β3S409A), or di-phospho-site (β3S408A,S409A) β3 mutants (Figure 4.5, lanes 1, 2, 4, 5 and 6, respecti vely). The e xpression le vels of these mutants were similar to those in wt β3 transfected cells (Figure 4.5). Some weak nonspecific bands of higher molecular weight were detected with the anti-P β3 Ab in COS-7 cell lysates, but these bands did not represent GABAA receptor β3-specifi immunoreactivity as they were also detected in extracts from mock-transfected cells. These results ha ve pro vided us with the k ey evidence for a special feature of the anti-P-β3 antibody to recognize phosphorylated β3 subunit, but only when specifi cally phosphorylated at both Ser408 and Ser409 residues. The foregoing type of analysis has pro ven to be a v ery important step during the characterization of a ne wly raised phosphorylation state-specific antibod because it can provide clear evidence for the specificity of the antibody for particula phosphorylated residues within the full-length sequence of the protein of interest. However, the successful application of this type of analysis is dependent on the level of endogenous phosphorylation of a protein of interest in transfected cells, which can often be too lo w to detect under basal conditions. This problem could be circumvented by either acti vating the endogenous kinase acti vity responsible for phosphorylation (if known) using specific act vators, or by co-transfecting a constitutively active form of the kinase together with the protein of interest. Alternatively, cells can be treated with a broad spectrum phosphatase inhibitor, such as high doses of okadaic acid, resulting in an enhancement of the endogenous phosphorylation state of a protein of interest, thereby increasing the probability of detection by a phosphorylation state-specific antibod . Further important information that can be obtained from this type of assay is the determination as to whether the ne wly raised phospho-specific antibody can b reliably emplo yed as a quantitati ve tool for analyzing the in situ phosphorylation state of the protein of interest. In this conte xt, noting here that a significant numbe of attempts to raise ne w phosphorylation state-specific antibodies ail to pass this important step of characterization is important. 4.2.4.4 Fourth Screen: Dephospho/Phospho Peptide Block Our final goal is to obtain a phosphorylation state-specific antibody that could utilized as a sensiti ve, specific and quantitat ve tool with which we can detect the endogenous phosphorylation le vel of a protein of interest in tissue or cells where this protein normally operates. Therefore, the final checkpoint for a n wly raised

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3rd screen 1

2

3

4

5

6

− +

− +

− +

− +

− +

− +

Forsk

66 P-β3 Ab

46 66

Tot-β3 Ab

46 1-mock 2-α1/γ2S 3-β3 wt / α1/γ2S 4-β3 A408 / α1/γ2S 5-β3 A409 / α1/γ2S 6-β3 A408/409 / α1/γ2S

FIGURE 4.5 Third screen: Characterization of anti-P- β3 antibody using the recombinant wt β3 subunit and phosphorylation site Ser408/409 β3 mutants in COS-7 cells. Recombinant wt β3 subunit (lane 3) and its phosphorylation site mutants: S408-A (lane 4), S409-A (lane 5) and S408/409-A (lane 6) were transfected into the COS-7 cell line, and 48 hours later incubated in the absence (–) or presence (+) of forsk olin (20 µM). Protein samples were resolved by SDS-PAGE. Immunoblotting assays were carried out using 0.5 µg/ml of anti-Pβ3 antibody (upper panel) or 1 µg/ml of β3-specific antibody (l wer panel), follo wed by incubation with [ 125I]-labeled anti-rabbit secondary antibody . Binding w as visualized using Phosphoimager scanning. Note that anti-P-β3 Ab specifically recognizes a band of about ~5 kDa present only in lysates from COS-7 cells transfected with the wt β3 subunit, which was strongly enhanced by forsk olin, lanes 3 (–)(+) F orsk, respectively. Weak nonspecific band of higher molecular weight do not represent GABAAR β3-specific immunoreact vity because they were also detected in extracts from mock-transfected cells (lane 1) and α1/γ2 transfected cells (lane 2).

phospho-antibody is to determine if this antibody specifically detects the phospho ylated native form of the protein of interest. This analysis involves immunoblotting with phosphorylation state-specific antibody in the absence or presence of a dephos pho- or phospho-form of the peptide used as antigen. Here, this final step is describe with respect to anti-P- β3 antibody characterization that w as carried out by immunoblotting of protein extracts from cultured cerebrocortical neurons, incubated either under control condition or in the presence of BDNF (100 ng/ml). Anti-P-β3 antibody w as incubated in the absence or presence of dephospho Ser408/409-peptide antigen or phospho Ser408/409-peptide antigen (both in 1:1000 antibody-to-peptide ratio) prior to the immunoblotting step. Using this approach, we detected a single band of about ~58 kDa in SDS e xtracts from cortical neurons

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identical in molecular mass to the recombinant β3 subunit (Figure 4.6, left panel) when antibody was incubated in the absence of antigen peptides. In the presence of a synthetic peptide phosphorylated at both Ser408/409, detection of the β3 subunit by anti-P-β3 Ab was prevented (Figure 4.6, right panel). In contrast, the detection of β3 sub unit by anti-P- β3 Ab w as unaf fected by the presence of dephosphoSer408/409 peptide (Figure 4.6, middle panel). Note that the BDNF-dependent increase in β3 subunit phosphorylation detected with the anti-P-β3 antibody (approximately four -fold, Figure 4.6) appeared quantitati vely similar to the increase in phosphorylation detected by immunoprecipitation of β3 from lysates of [32P]-labeled neurons using a β3-specific antibody [41]. Together, these results from independent measures demonstrated that anti-P- β3 Ab specifically recognizes the nat ve GABAAR-β3 subunit only when phosphorylated on both Ser408/409 residues. 4th screen P-β3-Ab

P-β3-Ab + deP peptide

P-β3-Ab + P408/409 peptide

Con BDNF (5, 10 min)

Con BDNF (5, 10 min)

Con BDNF (5, 10 min)

97

66 P-β3

46

FIGURE 4.6 Fourth screen: Characterization of anti-P- β3 antibody using dephospho/phospho-peptide block approach. Cultured cerebrocortical neurons were incubated under control conditions (con), or in the presence of brain-deri ved neurotrophic factor (BDNF, 100 ng/ml) and protein e xtracts were resolv ed by SDS-PAGE. The immunoblotting with anti-P- β3 antibody was carried out in the absence (left panel) or presence of dephospho Ser408/409 antigen peptide (middle panel) or diphospho Ser408/409 antigen peptide (right panel). Note that antiP-β3 antibody detected a single band of about ~58 kDa corresponding to the β3 sub unit, when incubated in the absence of antigen peptide or in the presence of dephospho Ser408/409 antigen peptide. In the presence of diphospho Ser408/409 antigen peptide detection of the β3 subunit by anti-P- β3 Ab was abolished.

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Phosphorylation Site-Specific Antibodies as Research Tools

4.2.5 AFFINITY-PURIFICATION SPECIFIC ANTIBODIES

OF

75

PHOSPHORYLATION STATE-

Further purification of a subpopulation of phosphorylation state-specific IgGs fr the whole serum is often required due to the presence of nonspecific IgGs recognizin other proteins in cell e xtracts and potentially af fecting precise quantification of th phosphorylated-form of a protein of interest. The second common reason for further purification of phospho-specific IgGs is the presence of w titer IgGs cross-reacting with other epitopes present in the antigen peptide, giving rise to a low-level binding to dephospho form of the protein of interest in cell e xtracts. The isolation of a phospho-specific pool of IgGs from the whole serum com monly includes tw o steps of purification, the first step being purification of to IgGs from the antiserum follo wed by the second step of purification that is base on the affinity chromatograp y using phosphopeptide or dephosphopeptide columns [46]. 4.2.5.1 Purification of Total IgGs from the Phospho-Specific Antiserum Purification of total pool of IgGs from the antiserum is performed as the first st in order to pre vent rapid dephosphorylation as well as de gradation of the phosphopeptide antigen coupled to the af finity column by high act vities of phosphatases and proteases present in the whole serum. This purification step is carried out usin Protein A-Sepharose beads (Protein A Sepharose CL-4B, Pharmacia), which bind to the Fc re gion of IgGs. The binding capacity of this matrix is high (about 20 mg IgG/ml of gel). The required amount of beads is determined by the v olume of gel required for column preparation, with the assumption that 1 g of dry beads will produce about 4 to 5 ml of the final gel olume. Sepharose beads are supplied as freeze-dried gel in the presence of additi ves. Beads are therefore reconstituted in water and thoroughly washed to remove these additives. The column prepared with the washed bead slurry is equilibrated with the binding b uffer (TBS/T: 50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20) in a ratio of 75% settled gel to 25% b uffer. The protocol for purification is described bel w: 1. An aliquot of serum (10 ml) is thawed and filtered through a 0.45-µm filt . 2. Serum is diluted by the binding buffer to 20 ml and the following protease inhibitors are added to prevent proteolysis of antiserum: benzamidine, 25 mM; ethylene diamine tetraacetic acid (EDT A), 1 mM; eth ylene glycolbis-(beta-aminoethyl ether)-N,N- tetraacetic acid (EGTA), 1 mM; phenylmethilsulfonyl fluoride (PMSF), 100 µM; leupeptin and antipain, 2 µg/ml; pepstatin and ch ymostatin, 2 µg/ml. 3. Small aliquots of serum are loaded into the column. To increase the level of IgG binding to the column, fl w-through fraction is collected and the loading is repeated.

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4. The column is w ashed with the follo wing buffers (50 ml): 50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20 50 mM Na-borate, pH 8.5/500 mM NaCl/0.05% Tween-20 50 mM Na-acetate, pH 5.5/500 mM NaCl/0.05% Tween-20 50 mM Tris, pH 7.5/500 mM NaCl 5. Antibodies are eluted with 100 mM glycine, pH 2.5, and 1 ml fractions are collected and neutralized with 50 µl of 1 M Tris, pH 8.0. 6. The presence of IgGs is determined by measuring absorbance at 280 nm and fractions are pooled. The amount of purified IgGs is calculated kn wing that the absorbance of 1 mg/ml of IgGs (A 280) = 1.4. 7. The column is washed and stored in the binding b uffer containing 0.02% NaN2. 4.2.5.2 Purification of Phospho-Specific Antibodies Using Affinity Chromatography with Peptide Columns The second purification step is designed to all w purification of la ge quantities of a subpopulation of total IgGs that sho w specificity t ward the phosphorylated form of the protein of interest. Columns for af finity purification can be prepared usi the phosphorylated protein if sufficient amount of this protein is vailable. However, for the long-term use of an af finity column, use of phospho- or dephosphopeptid antigen for the column preparation is recommended. Also recommended is the preparation of both phospho- and dephosphopeptide columns for affinity purificati of each ne wly produced antiserum because the IgG pool isolated using the phosphopeptide column will likely contain a small fraction of nonspecific IgGs tha bind to phosphorylation-independent epitopes of the protein of interest. Preparation of phospho/dephospho-peptide af finity columns utilizes SulfoLin coupling gel (Pierce) for coupling of N- or C-terminal c ysteine-containing peptides or activated CH Sepharose 4B (Pharmacia) for coupling of peptides via N-terminal amino group. SulfoLink coupling gel is a support matrix formed by 6% cross-linked agarose with an iodoacetyl group immobilized at the end of a 12-atom spacer . The coupling procedure [46] is briefly summarized bel w: 1. Resin (about 5 ml) is w ashed thoroughly with about 100 ml of 50 mM Tris, pH 8.5/5 mM EDT A on a cintered glass filte . 2. The peptide (5 to 10 mg) is dissolv ed in 2 ml of the same b uffer and its pH and the presence of free sulfhydryl groups checked as described in an earlier section. 3. The resin is transferred into a 15-ml plastic tube and allo wed to settle. 4. Excess w ashing b uffer is remo ved and the peptide solution is added to the resin. 5. The mixture is incubated for 1 hour at room temperature with gentle rotation. 6. The resin is w ashed with about ~100 ml of the coupling b uffer.

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7. The resin is incubated with 50 mM c ysteine in the coupling buffer for 60 min with rotation to block remaining reacti ve groups. 8. The resin is poured into a Fle x column and w ashed with 200 ml of 1 M NaCl. 9. Column is stored at 4°C in 50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20, 0.02% NaN 3. The alternati ve method for preparation of phosphopeptide and dephosphopeptide affinity columns is based on use of act vated CH Sepharose 4B (Pharmacia). This type of resin is significantly more stable than SulfoLink and all ws multiple purifications to be carried out ver a long period of time. Activated CH Sepharose 4B is pre-activated gel for co valent immobilization of proteins or peptides containing primary amino groups. The matrix is formed by 4% ag arose with the acti ve group immobilized on a six-aminohe xanoic acid spacer. The active group N-h ydroxysuccinimide is formed by esterification of the carboxyl group of CH Sepharose 4B. The binding capacity of this matrix is high (about 6 to 16 mM of peptide/ml drained gel). The required amount of beads is determined by the v olume of gel required for column preparation, with the assumption that 1 g of dry beads will produce about 4 to 5 ml of the final gel olume. The resin is supplied as freeze-dried po wder in the presence of additi ves that must be remo ved before the coupling reaction. The protocol for peptide coupling is described here: 1. Weigh out the required amount of freeze-dried po wder and resuspend it in 1 mM HCl. 2. Use about 200 ml of 1 mM HCl to remo ve the additi ve by w ashing the gel on a cintered glass filte . 3. Dissolve peptide (5 to 10 mg) in coupling b uffer, 0.1 M NaHCO 3, pH 8.0/0.5M NaCl and check the pH of peptide solution as described in an earlier section. 4. Add the peptide solution to the gel to form a final mix with a gel: uffer ratio of 1:2, which is suitable for coupling. 5. Rotate the mixture for 1 to 2 hours at room temperature or 4 hours at 4°C (the use of magnetic stirrer is not recommended). 6. Remove the excess ligand with at least 5 gel v olumes of coupling buffer. 7. Block remaining acti ve groups with 0.1 M Tris-HCl buffer, pH 8.0, or 1 M ethanolamine, pH 8.0, for at least 1 hour . 8. Wash the gel with at least three c ycles of alterating pH, each c ycle including a wash with fi e times the gel v olume of 0.1 M acetate b uffer, pH 4.0/0.5 M NaCl and a w ash with fi e times the gel v olume of 0.1 M Tris-HCl, pH 8.0/0.5 M NaCl. 9. Wash the gel with 50 mM Tris, pH 7.5/0.5 mM NaCl/0.05% Tween-20. 10. Pour the resin into a Fle x column and store at 4°C. The af finity purification of the phospho-specific IgGs from the antiserum essentially carried out as described earlier . The eluted pool of IgGs should be dialyzed ag ainst PBS or 10 mM MOPS, pH 7.5/150 mM NaCl and concentrated by

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centrifugation using Centriprep concentrators (Amicon) to an optimal concentration of 1 mg/ml. To isolate the suf ficient quantity and quality of the phospho-specific IgGs affinity purification, each of these protocols will l ely include additional steps of purification determined by the titer as well as the specificity of the antiserum To optimize the purification procedure adequatel , all the fractions obtained during the affinity purification should be stored and tested as described in earler section

4.3 CONCLUDING REMARKS In recent years, the use of phosphorylation site-specific antibodies as tools fo quantitative in situ detection of specific phosphorylation sites in numerous protein have become the method of preference for man y investigators. Although a number of phosphorylation site-specific antibodies are n w commercially available, the need to develop new antibodies is e xpected to continue to rise. This trend is in line with the rapid development of proteomic techniques and our increased understanding of the principles of signal transduction pathw ays and their integration. The techniques currently used for the production and characterization of phosphorylation site-specific antibodies are described in this chapter with the aim to form a orking basis for the future de velopment of ne w approaches and applications for these sophisticated research tools.

4.4 ACKNOWLEDGMENTS I would like to thank Dr . Talvinder S. Sihra for critical reading of this manuscript and helpful discussions.

REFERENCES 1. Macdonald, R.L. and Olsen, R.W ., GABAA receptor channels, Ann. Rev. Neurosci., 17, 569–602, 1994. 2. Rabow, L.E., Russek, S.J. and F arb, D.H., From ion currents to genomic analysis: Recent advances in GABAA receptor research, Synapse, 21, 189–274, 1995. 3. Sieghart, W., Structure and pharmacology of g amma-aminobutyric acid A receptor subtypes, Pharm. Rev., 47, 181–234, 1995. 4. Whiting, P.J., Bonnert, T.P., McKernan, R.M., Farrar, S., Le Bourdelles, B., Heavens, R.P., Smith, D.W., Hewson, L., Rigby , M.R., Sirinathsinghji, D.J., Thompson, S.A. and Wafford, K.A., Molecular and functional di versity of the e xpanding GABA-A receptor gene f amily, Ann. NY Acad. Sci., 868, 645–653, 1999. 5. Unwin, N., Neurotransmitter action: Opening of lig and-gated ion channels, Cell, 72 Suppl., 31–41, 1993. 6. Homanics, G.E., DeLorey, T.M., Firestone, L.L., Quinlan, J.J., Handforth, A., Harrison, N.L., Kraso wski, M.D., Rick, C.E., K orpi, E.R., Mak ela, R., Brilliant, M.H., Hagiwara, N., Fer guson, C., Sn yder, K. and Olsen, R.W ., Mice de void of g ammaaminobutyrate type A receptor beta3 sub unit have epilepsy, cleft palate, and h ypersensitive behavior, Proc. Natl. Acad. Sci. USA , 94, 4143–4148, 1997.

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7. Gunther, U., Benson, J., Benke, D., Fritschy, J. M., Reyes, G., Knoflach, ., Crestani, F., Aguzzi, A., Arigoni, M., Lang, Y., Bluethmann, M., Mohler, H. and Lüzscher, B., Benzodiazepine-insensitive mice generated by tar geted disruption of the g amma 2 subunit gene of g amma-aminobutyric acid type A receptors, Proc. Natl. Acad. Sci. USA, 92, 7749–7753, 1995. 8. Rudolph, U., Crestani, F., Benke, D., Brunig, I., Benson, J.A., Fritschy, J.M., Martin, J.R., Bluethmann, H. and Mohler , H., Benzodiazepine actions mediated by specifi gamma-aminobutyric acid(A) receptor subtypes, Nature, 401, 796–800, 1999. 9. Low, K., Crestani, F ., Keist, R., Benk e, D., Brunig, I., Benson, J.A., Fritsch y, J.M., Rulicke, T., Bluethmann, H., Mohler , H. and Rudolph, U., Molecular and neuronal substrate for the selecti ve attenuation of anxiety , Science, 290, 131–134, 2000. 10. Luddens, H., Korpi, E.R. and Seeburg, P.H., GABAA/benzodiazepine receptor heterogeneity: Neurophysiological implications, Neuropharmacology, 34, 245–254, 1995. 11. Korpi, E.R., Mattila, M.J.,Wisden, W. and Luddens, H., GABA(A)-receptor subtypes: Clinical ef fica y and selecti vity of benzodiazepine site lig ands, Ann. Med ., 29, 275–282, 1997. 12. Luscher, B. and K eller, C.A., Re gulation of GAB AA receptor traf ficking, channe activity, and functional plasticity of inhibitory synapses, Pharm. Ther., 102, 195–221, 2004. 13. Essrich, C., Lorez, M., Benson, J.A., Fritsch y, J.M. and Luscher , B., Postsynaptic clustering of major GAB AA receptor subtypes requires the g amma 2 sub unit and gephyrin, Nat. Neurosci., 1, 563–571, 1998. 14. Kneussel, M., Brandstatter, J.H., Laube, B., Stahl, S., Muller , U. and Betz, H., Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice,J. Neurosci., 19, 9289–9297, 1999. 15. Kittler, J.T., Delmas, P., Jovanovic, J.N., Bro wn, D.A., Smart, T.G. and Moss, S.J., Constitutive endoc ytosis of GAB AA receptors by an association with the adaptin AP2 comple x modulates inhibitory synaptic currents in hippocampal neurons, J. Neurosci., 20, 7972–7977, 2000. 16. Kittler, J.T. and Moss, S.J., Modulation of GAB A-A receptor activity by phosphorylation and receptor traf ficking: Implications for the e fica y of synaptic inhibition, Curr. Opin. Neurobiol., 13, 341–347, 2003. 17. Brandon, N.J., Jovanovic, J.N., Smart, T.G. and Moss, S.J., Receptor for acti vated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABA(A) receptors with the acti vation of G-protein-coupled receptors, J. Neurosci., 22, 6353–6361, 2002. 18. Porter, N.M., Twyman, R.E., Uhler, M.D. and Macdonald, R.L., Cyclic AMP-dependent protein kinase decreases GAB AA receptor current in mouse spinal neurons, Neuron, 5, 789–796, 1990. 19. Moss, S.J., Smart, T.G., Blackstone, C.D. and Hug anir, R.L., Functional modulation of GAB AA receptors by cAMP-dependent protein phosphorylation, Science, 257, 661–665, 1992. 20. Robello, M., Amico, C. and Cupello, A., Regulation of GABAA receptor in cerebellar granule cells in culture: Dif ferential involvement of kinase acti vities, Neuroscience, 53, 131–138, 1993. 21. Poisbeau, P., Cheney, M.C., Bro wning, M.D. and Mody , I., Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons, J. Neurosci., 19, 674–683, 1999.

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The Dynamic Synapse 22. Flores-Hernandez, J., Hernandez, S., Sn yder, G.L., Yan, Z., Fienber g, A.A., Moss, S.J., Greeng ard, P. and Surmeier , D.J., D(1) dopamine receptor acti vation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/D ARPP-32/PP1 signaling cascade, J. Neurophysiol., 83, 2996–3004, 2000. 23. Kano, M. and K onnerth, A., Potentiation of GAB A-mediated currents by cAMPdependent protein kinase, Neuroreport, 3, 563–566, 1992. 24. Nusser, Z., Sie ghart, W. and Mody , I., Dif ferential re gulation of synaptic GAB AA receptors by cAMP-dependent protein kinase in mouse cerebellar and olf actory bulb neurones, J. Physiol, 521(Pt. 2), 421–435, 1999. 25. Brunig, I., Sommer , M., Hatt, H. and Bormann, J., Dopamine receptor subtypes modulate olfactory bulb gamma-aminobutyric acid type A receptors, Proc. Natl. Acad. Sci. USA, 96, 2456–2460, 1999. 26. Levitan, I.B., Modulation of ion channels by protein phosphorylation. Ho w the brain works, Adv. Sec. Mess. Phospho. Res ., 33, 3–22, 1999. 27. Fraser, I.D. and Scott, J.D., Modulation of ion channels: A “current” view of AKAPs, Neuron, 23, 423–426, 1999. 28. Stelzer, A., Intracellular re gulation of GAB AA-receptor function, Ion Channels , 3, 83–136, 1992. 29. Stelzer, A. and Shi, H., Impairment of GAB AA receptor function by N-meth yl-Daspartate-mediated calcium influx in isolated CA1 yramidal cells, Neuroscience, 62, 813–828, 1994. 30. McDonald, B.J. and Moss, S.J., Differential phosphorylation of intracellular domains of g amma-aminobutyric acid type A receptor sub units by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase, J. Biol. Chem., 269, 18111–18117, 1994. 31. Wang, J., Liu, S., Haditsch, U., Tu, W., Cochrane, K., Ahmadian, G., Tran, L., Paw, J., Wang, Y., Mansuy, I., Salter , M.M. and Lu, Y.M., Interaction of calcineurin and type-A GAB A receptor g amma 2 sub units produces long-term depression at CA1 inhibitory synapses, J. Neurosci., 23, 826–836, 2003. 32. Kano, M., Kano, M., Fukunaga, K. and Konnerth, A., Ca(2+)-induced rebound potentiation of g amma-aminobutyric acid-mediated currents requires acti vation of Ca2+/calmodulin-dependent kinase II. Proc. Natl. Acad. Sci. USA, 93, 13351–13356, 1996. 33. Wan, Q., Man, H.Y ., Braunton, J., Wang, W., Salter, M.W., Beck er, L. and Wang, Y.T., Modulation of GAB AA receptor function by tyrosine phosphorylation of beta subunits, J. Neurosci., 17, 5062–5069, 1997. 34. Moss, S.J., Gorrie, G.H., Amato, A. and Smart, T.G., Modulation of GABAA receptors by tyrosine phosphorylation, Nature, 377, 344–348, 1995. 35. Boxall, A.R., GABAergic mIPSCs in rat cerebellar Purkinje cells are modulated by TrkB and mGluR1-mediated stimulation of Src, J. Physiol., 524(Pt. 3), 677–684, 2000. 36. Moss, S.J. and Smart, T.G., Modulation of amino acid-gated ion channels by protein phosphorylation, Int. Rev. Neurobiol., 39, 1–52, 1996. 37. McDonald, B.J., Amato, A., Connolly, C.N., Benke, D., Moss, S.J. and Smart, T.G., Adjacent phosphorylation sites on GAB AA receptor beta sub units determine re gulation by cAMP-dependent protein kinase, Nat. Neurosci., 1, 23–28, 1998. 38. Krishek, B.J., Xie, X., Blackstone, C., Hug anir, R.L., Moss, S.J. and Smart, T.G., Regulation of GABAA receptor function by protein kinase C phosphorylation, Neuron, 12, 1081–1095, 1994.

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39. Brandon, N.J., Uren, J.M., Kittler , J.T., Wang, H., Olsen, R., P arker, P.J. and Moss, S.J., Subunit-specific association of protein kinase C and the receptor for act vated C kinase with GAB A type A receptors, J. Neurosci., 19, 9228–9234, 1999. 40. Brandon, N.J., Jovanovic, J.N., Colledge, M., Kittler, J.T., Brandon, J.M., Scott, J.D. and Moss, S.J., A-kinase anchoring protein 79/150 f acilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta sub units, Mol. Cell. Neurosci., 22, 87–97, 2003. 41. Jovanovic, J.N., Thomas, P., Kittler, J.T., Smart, T.G. and Moss, S.J., Brain-deri ved neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, acti vity, and cell-surf ace stability , J. Neurosci., 24, 522–530, 2004. 42. Nairn, A.C., Detre, J.A., Casnellie, J.E. and Greeng ard, P., Serum antibodies that distinguish between the phospho- and dephospho-forms of a phosphoprotein, Nature, 299, 734–736, 1982. 43. Czernik, A.J., Girault, J.A., Nairn, A.C., Chen, J., Sn yder, G., K ebabian, J. and Greengard, P., Production of phosphorylation state-specific antibodies, Meth. Enzymol., 201, 264–283, 1991. 44. Bassiri, R.M., Dvorak, J. and Utiger, R.D., in Methods of Hormone Radioummunoassays, Jaffe, B.M. and Behrman, H.R. (Eds.), Academic Press: New York, 1979. 45. Walter, G., Production and use of antibodies ag ainst synthetic peptides, J. Immun. Meth., 88, 161, 1986. 46. Czernik, A.J., Mathers, J., Mische, S.M., in Regulatory Protein Modificatio , Hemmings, H.C.J. (Ed.), Humana Press: Ne w Jersey, 1997. 47. Czernik, A.J., Mathers, J., Tsou, K., Greengard, P. and Mische, S.M., Phosphorylation state-specific antibodies: Preparation and applications, Neuroprotocols, 6, 56–61, 1995.

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Protein Palmitoylation by DHHC Protein Family Yuko Fukata, David S. Bredt, and Masaki Fukata

CONTENTS 5.1 5.2 5.3

Introduction ....................................................................................................83 Overview of PAT Screening ..........................................................................84 Protocols.........................................................................................................85 5.3.1 Transfection........................................................................................85 5.3.2 Metabolic Labeling ............................................................................86 5.3.3 SDS-PAGE and Fluorograph y ...........................................................86 5.3.4 Notes...................................................................................................86 5.4 Acknowledgments ..........................................................................................88 References................................................................................................................88

5.1 INTRODUCTION Palmitoylation is the post-translational modification of proteins with palmitic aci (16-carbon saturated f atty acid) and re gulates the membrane tar geting, subcellular trafficking and function of proteins [1]. almitoylation occurs either through amidelinkage (N-palmitoylation) or thioester linkages (S-palmitoylation). S-palmitoylation occurs on c ysteine residues in di verse sequence conte xts and is more commonly found in most palmito ylated proteins. Here, the term of protein palmito ylation will mean S-palmitoylation. Protein palmito ylation is the frequent lipid modification o neuronal proteins and modifies ma y important proteins, including synaptic v esicle proteins, ion channels, guanosine triposphate (GTP)-binding proteins, neurotransmitter receptors and synaptic scaf folding proteins [2]. Examples include PSD-95, a protein that scaf folds receptors and signaling enzymes at the post-synapse; NCAM140, a neural cell adhesion molecule that localizes at the gro wth cone and regulates neurite outgro wth; and SN AP-25, a t-SN ARE protein that re gulates neurotransmitter release [2]. PSD-95 palmitoylation is necessary for sorting of PSD-95 to dendrites and participates in the post-synaptic clustering of PSD-95 in dendritic spines, thereby re gulating the clustering of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors [3]. Unlike other irreversible lipid modifications such as myrist ylation and pren ylation, palmito ylation is relatively labile and palmitate on proteins turns o ver rapidly. Importantly, the specifi 83

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extracellular signal regulates protein-palmitoylation levels [4]. At post-synaptic sites, palmitate continuously turns o ver on PSD-95. Depalmito ylation of PSD-95 is enhanced by glutamate receptor -mediated synaptic acti vity, and this process dissociates PSD-95 and AMPA receptors from the postsynaptic sites [3]. Although the actions of enzymes that add or remo ve protein palmitate might mediate the dynamic re gulation of palmito ylation, the enzymes ha ve been elusi ve. Recent genetic studies in yeast have identified proteins that mediate palmit ylation. Erf2p [5–7] and Akr1p [8] are palmito yl-transferases (PATs) for yeast Ras2p and yeast casein kinase2 (Yck2p), respectively (Figure 5.1). Erf2p andAkr1p are integral membrane proteins harboring a cysteine-rich domain containing a conserved DHHC (Asp-His-His-Cys) motif. In genomes of human and mouse, 23 kinds of DHHCcontaining proteins are predicted ( Figure 5.2 ). To identify the p hysiological PATs for substrates, systemically evaluating functions of the family of 23 DHHC-containing proteins is necessary . For this purpose, we isolated all mouse DHHC proteins and established a screening method that allo ws us to identify the candidate P AT for specific substrates [9]. We found that a subset of DHHC proteins specifically palm itoylates PSD-95 (P-PATs), and that DHHC proteins have substrate specificity (Fig ure 5.2) [9]. P-P AT acti vity re gulates synaptic clustering of PSD-95 and AMPA receptors, as well as modulating AMPA receptor function in hippocampal neurons. Thus, the DHHC protein-mediated reaction is a potential general mechanism of protein palmitoylation in cells. In this chapter, we describe procedures to screen the DHHC protein f amily to identify the specific AT. The procedures include three steps.

5.2 OVERVIEW OF PAT SCREENING Transfection of the tar get substrate cDN A together with indi vidual DHHC clone into cultured cells (e.g., HEK293T cells and COS-7 cells) Metabolic labeling with [ 3H]palmitic acid SDS-PAGE and fluorograp y Yeast DHHC proteins

Substrates

Erf2p

Ras2p

Akr1p

Yck2pc

FIGURE 5.1 Domain structures of yeast DHHC proteins, Erf2p and Akr1p. Each has several transmembrane domains (black box es) and a conserv ed c ysteine-rich domain containing DHHC (Asp-His-His-Cys) motif (DHHC-CRD, gray box es; DHYC in Akr1p). The DHHC sequence is thought to be essential for palmito ylating acti vity. In addition, Akr1p has six ankyrin repeat sequences (open circles).

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Protein Palmitoylation by DHHC Protein Family

DHHC-20 DHHC-2

DHHC-15 DHHC-7

DHHC-3 DHHC-23 DHHC-16

DHHC-6

DHHC-21

DHHC-12

H-Ras

DHHC-11

Yeast Erf2p DHHC-9

DHHC-18

DHHC-14 DHHC-5

DHHC-8 DHHC-19

Yeast Akr1p

DHHC-17

DHHC-22

DHHC-4

DHHC-13

DHHC-10

DHHC-1

SNAP-25

85

Gαs SNAP-25 PSD-95 GAP-43

FIGURE 5.2 Phylogenetic tree of the mouse DHHC protein f amily and summary of screening. DHHC proteins apparently have substrate specificit . Among them, DHHC-3 and DHHC7 palmitoylate PSD-95, GAP43, G s, and SN AP-25, suggesting that DHHC-3 and DHHC-7 show broad substrate specificit . DHHC-3 is also known as GODZ. DHHC-17 is an orthologue of yeast Akr1p and also kno wn as Hip14 (Huntingtin-interacting protein 14).

5.3 PROTOCOLS 5.3.1 TRANSFECTION Seed HEK293T (or COS-7) cells (3 × 105 to 6 × 105 cells/well, six-well format) and incubate at 37°C for 16 to 20 hours in the gro wth medium without antibiotics. Transfect about 80% confluent cells using lipofectamine plus according t the manufacturer’s instructions (Invitrogen). Dilute the DNA mixtures (2 µg) in 100 µl of DMEM (or Opti-MEM from Invitrogen). The DNA mixture includes 1 µg each of plasmids for the substrate and individual DHHC clone. Add 6 µl of Plus reagent from Invitrogen and mix gently. Incubate for 15 min at room temperature. Combine the diluted DNA mixture with diluted lipofectamine (4 µl in 100 µl of DMEM or Opti-MEM). Total volume will be 200 µl. Incubate for 15 min at room temperature. Add 800 µl of DMEM or Opti-MEM to the tube containing the complexes (total volume will be 1 ml).

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Remove the cultured medium from cells and add 1 ml of the diluted complexes. Incubate cells at 37°C for 4 hours and then add 1 ml of gro wth medium including 20% serum (twice the normal concentration of serum). Incubate the cells at 37°C for 20 to 24 hours.

5.3.2 METABOLIC LABELING Dry [ 3H]palmitic acid (PerkinElmer, 45 Ci/mM, 5 mCi/ml in ethanol) under reduced pressure in a concentrator such as SpeedV ac and reconstitute in small amount of 100% of ethanol (e.g., 1 µCi in 10 µl ethanol). Preincubate transfected cells for 30 min in serum-free DMEM supplemented with fatty-acid free bo vine serum alb umin (10 mg/ml, Sigma). Label cells for 4 hours with 0.5 mCi [ 3H]palmitate diluted in 1ml of preincubation medium (for each well). Note that final concentration of ethano in medium must be less than 1%.

5.3.3 SDS-PAGE

AND

FLUOROGRAPHY

Wash labeled cells with PBS, collect cells in 1 ml of SDS-P AGE sampling buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.001% bromophenol blue) with 10 mM DTT. Pipette up and down cell suspension on the dish before transferring it to a tube to help cell lysis. Heat the suspension at 95°C for no more than 2 min. For fluorograp y, separate 60 µl of cell suspensions with SDS-P AGE (13.5 cm × 8.5 cm separating gel). After fixing the gels for 30 min in a fixi solution (isopropanol:w ater:acetic acid = 25:65:10), treat the gel with Amplify (Amersham) for 30 min, dry under vacuum and expose to (Kodak Biomax MS) at 80°C for 24 hours. Signals for autopalmito ylation of most DHHC clones and enhanced palmito ylation of substrates by some DHHC clones are usually detected within 24 hours, 72 hours at the most ( Figure 5.3). Scan the radio-labeled bands in the autoradiograph and analyze with NIH software. To quantitate the relati ve PAT activity of each DHHC clone, we quantitate the ratio of the palmito ylated protein to total amount of protein (measured by Coomassie staining or quantitati ve immunoblot).

5.3.4 NOTES A construct of substrate with mutations in palmito ylation sites (e.g., deletion or replacement of cysteine residues) is a good negative control (Figure 5.3; note that no palmitoylation signals around 28 kDa in SNAP-25 ∆Cys). For another negative control to see the basal le vel of palmito ylation, transfect the substrate with mock v ector but not with DHHC clones. As a positi ve

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∆Cys

Protein Palmitoylation by DHHC Protein Family

200

-

87

SNAP-25 WT -

-

1

2

3

4

5

6

7

8

9

10 11 12 : DHHCs

53

(3H)Palm

114 96

36

28

WB

28

FIGURE 5.3 Screening of SN AP-25 palmito ylating enzymes. Indi vidual DHHC clones (DHHC-1 through DHHC-12 among 23 DHHCs are sho wn) were transfected together with SNAP-25 wild-type (WT) or cysteine-deleted SNAP-25 (∆Cys) [10] into HEK293 cells. After metabolic labeling with [ 3H]palmitate, proteins were separated by SDS-P AGE. The upper panel shows the autoradiograph and the lower panel shows the western blot with anti-SNAP25 antibody. An arrow indicates the signal of palmito ylated SNAP-25. SNAP-25 ∆Cys failed to be palmito ylated. Open arro wheads indicate the auto-palmito ylated signals of DHHC clones. White asterisks correspond to the palmito ylated SN AP-25, which migrates more slowly than the non-palmito ylated one. Note that DHHC-3, DHHC-7 and DHHC-17 (data not shown, Figure 5.2) strongly increased incorporation of [ 3H]palmitate into SNAP-25.

control, PSD-95 and PSD-95-PATs P-PATs) such as DHHC-3 and DHHC15 might be useful [9]. Incubation time of transfection is critical. Longer transfection (over 24 hours) increases the basal level of palmitoylation in the mock cells (in the absence of DHHC clones) and more-or-less hinders DHHC protein-enhanced palmitoylation. Most cultured cells e xpress endogenous DHHC proteins. Efficien y of transfection (especially for co-transfection) is an important factor for screening. HEK293T cells usually give a better efficien y (about 90% co-transfection) than COS-7 cells (about 60% co-transfection). We usually do not immunoprecipitate the substrate to detect the DHHC cloneinduced palmitoylation. The immunoprecipitation method does not necessarily reflect the total palmit ylated proteins because usual e xtraction conditions using Triton X-100 or radio-immunoprecipitation assay (RIPA) buffer cannot extract all the palmitoylated proteins, which strongly associate with membrane and go to the insoluble fraction. If immunoprecipitation of the substrate proteins is necessary , 1% SDS (follo wed by Triton X-100 neutralization) might be suitable for the extraction of palmitoylated proteins [9].

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Treat the cell suspension carefully because thioester linkage is labile and sensitive to heat and reducing condition. Some palmito ylated proteins (such as PSD-95, SN AP-25 and so on) shift their apparent molecular weights on SDS- PAGE (Figure 5.3). To identify the ph ysiological enzymes, the follo wing e xperiments are suggested after this screening: Confirm that the palmit ylation signal for the substrate is not o verlapped with the autopalmito ylation signal of the DHHC protein (Figure 5.3; note the autopalmito ylated signals indicated by open arro wheads). Transfection of the DHHC clone without the substrate might be necessary. Examine whether the candidate DHHC clone palmito ylates the substrate in other cell lines. If viral vectors (e.g., Sindbis or Semliki Forest virus) of DHHC clones are available, seeing the DHHC clone-enhanced palmitoylation of endogenous proteins in primary hippocampal neurons is possible [9]. Examine whether the candidate DHHC clone e xpresses in tissues where the substrate express. Examine whether palmito ylation of the endogenous substrate is af fected when the candidate DHHC clone is knocked-down by RNAi or inhibited by dominant-negative mutants.

5.4 ACKNOWLEDGMENTS We thank Dr. Roger A. Nicoll and Dr. Hillel Adesnik of the University of California, San Francisco, for helpful suggestions and discussion, and Dr . Paul A Roche of the National Institute of Health for a kind gift of SN AP-25 cDNA. We also thank Dr . Josef Kittler of Uni versity College London for gi ving us this opportunity. Dr. Yuko Fukata is supported by a long-term fello wship of The International Human Frontier Science Program Organization. Our study is supported by grants from the National Institutes of Health, Christopher Ree ves Paralysis Foundation, the Human Frontier Science Program and the American Heart Association.

REFERENCES 1. Smotrys, J.E. and Linder , M.E., P almitoylation of intracellular signaling proteins: Regulation and function, Ann. Rev. Biochem., 73, 559–587, 2004. 2. El-Husseini Ael, D. and Bredt, D.S., Protein palmito ylation: A regulator of neuronal development and function, Nat. Rev. Neurosci., 3(10), 791–802, 2002. 3. El-Husseini Ael, D., Schnell, E., Dak oji, S., Sweene y, N., Zhou, Q., Prange, O., Gauthier-Campbell, C., Aguilera-Moreno, A., Nicoll, R.A. and Bredt, D.S., Synaptic strength regulated by palmitate c ycling on PSD-95, Cell, 108(6), 849–863, 2002. 4. Wedegaertner, P.B. and Bourne, H.R., Activation and depalmito ylation of Gs alpha, Cell, 77(7), 1063–1070, 1994.

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5. Bartels, D.J., Mitchell, D.A., Dong, X. and Deschenes, R.J., Erf2, a no vel gene product that af fects the localization and palmito ylation of Ras2 in Saccharomyces cerevisiae, Mol. Cell. Biol ., 19(10), 6775–6787, 1999. 6. Zhao, L., Lobo, S., Dong, X., Ault, A.D. and Deschenes, R.J., Erf4p and Erf2p form an endoplasmic reticulum-associated comple x in volved in the plasma membrane localization of yeast Ras proteins, J. Biol. Chem ., 277(51), 49352–49359, 2002. 7. Lobo, S., Greentree, W.K., Linder, M.E. and Deschenes, R.J., Identification of a Ra palmitoyltransferase in Saccharomyces cer evisiae, J. Biol. Chem ., 277(43), 41268–41273, 2002. 8. Roth, A.F., Feng, Y., Chen, L. and Davis, N.G., The yeast DHHC cysteine-rich domain protein Akr1p is a palmito yl transferase, J. Cell Biol ., 159(1), 23–28, 2002. 9. Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R.A. and Bredt, D.S., Identification o PSD-95 palmitoylating enzymes, Neuron, 44(6), 987–996, 2004.

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6

Studying the Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation Approaches I. Lorena Arancibia-Cárcamo, Benjamin P. Fairfax, Stephen J. Moss, and Josef T. Kittler

CONTENTS 6.1 6.2

6.3

Introduction ....................................................................................................92 Endocytosis ....................................................................................................92 6.2.1 Clathrin and Non-Clathrin Mediated Endoc ytosis ............................92 6.2.2 The Endosomal System and the Rab GTP ases .................................94 6.2.2.1 Rab GTPases.......................................................................95 6.2.3 Signals for Endoc ytosis and Post-Endoc ytic Sorting ........................96 Methods for Studying Endoc ytosis................................................................98 6.3.1 Antibody Feeding...............................................................................98 6.3.2 Cell Surface Biotinylation................................................................103 6.3.2.1 Biotinylation Method ........................................................104 6.3.2.2 Quantification and Verificatio .........................................106 6.3.2.3 Endocytosis .......................................................................106 6.3.2.4 Further Trafficking Events................................................107 6.3.2.5 Biotinylation of Slices ......................................................108 6.3.2.6 Degradation Assays ..........................................................109 6.3.3 Other Useful Tools for Studying Neurotransmitter Receptor Endocytosis and Post-Endoc ytic Sorting .........................................110

91

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6.4 Conclusion....................................................................................................112 References..............................................................................................................113

6.1 INTRODUCTION Alterations in the acti vity of post-synaptic neurotransmitter receptors including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors, N-methyl-D-aspartate (NMDA) receptors, kainate receptors and g amma h ydroxybutric acid type A (GABAA) receptors make an important contribution to modulation of synapse strength, neuronal excitability and the plasticity of synapses. Changes in the strength of neurotransmitter receptor signalling are thought to be achie ved, in part, by the modulation of channel g ating and conductance or by re gulating the number or location of receptors e xpressed on cell surface and synaptic membranes. Neurotransmitter receptor traf ficking is n w recognized as a k ey mechanism for altering the strength of synapses during synaptic plasticity by changing synaptic receptor number [1]. Here, we focus on receptor membrane traf ficking and, i particular, receptor endoc ytosis as a mechanism to re gulate the number of surf ace and synaptic neurotransmitter receptors. We briefly r view some general cellular mechanisms that underlie surface membrane protein internalization and then discuss in detail some of the cell biological approaches that ha ve been important for studies of neurotransmitter receptor traf ficking

6.2 ENDOCYTOSIS Endocytosis is the process by which cells internalize membrane proteins, lipids, extracellular lig ands and soluble molecules. Probably the most well-characterized pathway of internalization is clathrin-mediated endocytosis. In addition, a variety of other clathrin-independent pathways have also been characterized including phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake and a poorly characterized form of constituti ve clathrin-independent endoc ytosis [2,3].

6.2.1 CLATHRIN

AND

NON-CLATHRIN MEDIATED ENDOCYTOSIS

A major route for receptor internalization is clathrin-mediated endoc ytosis. Soluble clathrin consists of a 190-kDa hea vy chain associated with a 25-kDa light chain (for re views on clathrin structure and clathrin mediated endoc ytosis see [3–6]). The heavy and light chain complex can form a clathrin triskelion that can in turn assemble into a polygonal lattice, which aids to deform the plasma membrane into a b ud or clathrin-coated pit and subsequently a clathrin coated vesicle (CCV). Recruitment of clathrin to the plasma membrane is thought to be achieved by the interaction of clathrin with theβ subunit of the tetrameric adaptor protein (AP) complex AP2. Whereas four distinct adaptor protein complexes exist (AP1-4), AP2 has been sho wn to play a k ey role in the formation of CCVs at the plasma membrane [7,8]. In contrast, AP1 is associated with Golgi-deri ved CCVs and AP3 and AP4 have not yet been fully characterized, although it has

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been ssuggested that AP3 plays a role in trafficking from early endosomes to lat endosomes [8]. The AP2 complex exists as a hetero-tetrameric assembly of tw o large (approximately 100 to 130 kDa) sub units ( α and β2), a 50-kDa medium subunit termed µ2 and a small 20-kDa sub unit, α2. While the β subunit of AP2 interacts with clathrin-heavy chains, recruitment of AP2 to the plasma membrane is thought to be achie ved in part by the ability of both the α and µ2 subunits to bind membrane polyphosphoinositides (in particular , PIP2) in addition to the ability of the µ2 sub unit (and potentially also the β subunit) to interact with specific amino acid signal sequences within the ytoplasmic domains of membrane protein cargo [9]. For example, the direct interaction of several neurotransmitter receptors, including GABAA receptor, AMPA receptors and NMDA receptors with AP2 has been demonstrated. In addition to binding phospholipids, receptor/membrane protein car go and clathrin, AP2, via its α subunit, has the ability to bind a number of accessory proteins that comprise important components of the endoc ytic machinery including AP180/CALM, epsin/eps15, amphiphysin and auxilin [10]. These four accessory proteins ha ve all been found to also bind clathrin and bind the same site of the AP2 α subunit, indicating that perhaps these proteins act at different steps during CCV formation and life-cycle [11]. Interestingly, amphiphysin has an SH3 domain (a domain that binds short proline-rich amino acid stretches) that has been sho wn to bind a poly-proline domain in the lar ge GTPase dynamin [12]. Dynamin plays a critical role in the conversion of the nascent invaginated clathrin-coated pit into a fully formed and detached CCV by acting as a “pinchase” to f acilitate the scission of the CCV from the plasma membrane [11–14]. The critical role of dynamin in endoc ytosis from the plasma membrane has been ef fectively used to demonstrate the impor tance of receptor internalization in re gulating the strength and plasticity of synapses (see section 6.3.3). Once the CCV is formed, it sheds its coat by action of hsc70 and auxilin and fuses with the endosomal system where receptor car go is either sorted for rec ycling back to the plasma membrane or tar geted for degradation [5]. Clathrin-mediated endoc ytosis has been demonstrated to be a major route of neurotransmitter receptor remo val from the cell surf ace and a critical mechanism for certain forms of synaptic plasticity such as long-term depression (LTD) of e xcitatory synaptic transmission [1]. It is no w well established that AMPA receptors can be internalized in response to a number of stimuli, including ligand binding, neuronal activity, NMDA receptor activation and treatment with insulin and experiments in recombinant systems, and primary neurons have revealed these processes are dependent on the clathrin-mediated endoc ytic machinery [15–17]. Similarly , GABAA receptors, NMDA receptors and kainate receptors ha ve been found to endoc ytose in a clathrin-dependent manner in HEK293 cells [18–20] and cultured neurons [21–24]. Although the majority of surf ace membrane receptors, including se veral neurotransmitter receptors, ha ve been reported to be internalized via a clathrin-dependent pathw ay, several other pathw ays of internalization from the cell surf ace also exist, the best characterized of these being caveolin- or lipid raft-dependent pathways [2,3,25]. Caveolae are flask shaped, noncoated pits identified by the presence of t transmembrane protein caveolin-1 [26,27]. Lipid rafts describe the accumulation of

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saturated lipids and cholesterol into microdomains within the plasma membrane and can be disrupted by depletion of cholesterol. Importantl y, proteins can also be partitioned into lipid rafts, an example being cell surface glycosylphosphatidylinositol (GPI)–anchored proteins that interact with cholesterol and sphingolipids via their saturated acyl chain. The presence of c aveolin in lipid rafts supported the idea that internalization via lipid rafts and caveolae was one unique pathway. However, recent observations suggest that c aveolae are a distinct subtype of raft/membrane subdomain, as the upta ke of some proteins enriched in rafts is possible in the absence of caveolin and, hence, caveolae structures [28,29]. Therefore, separate caveolin-dependent and raft-dependent internalization path ways appear li kely to exist. Little is known regarding the molecular mechanism that underlies raft-mediated internalization of proteins from the cell sur face; h owever, the obser vation that lipid raftassociated proteins often internalize through a non–clathrin-dependent path way and the fact that this internalization is particularly sensitive to cholesterol depletion points to the existence of this alternat ive raft-dependent internalization path way [2,3,25]. The ability of lipid rafts to partition/enrich some membrane proteins could facilitate these proteins being internalized via non-clathrin path ways, as is the case with the interleukin-2 receptor and EGF receptor [30–32]. Interestingly, both GABAA receptors and AMPA receptors have recently been detected in neuronal rafts [30], and the observation that disruption of rafts in cultured hippocampal neurons appears to alter AMPA receptor surface stability suggests that under some circumstances, raft-mediated internalization could be important for removing neurotransmitter receptors from the cell sur face [30].

6.2.2 THE ENDOSOMAL SYSTEM

AND THE

RAB GTPASES

Once receptor cargo has been internalized, it enters the endocytic pathway. Evidence is found to suggest that both the clathrin-dependent and c aveolin-dependent internalization path ways me rge at the l evel of the early endosome (also kn own as the sorting endosome) [33], although caveolin has also been involved in direct targeting to the endoplasmic reticulum (ER) and Golgi [34,35]. Once receptor ca rgo has entered the endosomal system, it can be sorted for d egradation or re cycled back to the plasma membrane, in part dependent upon signals within the protein sequence of receptor ca rgo and the machinery assembled around it. Endosomes comprise a system of heterogeneous compartments that h ave generally been characterized as early or late endosomes, depending on the kinetics with which the compartments are loaded with endo cytosed material. Early endosomes are tu bular and located toward the cell peripher y. Receptor cargo can then be sorted from early endosomes to late endosomes, which are more spherical, and oriented closer to the nucleus. In addition, a subset of late endosomes have a multivesicular appearance and are termed multi-vesicular bodies [36]. Late endosomeseventually mature into lysosomes where their cargo is d egraded by lysosomal proteases. S everal components of the protein machinery thought to be i nvolved in sorting receptor ca rgo towards the degradative pathway h ave recently been identified, including certain Rab isoforms and th proteins Hrs/S TAM, TSG101 and the ESC RT compl exes ( Figure 6.1 ); however, whether these proteins are important for the postendoc ytic sorting of

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Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors 95 DYNAMIN

B4 RA

AP2 CCV

CLATHRIN

RA

B1

1

CAVEOSOME

R. E.

R AB 5

SORTING ENDOSOME pH ~ 6

pH ~ 6.4

11

HRS/STAM

RA

B

ESCRT II

ESCRT I

ESCRT III

R AB 9

MVB/LATE ENDOSOME pH ~ 5-6

R AB 7

LYSOSOME

pH ~ 5-5.5

FIGURE 6.1 Overview of the steps involved in the clathrin mediated endocytic pathway and the role of Rab GTP ases. Recruitment of AP2 and clathrin to the cell membrane causes this to deform and in vaginate. The accessory protein amphiph ysin recruits the GTP ase dynamin, which acts as a “pinchase” to release the clathrin-coated vesicle. Upon shedding of the clathrin coat, the car go containing v esicle fuses with the sorting endosome by action of Rab 5 and the aid of accessory endosomal proteins such as EEA1. Car go can then be rec ycled via the recycling endosome, an e vent mediated by Rab 4 or Rab11, or transported to late endosomes/multivesicular bodies (MVBs) with the aid of Hrs and the ESCR T machinery. From late endosomes proteins can be rec ycled via the Golgi or de graded in the lysosome. The diagram also sho ws how proteins internalized in a ca veolin dependent manner can join the clathrin endocytic pathway at the le vel of the sorting endosome and are also dependent on dynamin activity.

neurotransmitter receptors has yet to be determined. Receptor ca rgo can also be sorted from early endosomes to recycling endosomes from where they can be sorted into vesicles which will ta ke them back to the cell sur face for re-insertion into the plasma membrane. Similarly, specific Rab isoforms appear to play a critical role i this process (see section 6.2.2.1) in addition to other essential components of the recycling machinery such as Rme1. 6.2.2.1 Rab GTPases Rab GTP ases are members of the ras superf amily of small GTP ases and are found expressed in all eukaryotes. Rab GTP ases exert a k ey regulatory function in

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membrane trafficking by their ability to recruit critical endo ytic effector proteins to the Rab-specific subcompartments of the endosomal system [37,38] and play a important role in regulating membrane transport between different endosomal compartments. The Rab GTP ases act as molecular switches, which re gulate a number of cellular events including vesicle budding, interaction between motile vesicles and the c ytoskeleton, fusion between transport v esicles and tar get or ganelles and organelle architecture [37–39]. Rabs 4, 5, 7, 9 and 11 ha ve all been sho wn to play key roles within the endosomal system. Rab 5 is mainly found on the membrane of early endosomes and re gulates the binding and recruitment of a lar ge number of effector proteins to early endosomes, including early endosomal antigen 1 (EEA1) and Rabenosyn 5, by controlling the generation of phosphatidylinositol-3-phosphate (PI3P) on early endosomal membranes. Together, Rab 5 and its ef fectors act as a regulatory system to allow vesicle docking and fusion to early endosomes [37,39,40]. Both Rab 4 and 11 are implicated in the rec ycling of car go back to the plasma membrane through re cycling endosomes ( Figure 6.1 ). Interestingly, fast re cycling of the transferrin receptor (and also AMPA receptors) has been sho wn to be dependent on Rab 11 [41]. Late endosomes, on the other hand, are enriched in Rab 7 and Rab 9 although the y mediate distinct actions [42,43]. Rab 9 has been found to regulate transport from the late endosome to the trans-Golgi netw ork, whereas Rab 7 has recently been sho wn to be responsible for endosome fusion and the maintenance of lysosomal structures [43,44]. Therefore it is clear that Rabs play an important role in defining subcompartments of the endo ytic system. The critical role of Rab proteins in regulating the post-endocytic sorting of some neurotransmitter receptors has recently been demonstrated by se veral groups, and these proteins or their mutants have been ele gantly used as endoc ytic markers and also to further defin the importance of neurotransmitter receptor sorting in synaptic plasticity (see Table 6.1 and section 6.3.3).

6.2.3 SIGNALS

FOR

ENDOCYTOSIS

AND

POST-ENDOCYTIC SORTING

A number of sequences for the endoc ytosis of cell surf ace receptors and other membrane proteins ha ve been identified [45]. Most of the signals so ar identifie have been classified into tyrosine-based signals, dileucine based signals and motif that are tar gets for post-translational modifications The sequence motif NPXY w as one of the first signals for internalization to b identified. It as originally identified in the LDL recepto , but later studies found this motif in v arious cell surf ace proteins including inte grins and the α-amyloid precursor protein. In addition to the NPXY motif, another important f amily of tyrosine-based signal are the YXXØ signals, where Ø represents an y bulky hydrophobic amino acid [45]. These tyrosine-based internalization signals ha ve been shown to be important for the internalization of a v ariety of membrane proteins and receptors including the transferrin receptor . Tyrosine-type sorting signals ha ve not only been implicated in internalization b ut have also been found to be important in other sorting e vents, such as lysosomal tar geting from endosomes and the transGolgi network (TGN) [45–47]. Analysis of the cytosolic domain of the CD3 receptor in T-cells led to the disco very of dileucine-based signals [48]. The [DE]XXXL[LI]

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Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors 97

motif is found to be highly conserv ed, and as with the YXXØ motif, its function is not restricted to internalization b ut can also mediate other membrane traf fickin events such as lysosomal tar geting [45]. Tyrosine and dileucine-type endoc ytosis signals have also been implicated in the internalization of se veral neurotransmitter receptors including GABAA receptors and NMDA receptors. Studies using chimeras of Tac and the cytoplasmic tail of the NR2B subunit revealed that the NR2B subunit contained a YXXØ motif that was responsible for internalization [22]. Furthermore, internalization of the Tac-NR2B chimera was prevented using dynamin K44A mutant (see section 6.3.3) that abolishes the activity of the GTPase dynamin, indicating that the internalization of Tac-NR2B is dynamin-dependent. NR2A also contains a YXXØ motif within its intracellular tail. Ho wever, Lavezzari et al. recently showed in a chimeric study that this motif w as not involved in the internalization of NR2A, and instead mutation of a dileucine type motif (L1319 and L1320) resulted in a significant reduction of NR2A endo ytosis [23]. In this same study, they found that both NR2A and NR2B are able to bind to the µ2 sub unit of AP2 although NR2B binds with much higher af finit . Dileucine-type signals ha ve also been implicated in internalization of GAB AA receptors from the cell surf ace of HEK293 cells. Herring et al. reported that a dileucine motif within the GABAA receptor β2 subunit is critical for endoc ytosis and that internalization of GAB AA receptors lacking this motif is dramatically inhibited [49]. In addition, se veral atypical endoc ytosis and AP2 adaptor binding motifs ha ve been identified in neurotransmitter receptor intra cellular domains. These include basic amino acid rich atypical AP2 binding motifs in both GAB AA receptor [50] and AMPA receptor [51] intracellular domains conserved with a pre viously identified atypical motif in synaptotagmin 1 [52]. AMPA receptors also contain other atypical amino acid motifs important for their internalization [17,53]. In addition, Scott et al. were able to sho w using tagged NMD A receptor sub units and chimeric constructs that the NR1 sub unit also contains a number of endocytosis signals in the membrane proximal domain of its C-terminal tail, which defined a n vel set of internalization motifs and which contrib ute in an additive manner to NMDA receptor endocytosis when expressed with NR2B subunits [54]. In addition to non-classical endocytosis motifs, another recently defined atypica signal for internalization is co valent modification with the polypeptide ubiquitin which has also been suggested to act as an endocytosis and membrane sorting signal. Ubiquitin is a highly conserv ed polypeptide that can be conjug ated to lysine chains via its carboxyl-terminal glycine. Ubiquitylation of proteins occurs in three steps: activation, conjug ation and lig ation. These steps are mediated by E1, E2 and E3 enzymes, respectively [55]. The presence of several endogenous lysines in ubiquitin allows for it to self-ubiquitinate, generating poly-ubiquitin chains. Evidence suggests that mono-ubiquitination or small ubiquitin chains act as signals for endoc ytosis [56,57]. Evidence of ubiquitin functioning as an internalization signal initially came from studies in yeast where a number of plasma membrane proteins are internalized and tar geted for v acuolar de gradation by a ubiquitin-dependent mechanism [57]. More recently, ubiquitin acting as a signal for endoc ytosis has also been demonstrated for a number of mammalian receptors including the EGF receptor [58–60], the epithelial sodium channel [61], E-cadherin [62] and potentially glycine receptors

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[63]. Interestingly, a recent study by Burbea et al. found that ubiquitin also plays an important role in the internalization of GluR1 receptors in C. elegans and that this internalization is dependent on the function of the clathrin adaptor AP180 [64]; however, a direct role for ubiquitin in the internalization of mammalian AMPA receptors has not yet been identified. The exact mechanims by which ubiquitination serves as a signal for endoc ytosis are still incompletely understood [55]. Similarly to tyrosine and dileucine-type signals, ubiquitin modification of membrane protein is also implicated in further traf ficking vents down the endosomal system through interaction with proteins such as Hrs and the ESC RT compl exes ( Figure 6.1 ), including tar geting to multi vesicular bodies and late endosomes suggesting that ubiquitination plays an important role in the late stages of the endoc ytic pathway [65,66].

6.3 METHODS FOR STUDYING ENDOCYTOSIS The critical role played by neurotransmitter receptor endocytosis and post-endocytic sorting in re gulating the number of surf ace and synaptic receptors and, hence, the strength of the synapse has lead to a significant interest in approaches for studyin the mechanisms of neurotransmitter receptor endocytosis. Two techniques have been used in ab undance for studies of neurotransmitter receptor internalization and trafficking: a biochemical approach based on the c valent modification of sur ace receptors with biotin, which has pro ved particularly useful for studies of receptor endocytosis kinetics and turno ver rate; and an immunofluorescence labelin approach for following receptors down the endosomal pathway using antibodies that recognize epitopes in the e xtracellular domains of the receptors being studied.

6.3.1 ANTIBODY FEEDING Antibody feeding is a po werful technique that has been ele gantly used to visualize neurotransmitter receptor endocytosis and trafficking. It i volves the use of antibodies that recognize specific xtracellular epitopes on the receptor of interest to label these receptors in li ve cells. The target epitopes can either be endogenous signals (i.e., native amino acids) within the e xtracellular domain of the receptor sub unit, or they can be an engineered extracellular tag on a recombinant receptor (for example, a myc epitope). Under non-permeabilizing conditions in li ve cells, only receptors on the surf ace membrane will be labeled when cells are e xposed to the antibody . Once the antibody has bound a surface-expressing receptor, the antibody is generally assumed to stay bound to the receptor throughout the endosomal pathw ay until the acidic pH of the lysosome dissociates the antibody/receptor comple x and de grades both receptor and antibody. Also, it is assumed by several groups that if the receptor is targeted for rec ycling, the antibody will remain bound to the receptor , allowing this technique to be modified to also permit the study of receptor re ycling [67]. Antibody feeding has been used extensively for the study of neurotransmitter receptor trafficking in both heterologous xpression systems and cultured neurons. Generally, three main steps e xist in antibody feeding protocols: binding of the antibody to the target receptor of interest, internalization of the antibody-receptor comple xes

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and immunoc ytochemistry. Here, we describe these steps and also some of the variations that ha ve been reported for ho w some of these steps are performed. Antibody binding to cell surf ace receptors in cultured cells is generally carried out at 4°C (which blocks membrane traf ficking) to inhibit receptor endo ytosis during the antibody binding step. Both cell lines and cultured neurons appear to tolerate exposure to low temperatures [18,21,54]. Antibody labeling can be carried out in media b uffered with HEPES (for e xample, we routinely use a solution containing 25 mM HEPES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2, 15 mM glucose, pH 7.4, or DMEM supplemented with 25 mM HEPES and 0.5% BSA, pH 7.4) [18,21,49]. Because primary cultured neurons can be more sensiti ve to exposure to low temperatures for prolonged periods of time, se veral groups ha ve performed antibody binding to surf ace receptors at 37°C [20,68]. In this case, antibody can simply be added directly to the culture medium [20,69]; ho wever, using this approach, antibody labeled receptor comple xes can start to internalize during the labeling step. When looking at activity or ligand-induced internalization of receptors that do not e xhibit significant basal endo ytosis, this characteristic does not pose too significant a problem as sur ace receptors can be labeled at 37°C with very little receptor internalization until endocytosis is triggered. Similarly, for qualitative studies of constitutive receptor endocytosis it is also possible to allo w antibody binding and internalization to occur simultaneously . Ho wever, for co-localization studies, blocking receptor uptak e during the initial stage of antibody binding could pro ve advantageous by increasing the number of receptors found at specific o ganelles at a particular time point during the subsequent endoc ytosis stage of the protocol. Similarly, when looking at rates or time course of constituti ve endocytosis in neurons, labeling surf ace receptors at 37°C might gi ve rise to inaccurate results. F or this reason, some groups have elected to overcome the problem of receptor internalization during antibody labeling at 37°C by using a re versible block of endoc ytosis during the antibody incubation period. One such approach is the use of high (h ypertonic) concentrations of sucrose (350 to 500 mM) in the medium [68] during antibody binding. High sucrose incubation has been sho wn to reduce the number of clathrincoated pits on the plasma membrane and cause the polymerization of clathrin below the cell surf ace, inhibiting internalization [70], and has been commonly used as a method to block receptor internalization via the clathrin pathw ay [20,71]. However, one disadvantage of h ypertonic sucrose treatment of neurons is that it is also commonly used to elicit neurotransmitter release, and therefore is lik ely to cause substantial exocytosis of presynaptic neurotransmitter containing vesicles that can confound or complicate the interpretation of studies of acti vity-dependent receptor internalization [72]. Once the antibody has bound surf ace receptors, cells are returned (in the case where antibody was bound in binding media) to conditioned culture media to allo w receptor endocytosis. Receptors are allo wed to internalize for the desired period of time and then cells are placed on ice to stop endoc ytosis and fi ed (for e xample, using 2 to 4% paraformaldehyde). Incubating the cells with a fluorophore-conju ated secondary antibody prior to permeabilization will allo w the detection of surf ace receptor populations. Cells can then be permeabilized (for e xample, using 0.02% Triton) and internalized receptors detected with a secondary antibody conjug ated to

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Internalization

Fix

Permeabilization

37oC

Secondary

Secondary

(a)

Internalization 37oC

Permeabilization Fix

Secondary

(b)

FIGURE 6.2 Schematic diagram of the antibody feeding technique. (a) Diagram sho wing the steps in volved in the labeling of surf ace and internalized receptors. Primary antibody to an extracellular epitope is allo wed to bind the receptor in conditions that block endoc ytosis. Cells are then returned to conditioned media where receptors are allowed to internalize. After fixation addition of a fluorophore conj ated secondary antibody (white) labels all remaining surface receptors. Cells are then permeabilized and incubated with a dif ferent fluorophor conjugated secondary antibody (black) to label the pool of internalized receptors. (b) Schematic diagram sho wing the use of antibody feeding to label internalized receptors only . Binding and internalization of receptor -antibody complexes is carried out as abo ve. Before fixation, cells are ashed in 0.2 M acetic acid/0.5 M NaCl in PBS to remo ve any surfacebound primary antibody . Cells are then permeablized and incubated with a fluorophore conjugated secondary antibody to identify internalized receptors.

a different fluorophore. Using this approach, it is possible to distinguish betwee surface and internalized receptors (Figure 6.2). Recent adv ances in digital image processing have allowed for the quantification of internalized receptor l vels normalized to surf ace receptor or total receptor number . In general, this is done by comparing the intensities of the dif ferent fluorophores representing the di ferent receptor populations, although it can also be done by analyzing the surf ace area occupied by each fluorophore. Although an intensity measurement will be more accurate, the parameters for image acquisition must be k ept constant from cell to cell. The use of a single fluorophore to label both populations (e.g., the total recepto population and the internalized receptor population), in combination with a second fluorophore that ta gets only one of the receptor populations (for example, remaining surface receptors), allows for a control intrinsic within the experiment. For example, Herring et al. follo wed constitutive GABAA receptor endocytosis in HEK293 cells with antibody feeding e xperiments [49] using monoclonal myc (9E10) antibody , which recognized a myc epitope tag engineered into the e xtracellular domain of the

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GABAA receptor α1 subunit. After binding of the myc antibody to the receptor at 4°C for 1 hour , they incubated the cells with a Texas Red conjug ated anti-mouse secondary for another hour on ice. F ollowing this, cells were returned to 37°C and receptors allowed to be internalized. After 20 min, membrane traf fic as blocked by incubating cells ag ain at 4°C (on ice). At this point, both internalized and remaining surface receptor populations were labeled with the Texas Red secondary antibody. Following this, an Alexa-488 conjugated secondary antibody w as used to label remaining surf ace antibodies (under non-permeabilizing conditions). Texas Red-labeled receptors therefore represent the total number of surf ace and internalized receptors, whereas Texas Red and Alexa-488 co-labeled receptors represent those remaining at the cell surf ace. Using this approach, carrying out a direct comparison of the intensities of the dif ferent receptor subpopulations is possible, as they are both labeled with the same fluorophore. Where recombinant receptors are used, another approach is to use GFP-labeled receptors (the GFP signal will gi ve a value for all receptors in the cell) and a primary antibody to label internalized receptors, which can then be detected with a fluorophore that does not verlap with GFP and which will allo w values to be determined for the number of internalized receptors (this can then be normalized to the GFP signal, representing the total receptor population). Not in all cases does one want to label the remaining surface receptors. In some cases, one might want to carry out co-localization studies where a number of dif ferent fluorophores are required; in other cases, especially in neuronal dendrites, xtensive receptor expression at the cell surface could mask the signal received from internalized receptors. In addition, for the sake of quantitation, detecting only the internalized receptor population (for e xample, when normalizing to an intrinsic control such as GFP or other parameter such as cell v olume or the signal from a c ytosolic marker) might be preferable [73]. In such cases, selectively stripping any remaining surfacebound primary antibody and leaving intracellular antibody-receptor complexes intact by washing the cells at l ow pH prior to fixation is possible Figure 6.2 ). The intracellular antibody labeled receptors that ha ve been internalized can then be detected with a secondary antibody after cell permeabilization. Although this procedure has been e xtensively used in a number of cell lines, Carroll et al. first use it on cultured hippocampal neurons for the study of AMPA receptor endoc ytosis [69] and several other groups have also used antibody stripping protocols to monitor receptor internalization in cultured neurons [67,73]. Using antibody stripping, Ehlers and colleagues have further developed a version of the antibody feeding technique to allo w the study of receptor rec ycling [54]. In this method, surface receptors are antibody labeled and allowed to internalize. After internalization, cells are placed on ice and remaining surf ace bound antibodies are stripped by exposing the cells to 0.2 M acetic acid/0.5 M NaCl in phosphate b uffer solution (PBS), gi ving rise to a solution of approximately pH 3.0). The cells are then incubated at 37°C in the presence of a fluorophore-conju ated antibody. This secondary antibody will then bind to an y receptor-antibody complex that returns to the plasma membrane. After the receptor rec ycling step, the cells are then fi ed, permeabilized and incubated with a second fluorophore-conju ated antibody to label internalized receptors that f ailed to rec ycle. Recycled receptors will be co-labeled

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Acid Strip 4 °C

Internalization 37°C

(a)

(b)

Internalization and Recycling

Fix/Permeabilization

37°C

Secondary Antibody

(d)

(c)

(e)

FIGURE 6.3 The use of antibody feeding to study receptor rec ycling as carried out by Scott et al. [54] (a) Primary antibodies bind surf ace receptors. (b) Cells are then placed in conditioned media at 37°C to allow receptor-antibody complexes to internalize. (c) Any remaining surface-bound antibody is then stripped by washing the cells in 0.2 M acetic acid/0.5 M NaCl in PBS at 4°C. (d) Cells are then returned to 37°C where further internalization and recycling events take place. The cells are placed in conditioned media containing a fluorophore conju gated secondary antibody (white) to bind an y receptor-antibody complexes that return to the surface. (e) Finally, the cells are fi ed, permeabilized and incubated with a dif ferent fluoro phore conjugated antibody (black) to label an y internalized receptors that f ailed to rec ycle.

with two fluorophores, whereas remaining internalized receptors will only be labele with the fluorophore used under non-permealizing conditions. A schematic diagram is shown in Figure 6.3. As an alternative recycling assay, receptor recycling has also been measured as a decrease in signal from an antibody-labeled internalized receptor pool over time [67]. The simplicity of the antibody feeding technique has made it a popular method for studying the molecular determinants of receptor endoc ytosis. It has been used to determine the route taken by internalized receptors through the endocytic pathway by visualization of labeled receptors as the y progress do wn the endoc ytic pathway by co-labeling with antibodies to endogenous mark ers of dif ferent or ganelles or transfected GFP-tagged mark ers to label structures including CCVs, early endosomes, recycling endosomes, late endosomes and lysosomes ( Table 6.1) [74]. This approach has also been used to further study the mechanisms and factors controlling surface receptor internalization, and the molecular signals within receptor intracellular domains responsible for receptor endoc ytosis and post-endoc ytic sorting.

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TABLE 6.1 Commonly Used Markers for the Identification of Endosomal Compartments Compartment CCV Early Endosome

Recycling Endosome

Late Endosome

Lysosome

Markers Used

References

Clathrin, GFP-Clathrin AP2 EEA1 Rab 5, GFP-Rab 5 Transferrin receptor Fluorophore conjugated transferrin Transferrin receptor Flurophore conjugated transferrin Rab 4, YFP-Rab 4 Rab 11, GFP-Rab 11 GFP-Rme I Syntaxin 13 LAMP 1, GFP-LAMP 1 Rab 7, GFP-Rab 7 Rab 9, GFP-Rab 9 LAMP 1, GFP-LAMP 1

[101] [21] [17,74,102,103] [23,54,74] [74] [101] [17,74] [54,67,102] [74] [23,67] [67] [17,103] [74] [54] [23] [17,74,103]

6.3.2 CELL SURFACE BIOTINYLATION The covalent modification of cell sur ace receptors with biotin is another v aluable technique for monitoring receptor traf ficking. Bioti ylation enables the cell surf ace receptor complement at a particular time point to be labeled, b ut instead of using a specific antibod , all surface proteins are tagged with biotin. The subsequent fate of biotinylated proteins can then be determined through af finity purification fol wed by SDS-PAGE and Western blotting. Biotin is a 244.3 Da w ater-soluble vitamin (vitamin H) that covalently bonds to lysine groups at temperatures lo w enough to inhibit endoc ytosis. A number of commercially available biotin derivatives that react with primary amines ha ve been developed that incorporate link er regions, so as to minimize steric interference and also allow the clea vage of the biotin ylated molecule. The egg-white protein a vidin has a high af finity for biotin, and hence bioti ylated proteins can be ef ficientl purified from lysates with vidin conjug ated to silica beads (most commercially available kits utilize bacterially derived streptavidin). Indeed, the interaction between biotin and a vidin is one of the tightest kno wn with a Kd of ~10 –15 M, and the robustness of this binding permits purification of bioti ylated proteins from solutions containing denaturing agents such as SDS. Sulfo-NHS-biotin (N-h ydroxysulfosuccinimidobiotin) is a biotin deri vative that is commonly used in neuronal receptor trafficking xperiments (a vailable from Pierce). It contains a ne gatively char ged sulphate group which confers membrane impermeablity , thus minimizing labeling of intracellular proteins, and it has low cytotoxicity and so is suitable for experiments lasting many hours.

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Although biotinylation is a biochemical technique that does not permit visualization of receptor location, it has many advantages over techniques that label surface receptors with specific antibodies. It is particularly useful if antibodies to the protei in question are not suitable for microscopy, or are raised to intracellular and therefore inaccessible domains. Biotin ylation also af fords the luxury of being able to study the traf ficking of multiple di ferent receptors/membrane proteins within the same experiment. This is easily achieved if the proteins of interest are of dif ferent molecular weights, in which case different parts of the same Western blot membrane (i.e., representing different molecular wieghts) can be separately probed with antibodies recognizing different proteins of interest. Alternatively, this can also be achieved by stripping and reprobing the Western blot membrane with a dif ferent antibody. The small size of the biotin molecule, in contrast to an antibody, means that the properties of labeled receptors are less likely to be affected than when antibody tagged, whereas the univalency of biotin derivatives means that the cross-linking of receptors through the biotin molecule will not occur . During biotin ylation experiments, the receptors on millions of cells are assayed simultaneously, giving greater quantitative sensitivity than that appreciable using immunofluoresence techniques, and so subtle diferences in endocytosis and trafficking rates can be delineated 6.3.2.1 Biotinylation Method The technique described here is modified from that used by Mammen et al. [75]. Dishe of cultured neurons are remo ved from the incubator and cooled on ice with all further steps carried out on ice and using solutions at 4 °C so as to prevent receptor endocytosis and membrane trafficking during the biotin labeling step [75]. Culture media is rem ved and dishes are then rinsed twice with PBS supplemented with 0.5 mM MgCl 2 and 1 mM CaCl 2 prior to e xposure of cells to biotin, dissolv ed in the supplemented PBS to a concentration of 1 mg/ml. The biotinylation reaction should be carried out for a period of 10 to 30 min (we routinely biotinylate for 12 min) with continuous or periodic gentle agitation to ensure equal co verage of all neurons. We ha ve found that 2 to 4 ml of solution is enough to coat 10-cm dishes of cultured neurons, and for 6-cm dishes, 0.5 to 1 ml should suf fice. After biotinylation, removing all unreacted biotin is important, and this can be achie ved through three 5-min w ashes with a quenching agent such as 50 mM lysine or glycine and 0.5% BSA dissolved in PBS/Mg2+/Ca2+. After quenching, cells are washed two further times for 3 min in PBS/Mg 2+/Ca2+. At this point, cells can be lysed to identify the surf ace receptor pool, or the media returned to the dishes containing cells with biotinylated membrane proteins and replaced in the incubator for trafficking and d gradation experiments. Efficient solubilization of most receptor proteins is achi ved when cells are lysed in standard radio-immunoprecipitation assay b uffer (RIPA) containing 0.1% SDS (RIPA: 50 mM Tris, pH 7.4, 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS). Neurons should be scraped of f dishes in this b uffer, the lysates placed in microcentrifuge tubes and then left to solubilize with rotation on a wheel at 4°C for 1 hour . After solubilization, cell and nuclear debris are remo ved by centrifugation at 14,000 G for 15 min. Biotinylated proteins can then be captured from the supernatant using af finity chromatograp y with avidin slurry at 4°C for 2

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(a)

Internalization 37°C

Recycling 37°C

Cleavage Buﬀer 4°C

Cleavage Buﬀer in media

(b)

(c)

. Permeabilization . Streptavidin Precipitation

SDS PAGE Immunoblotting

FIGURE 6.4 The use of biotin to study (a) surf ace numbers, (b) internalization and (c) recycling of receptors. (a) Biotin is allo wed to bind surf ace proteins in conditions that block endocytosis. (b) Cells are placed in conditioned media at 37°C to allo w internalization of surface receptors. The cells are then w ashed twice with cleavage buffer (50 mM glutathione, 75 mM NaCl, 10 mM EDT A, 0.075 N NaOH) at 4°C to remo ve any surface bound biotin. (c) To study recycling events as a loss of internalized signal, cells are once ag ain returned to conditioned media containing 50 mM glutathione to clea ve an y receptor -biotin comple xes that return to the surf ace. Depending on whether the biotin protocol is being used to look at surface receptor numbers, receptor internalization or receptor rec ycling, cells will be solubilised in RIP A after steps (a), (b) or (c), respecti vely. Biotin ylated proteins can then be precipitated using streptavidin beads. Finally, the precipitated complexes are resolved by SDSPAGE and the receptors of interest identified by immunoblotting

hours (we routinely use a ratio of 3:1 of cell lysate and a vidin beads; approximately 100 µl of Neutra vidin beads, a vailable from Pierce). The slurry is then spun do wn from the supernatant and should be w ashed twice for 5 min per w ash in RIP A containing 500 mM NaCl follo wed by a further w ash in standard RIPA. The slurry is then boiled in Laemmeli gel loading b uffer for 5 min prior to SDS-P AGE and Western blotting (Figure 6.4a). The above or similar protocols have been effectively used for surf ace biotin ylation studies of the traf ficking of ma y neurotransmitter receptors including AMPA receptors, NMDA receptors, kainate receptors, GAB AA receptors and GABAB receptors, among others [22,24,74,76,77]. One common difficulty occurring during the bioti ylation protocol is the detachment of neurons from the dishes and thus the prevention of further experimentation. This is usually attrib utable to too vigorous rinsing and is pre ventable by ensuring minimal trauma to neurons by careful solution changes during the biotin ylation protocol. To v erify membrane inte grity during the biotin ylation protocol and that only surface proteins ha ve been biotin ylated, Western blots of the precipitated

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biotinylated proteins can also be probed with antibodies to control intracellular proteins such as for example 14-3-3, actin, tubulin or MAP1a. No signals should be detected to intracellular proteins. 6.3.2.2 Quantification and Verification The simplest use of biotin ylation is to quantify the total amount of receptor at the cell surface [76]. This is useful when comparing treated v ersus untreated groups of cells and can also be used to determine the proportion of the total receptor pool that resides at the cell surf ace [76]. Biotin ylation has been used in this manner to demonstrate the relative increase in proportion of GluR1 receptor subunits that reside at the neuronal surf ace with increasing maturity in spinal cord neurons [75]. As mentioned previously, biotinylation is especially suitable for identifying small differences between treatment groups that might not be apparent using immunofluo resence techniques. Biotinylation is also suitable to demonstrate the surface traffick ing of an e xperimentally expressed tagged protein in neurons. An example of this use has been to demonstrate the ef ficient accumulation of a myc-tagged GluR receptor subunit at the surf ace of virally infected neurons e xpressing a myc-GluR2 construct. Here, biotinylation allowed the demonstration of motifs within the GluR2 subunit required for cell surf ace accumulation of the sub unit [78]. 6.3.2.3 Endocytosis The biotin deri vative sulfo-NHS-SS-biotin contains a disulphide bond between the amino-terminal reactive linker group and biotin that is brok en upon the addition of a reducing agent such as glutathione (a vailable from Pierce). In effect, this adds the facet of reversibility to the biotinylation technique, which can be effectively applied to studying the endoc ytosis of neurotransmitter receptors. To determine whether a receptor of interest is being endoc ytosed (and therefore the rates of endoc ytosis), neuronal cultures can first be pretreated with the lysosomal inhibitor leupeptin a 100 µg/ml for 1 hour to pre vent lysosomal proteolysis of internalized proteins [74,77]. Labeling of surface receptors with sulfo-NHS-SS-biotin is then carried out following the protocol described above for the standard surface receptor biotinylation (retaining the culture media from the dishes of neurons at the be ginning of the experiment is useful). Upon completion of biotin labeling of the surf ace receptor population (in PBS/Mg2+/Ca2+ at 4°C, as above) and subsequent blocking, the media (at 37°C) is returned to dishes that are then returned to 37°C incubator. Upon return of labeled cells to 37°C, membrane trafficking of receptors will resume (at this poin receptor agonists/antagonists can also be added for studies of agonist induced inter nalization). Importantly, any biotin labeled receptors that are internalized during this period will be protected from the non-membrane permeable biotin clea vage reagent (see below). Control dishes (usually tw o) can be k ept on ice (to pre vent endoc ytosis) for some useful controls. One of these plates can be used to obtain a reference v alue for the total number of surf ace receptors (i.e., total surf ace receptor number at time zero, prior to internalization) e xactly as in standard surf ace receptor biotin ylation

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described above. Another dish can be used to control for the ef fica y of the biotin stripping protocol. The remaining biotin labeled dishes can be incubated at 37°C for set time-points to produce a time course of endoc ytosis (when a particular time point has ended, dishes can be returned to 4°C to block an y further membrane trafficking and internalization). or most receptors, suggested time-points are usually 0, 5, 15, 30 and 60 min. After the internalization step, media is removed from dishes and they are twice treated with 50 mM glutathione (dissolv ed in 75 mM NaCl, 10 mM EDTA and 0.075 N NaOH to adjust the stripping solution to between pH 7.5 to 8.0) for 15 min per treatment at 4°C with gentle agitation. All remaining surface sulfo-NHS-SS-biotin is clea ved (stripped) from the remaining surf ace proteins by the reducing agent during this step. In contrast, because glutathione (the reducing agent used for the clea vage step) is not permeable across intact cells membranes, any surface biotinylated receptors that were endocytosed during the endocytosis time course are protected from clea vage. After three to fi e further w ashes in PBS/Mg 2+/Ca2+ to remove any remaining glutathione from the dishes and neuronal surf ace, remaining protected, internalized biotinylated proteins can then be captured using solubilization of cells in RIP A buffer and strepta vidin af finity chromatograp y, SDS-P AGE and Western blotting as described above for the standard surface biotinylation protocol. This protocol therefore gives a direct read-out for the number of internalized receptors ( Figure 6.4b), which can be compared under dif ferent treatment conditions [79] or which can also be expressed as a percentage of the total surf ace receptor population at the start of the protocol (using the v alues deri ved from the control plate described abo ve). Although glutathione clea vage of surf ace biotin is highly ef ficient, erifying the extent of clea vage within each e xperiment is wise by also glutathione-stripping a control dish of neurons that have remained on ice and thus will have no intracellular accumulation of protected biotin ylated proteins [74,77]. This control should gi ve only a v ery lo w background signal le vel for the receptor of interest because no receptors are internalized and protected when membrane traf fic is bloc ed at 4°C [74,77]. This type of approach has been ele gantly used to study the kinetics and control mechanisms of AMPA receptor internalization in cultured cortical neurons [74]. Similarly , this approach has also been used to study the internalization of several other neurotransmitter receptors including GAB AA receptors [77], GAB AB receptors [76], NMD A receptors [22,74] and kainate receptors [24] in cultured cortical neurons. 6.3.2.4 Further Trafficking Events Sulfo-NHS-SS-biotin can also be effectively used to test whether internalized receptors are returned to the plasma membrane for re-insertion using a “rec ycling” assay. When the rate of internalization and membrane re-insertion of biotin ylated proteins has equalized, then the internalized pool of biotin ylated proteins will be observ ed to plateau at an equilibrium point [74,77]. At this point of maximal intracellular accumulation, analyzing the rate of re-insertion of endocytosed receptors and hence studying f actors that control re-insertion is possible. To do this, biotin ylation should be carried out in a manner identical to that described for studying receptor

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endocytosis above: surface receptor pools are labeled with “re versible” sulfo-NHSSS-biotin at 4°C, followed by incubation at 37°C to allo w internalization of biotinlabeled receptors and then cleavage of remaining surface receptors to give a protected population of biotin ylated, internalized receptors (follo wing the method described above). However, after neurons are treated with glutathione to remove the remaining surface biotin, instead of subsequent lysis in RIP A b uffer (as in the endoc ytosis assay) an additional live cell glutathione biotin cleavage step is carried out whereby neurons are incubated at 37°C for further time periods with glutathione in thexternal e culture medium to clea ve an y internalized biotin ylated receptors rec ycling to the cell surface. For this modified protocol, after the 4°C biotin cle vage step, cells are returned to culture medium containing 50 mM glutathione (with NaOH to pH 7.5 to 8.0) and returned to 37°C to allo w receptor re-insertion. A control dish should be kept on ice, which will serv e as the time zero control for the rec ycling assay. If any sulfo-NHS-SS-biotinylated receptors in the internalized pool are reinserted in the membrane, they will be e xposed to the glutathione within the media and hence cleaved. The loss of biotnylated internalized receptors after the second biotin clea vage provides a measure of receptor recycling [74,77]. At the end of the time course, biotinylated proteins are purified using strept vidin af finity chromatograp y and analyzed using SDS-PAGE and Western blotting as usual. The rate of re-insertion will be equivalent to the rate of signal diminishment with time for the protein being detected by Western blotting (Figure 6.4c) [74,77]. The recycling protocol has been effectively used to demonstrate the rec ycling of internalized receptor populations including those of AMPA receptors, kainate receptors and GAB AA receptors [24,74,77]. 6.3.2.5 Biotinylation of Slices A modification to the bioti ylation protocol has recently been reported that allo ws for the biotinylation of organotypic and acute hippocampal slices [80], which therefore has the adv antage of using a neuronal preparation that maintains neuronal connectivity and correlates better with other ph ysiological experiments using brain slice preparations (such as electroph ysiological recordings). Kaila and colleagues have described a method for labeling 350 µM acute hippocampal slices using sulfoNHS-SS-biotin at 100 µM dissolv ed in standard ph ysiological solution (artificia cerebro-spinal fluid,ACSF) [80,81]. To allow complete penetration of the slices with biotin, and thus the labeling of cells within the slices, the reaction should last for 45 min. Using this technique, demonstrating that biotin ylated slices sho w normal evoked field responses and that glutamate receptors within the slices are subject t ligand mediated de gradation lik e that noted in cultures w as possible [74,80]. The maximal viability of slices is 5 to 6 hours, ho wever, meaning that this technique is only suitable to measure acute changes in surf ace receptor numbers in tandem with electrophysiologal e vents. Also questionable is whether this technique will pro ve suitable for endoc ytosis assays where the ef ficien y of biotin clea vage might v ary according to the depth of the cell within the slice.

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6.3.2.6 Degradation Assays A final application of sur ace receptor biotinylation is the determination of membrane receptor degradation rate. This technique in volves the labeling of surf ace receptors in live cells with biotin and then follo wing the loss of the biotin ylated receptor pool (biotinylated receptors are purified and analyzed using strept vidin, SDS-PAGE and Western blotting as described abo ve) after defined times of incubation (in cultur media at 37°C) and comparing to the level of surface receptors at a time zero control. In this type of approach, biotin labeled receptors are assumed de graded and will no longer be captured by streptavidin chromotagraphy. Therefore, degradation of receptors is observed as a loss of the biotinylated receptor population over time, which can be quantified from the Western blot signals. Briefl , media is remo ved and set aside from dishes of cultured neurons that are then surf ace biotinylated on ice with sulfoNHS-biotin following the procedures described abo ve. After rinsing and quenching as detailed above, the cells (which now have biotinylated surface membrane proteins) are returned to culture media and placed at 37°C for defined periods of time (on dish is kept as a time zero total surf ace receptor control). When the time has elapsed for a particular time point, the sample can then be snap frozen until all the time points are collected. When all required time points ha ve been collected, lysates are tha wed at 4°C and the a vidin purification of bioti ylated proteins can proceed as normal followed by SDS-PAGE and Western blotting. Lysosomal inhibitors (e.g., leupeptin, chloroquine) or inhibitors of other de gradative pathways (such as proteosome inhibitors) can be included in duplicate time points to allo w determination of the mechanism and pathw ay of de gradation [24,74,77]. Similarly , agonists and antagonists of receptors or signalling pathways can also be included in the assays to identify mechanisms important for controlling rates of receptor de gradation. The adv antage of this technique o ver conventional metabolic labeling e xperiments is that it allo ws the determination of half-life of surf ace and therefore presumably acti ve receptors as opposed to the total receptor pool. Because surf ace trafficking of receptors relies upon correct assembl , the calculated degradation will not include inappropriately folded receptors that w ould be normally tar geted for degradation directly from the endoplasmic reticulum. This technique does not identify the location of the receptor at the time of cell lysis. This technique also assumes that biotinylation per se has no effect on receptor degradation rates. When employing this technique to analyze effects of agonist on degradation rate, ruling out any change in pharmacology of biotinylated receptor that could occur if the site of biotinylation is close to the site of agonist binding is important [76]. The surface receptor biotinylation de gradation approach has been used to demonstrate v arious aspects of neurotransmitter receptor degradation [24,74,77], including the variable degradation rate of glutamate receptors according to age of neuronal culture [75] and also the agonist-induced de gradation of GAB AB receptors [76] with both of these studies demonstrating efficient purification of biot ylated proteins at times greater than or equal to 24 hours post biotin ylation

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6.3.3 OTHER USEFUL TOOLS FOR STUDYING NEUROTRANSMITTER RECEPTOR ENDOCYTOSIS AND POST-ENDOCYTIC SORTING The identification during the last decade of ma y of the core components of the machinery important for membrane protein endoc ytosis and postendoc ytic sorting has allowed the production of an e xpanding molecular genetic tool box that can be used to alter or block specific steps in the endo ytic pathway. Several of these tools have proved very useful for g aining additional insights into the molecular mechanisms that underlie the re gulation of neurotransmitter receptor internalization and membrane trafficking and the role of these tra ficking vents in synaptic plasticity . These tools have often been used in conjunction with labeling techniques to follo w neurotransmitter receptors while simultaneously interfering with the endoc ytic machinery. Here we discuss a fe w examples of their use. One of the most widely used genetically encoded dominant ne gative cDN A constructs applied to studies of membrane protein internalization has been dominant negative dynamin. Dynamin family GTPases play a critical role in membrane traffi and vesicle endocytosis by acting as “pinchases” to allo w the budding and scission of nascent car go vesicles from the plasma membrane or other membrane compartments [12]. Importantly , the identification of mutations that abolish the act vity of dynamin family members, and in particular mutations that impair dynamin GTP ase activity or GTP binding, have been helpful for better understanding the role of these proteins in receptor endocytosis [12]. In particular, mutation of Lys 44 (a conserved amino acid throughout all GTP ases, mutation of which impairs GTP binding) in dynamin 1 was found to block endoc ytosis and the internalization of se veral membrane proteins including transferrin receptor and the EGF receptor . More recently , the equi valent mutation in dynamin 2 (dynamin 2 K44A mutant) w as sho wn to inhibit receptor endoc ytosis even more potently than the dynamin 1 K44A mutant. The dynamin 1 and 2 K44A mutants ha ve been extensively used for elucidating the mechanisms (and route) of internalization of man y membrane proteins, including several neurotransmitter receptors (such as AMPA receptors, NMDA receptors, and GABAA receptors) [12,21,22,49,69]. Importantly , interfering with the acti vity of dynamin has also been critical for demonstrating that neurotransmitter receptor internalization directly underlies the ph ysiological decrease in the strength of synaptic responses in se veral forms of synaptic plasticity [1,82,83]. Although dynamin plays an important role in clathrin mediated endocytosis and dynamin mutants have commonly been used to sho w that a receptor is internalized by a “dynamin-dependent clathrin mediated” internalization pathw ay, dynamin has also more recently been sho wn to be required for the b udding of ca veolae from purified endothelial plasma membranes [84]. The use of anti-dynamin antibodies in studies of cholera toxin B internalization ha ve further supported the idea that dynamin function is not restricted to clathrin dependent endocytosis [85]. Since these initial findings, dynamins h ve been shown to be involved in the caveolin-dependent uptake of various molecules including the SV40 virus [86], echo virus 1 [87], muscarinic choliner gic receptors [88] and interleukin 2 receptors [12,32]. Dynamin, however, has not been found to be necessary for all caveolae-dependent endocytosis, suggesting that dynamin mutants are not just general blockers of endocytosis but do

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allow for the identification of specific upt e pathways [12]. However, care must be taken in the interpretation of results obtained using dominant ne gative dynamin constructs with respect to the particular internalization pathw ay (e.g., clathrin or non-clathrin dependent) tak en by a particular surf ace membrane protein. Whereas dynamin mutants ha ve pro ved useful for better understanding the initial steps of neurotransmitter receptor endoc ytosis, other dominant ne gative constructs are increasingly being used to obtain clues as to the post-endoc ytic sorting f ate of internalized receptors and the mechanisms important for controlling this. For example, as described above, the Rab GTPases play a particularly important role in the re gulation of membrane transport between intracellular compartments within the endoc ytic pathw ay and dif ferent isoforms are enriched in particular endocytic subcompartments [43]. Hence, Rab proteins ha ve been widely used as markers to identify specific subcompartments of the endo ytic pathway, particularly different types of endosome (seeTable 6.1). Although antibodies specific for dfferent Rab isoforms are a vailable, GFP fusions of the dif ferent Rabs ha ve also been extensively used as subcellular mark ers, and the use of GFP mark ers often a voids problems arising from IgG subtype limitations [37,40,89,90]. More recently, because of the essential role of Rab proteins in post-endoc ytic trafficking, a number of mutant Rab proteins h ve been used to block or alter the transport of receptors between dif ferent sub-compartments of the endoc ytic system and further elucidate the ph ysiological role of receptor post-endoc ytic sorting in regulating receptor trafficking and plasticit . In an elegant study carried out by Brown et al., for example, Rab 5 was found to drive the internalization of AMPA receptors upon NMDA receptor activation during hippocampal LTD [89]. In this study, overexpression of GFP-Rab 5 resulted in a significant reduction of AMPA EPSCs b ut not of NMDA receptor currents, whereas the use of a dominant ne gative GFP-Rab 5 construct with a mutation in its GTP binding domain (GFP-Rab 5, S34N) had no effect on either AMPA or NMD A EPSCs. In addition, NMD A receptor-dependent LTD caused an increase in active Rab 5, and cells expressing the dominant negative form of Rab 5 f ailed to e xhibit LTD. Importantly , this same study re vealed the importance of Rab5 in AMPA receptor removal upon NMDA receptor activation but not in constitutive endocytosis [89]. In another e xample of the use of GFP-Rabs as tools for ph ysiological studies of receptor endocytosis, Park et al. used dominant ne gative GFP-Rab 11 (S25N) to prevent the endocytic recycling of AMPA receptors and observ ed an important link between AMPA receptor rec ycling and L TP [67]. They showed that when Rab 11 function is impaired, internalization ofAMPA receptors and transferrin is unaffected; however, receptor cargo fails to return to the surface and remains trapped within the recycling compartment resulting in a reduction in cell surface receptor numbers [67]. Furthermore, cells transfected with wild-type GFP-Rab 11 sho wed no difference in AMPA receptor recycling compared to those transfected with GFP only . The use of this construct allo wed the authors to then in vestigate the ph ysiological ef fects of AMPA receptor recycling by carrying out electrophysiological studies in hippocampal neurons transfected with GFP-Rab 11 (S25N). Using this approach, the authors elegantly sho wed that AMPA receptors important for L TP of e xcitatory synaptic transmission are derived from a recycling pool of previously internalized receptors.

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In this same study , P ark et al. confirmed their results by ta geting the acti vity of another protein involved in the recycling pathway [67]. Rme I is an Eps15-homology domain containing protein involved in the exit of membrane proteins from recycling endosomes [91,92]. Mutation studies ha ve sho wn that Rme I is responsible for receptor recycling in mammalian cell lines and that mutation of a single amino acid within its identified Eps15-homology domain is able to pr vent rec ycling of the transferrin receptor, without ha ving an ef fect on membrane protein transport from sorting endosomes to late endosomes [67,91,92]. In the study by P ark et al. [67], the authors demonstrated that dominant negative Rme1 blocked recycling of AMPA receptors and also LTP. From the above examples, the use of mutant versions of key components of the endoc ytic pathway have clearly pro vided fundamental information about the molecular mechanisms and protein machinery that underlie the membrane trafficking of neurotransmitter receptors and the role this receptor traf ficking plays in arious aspects of synaptic plasticity .

6.4 CONCLUSION The importance of neurotransmitter receptor traf ficking as a ey mechanism for regulating the number of surface and synaptic receptors and hence synaptic strength and plasticity is no w clear [1,93]. Cell biological and biochemical approaches, in particular receptor biotinylation and antibody labeling methods, ha ve played a critical role in establishing the dynamic nature of neurotransmitter receptor traf ficking These approaches will continue to be important as researchers further elucidate the molecular machinery underlying receptor internalization, post-endocytic sorting and, in particular, the signalling mechanisms that converge on this machinery to regulate receptor trafficking. Similarl , these approaches will also be instrumental in further addressing ho w altered neurotransmitter receptor traf ficking could contri ute to alterations in synapse strength and neuronal viability during diseases processes [79,94]. In the future, biotinylation and antibody labeling techniques will be increasingly combined with loss of function approaches aimed at altering the molecular constituents of neurons, such as neuronal transfection with constructs encoding dominant negatives or shRNAis. Similalrly, the combination of antibody feeding or biotinylation with transduction of neurons with membrane permeant function blocking peptides and proteins will also be a powerful strategy. Increasingly, biotinylation will be used to label receptors in or ganotypic and acute brain tissue slices, which has several advantages over dissociated cultured neurons such as maintaining more physiological neuronal connectivity. Finally, novel methods for the labeling of neurotransmitter receptors with biotin or no vel fluorescent probes are eme ging. Of particular interest is the recently described site-specific labeling of cell sur ace proteins, including neurotransmitter receptors, with biotin lig ase [95–97]. In addition, the use of an increasing number of genetically encodable tags for the binding of fluorescent probes such as labeled ungarotoxin, the biarsenical dyes ReAsHEDT2 and FlAsH-EDT 2 or semiconductor Quantum Dots will further f acilitate the microscopic visualization of neurotransmitter receptor traf ficking vents along the endocytic and rec ycling pathways [98–100].

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Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors 117 75. Mammen, A.L., Huganir, R.L. and O’Brien, R.J., Redistrib ution and stabilization of cell surf ace glutamate receptors during synapse formation, J. Neurosci., 17(19), 7351–7358, 1997. 76. Fairfax, B.P., Pitcher, J.A., Scott, M.G., Calver, A.R., Pangalos, M.N., Moss, S.J. and Couve, A., Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability, J. Biol. Chem., 279(13), 12565–12573, 2004. 77. Kittler, J.T., Thomas, P., Tretter, V., Bogdano v, Y.D., Hauck e, V., Smart, T.G. and Moss, S.J., Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating gamma-aminobutyric acid type A receptor membrane trafficking, Proc. Natl. Acad. Sci. USA , 101(34), 12736–12741, 2004. 78. Osten, P., Khatri, L., Perez, J.L., Kohr, G., Giese, G., Daly, C., Schulz, T.W., Wensky, A., Lee, L.M. and Zif f, E.B., Mutagenesis re veals a role for ABP/GRIP binding to GluR2 in synaptic surf ace accumulation of the AMPA receptor , Neuron, 27(2), 313–325, 2000. 79. Snyder, E.M., Nong, Y., Almeida, C.G., Paul, S., Moran, T., Choi, E.Y., Nairn, A.C., Salter, M.W., Lombroso, P.J., Gouras, G.K. and Greengard, P., Regulation of NMDA receptor trafficking by amyloid-beta, Nat. Neurosci., 8(8), 1051–1058, 2005. 80. Thomas-Crusells, J., Vieira, A., Saarma, M. and Ri vera, C., A no vel method for monitoring surface membrane trafficking on hippocampal acute slice preparation, J. Neurosci. Meth., 125(1-2), 159–166, 2003. 81. Rivera, C., Voipio, J., Thomas-Crusells, J., Li, H., Emri, Z., Sipila, S., P ayne, J.A., Minichiello, L., Saarma, M. and Kaila, K., Mechanism of acti vity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2, J. Neurosci., 24(19), 4683–4691, 2004. 82. Morishita, W., Marie, H. and Malenka, R.C., Distinct triggering and e xpression mechanisms underlie LTD of AMPA and NMDA synaptic responses, Nat. Neurosci., 8(8), 1043–1050, 2005. 83. Luscher, C., Xia, H., Beattie, E.C., Carroll, R.C., v on Zastrow, M., Malenka, R.C. and Nicoll, R.A., Role of AMPA receptor c ycling in synaptic transmission and plasticity, Neuron, 24(3), 649–658, 1999. 84. Oh, P., McIntosh, D.P. and Schnitzer, J.E., Dynamin at the neck of caveolae mediates their b udding to form transport v esicles by GTP-dri ven fission from the plasm membrane of endothelium, J. Cell Biol., 141(1), 101–114, 1998. 85. Henley, J.R., Krue ger, E.W., Oswald, B.J. and McNi ven, M.A., Dynamin-mediated internalization of ca veolae, J. Cell Biol., 141(1), 85–99, 1998. 86. Pelkmans, L., Puntener, D. and Helenius, A., Local actin polymerization and dynamin recruitment in SV40-induced internalization of ca veolae, Science, 296(5567), 535–539, 2002. 87. Pietiainen, V., Marjomaki, V., Upla, P., Pelkmans, L., Helenius, A. and Hyypia, T., Echovirus 1 endoc ytosis into ca veosomes requires lipid rafts, dynamin II, and signaling events, Mol. Biol. Cell , 15(11), 4911–4925, 2004. 88. Dessy, C., K elly, R.A., Ballig and, J.L. and Feron, O., Dynamin mediates ca veolar sequestration of muscarinic choliner gic receptors and alteration in NO signaling, EMBO J., 19(16), 4272–4280, 2000. 89. Brown, T.C., Tran, I.C., Backos, D.S. and Esteban, J.A., NMD A receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD, Neuron, 45(1), 81–94, 2005. 90. Trischler, M., Stoorvogel, W. and Ullrich, O., Biochemical analysis of distinct Rab5and Rab11-positi ve endosomes along the transferrin pathw ay, J. Cell Sci ., 112(Pt. 24), 4773–4783, 1999.

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91. Lin, S.X., Grant, B., Hirsh, D. and Maxfield, .R., Rme-1 re gulates the distrib ution and function of the endoc ytic recycling compartment in mammalian cells, Nat. Cell Biol., 3(6), 567–572, 2001. 92. Grant, B., Zhang, Y., Paupard, M.C., Lin, S.X., Hall, D.H. and Hirsh, D., Evidence that RME-1, a conserv ed C. ele gans EH-domain protein, functions in endoc ytic recycling, Nat. Cell Biol. , 3(6), 573–579, 2001. 93. Kittler, J.T. and Moss, S.J., Neurotransmitter receptor traf ficking and the r gulation of synaptic strength, Traffi , 2(7), 437–448, 2001. 94. Goodkin, H.P., Yeh, J.L. and Kapur , J., Status epilepticus increases the intracellular accumulation of GABAA receptors, J. Neurosci., 25(23), 5511–5520, 2005. 95. Howarth, M., Takao, K., Hayashi,Y. and Ting, A.Y., Targeting quantum dots to surface proteins in li ving cells with biotin lig ase, Proc. Natl. Acad. Sci. USA , 102(21), 7583–7588, 2005. 96. Chen, I., Ho warth, M., Lin, W. and Ting, A.Y., Site-specific labeling of cell sur ace proteins with biophysical probes using biotin ligase, Nat. Meth., 2(2), 99–104, 2005. 97. Chen, I. and Ting, A.Y., Site-specific labeling of proteins with small molecules i live cells, Curr. Opin. Biotec h., 16(1), 35–40, 2005. 98. Dahan, M., Levi, S., Luccardini, C., Rostaing, P., Riveau, B. and Triller, A., Diffusion dynamics of glycine receptors re vealed by single-quantum dot tracking, Science, 302(5644), 442–445, 2003. 99. Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T.J., Adams, S.R., Garner, C.C., Tsien, R.Y., Ellisman, M.H. and Malenka, R.C., Activity-dependent re gulation of dendritic synthesis and trafficking ofAMPA receptors, Nat. Neurosci., 7(3), 244–253, 2004. 100. Sekine-Aizawa, Y. and Huganir, R.L., Imaging of receptor trafficking by using alpha bungarotoxin-binding-site-tagged receptors, Proc. Natl. Acad. Sci. USA , 101(49), 17114–17119, 2004. 101. Blanpied, T.A., Scott, D.B. and Ehlers, M.D., Dynamics and re gulation of clathrin coats at specialized endocytic zones of dendrites and spines, Neuron, 36(3), 435–449, 2002. 102. Washbourne, P., Liu, X.B., Jones, E.G. and McAllister , A.K., Cycling of NMD A receptors during trafficking in neurons before synapse formation,J. Neurosci., 24(38), 8253–8264, 2004. 103. Lee, S.H., Simonetta, A. and Sheng, M., Sub unit rules go verning the sorting of internalized AMPA receptors in hippocampal neurons, Neuron, 43(2), 221–236, 2004.

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7

Visualization of AMPAR Trafficking and Surface Expression Pavel V. Perestenko and Jeremy M. Henley

CONTENTS 7.1

Introduction ..................................................................................................120 7.1.1 AMPARs ..........................................................................................120 7.1.2 Transport and Targeting of AMPARs within Neurons ....................121 7.1.3 Dynamic Regulation of Functional Synaptic AMPARs..................122 7.2 Fluorescent Protein Tools ............................................................................122 7.2.1 Green Fluorescent Proteins ..............................................................122 7.2.2 Construction and Use of Fluorescently Labeled Proteins ...............123 7.2.3 Imaging Hardware............................................................................124 7.2.4 Viral Transduction............................................................................125 7.2.5 General Applications of XFPs in Confocal Microscop y ................125 7.3 Application of XFP Imaging to AMPARs ..................................................126 7.3.1 Visualizing Intracellular Mo vement of GFP-GluR1 and GluR2 ...............................................................................................126 7.3.2 pH-Sensitive GFP (pHluorin) to Monitor AMPAR Surface Expression and Diffusion.................................................................130 7.4 Future Imaging Possibilities ........................................................................132 7.4.1 pH Sensors .......................................................................................132 7.4.2 Photoactivatable GFP .......................................................................133 7.4.3 Bimolecular Fluorescence Complementation Analysis (BiFC) ...............................................................................133 7.4.4 Fluorescent Timer ............................................................................133 7.4.5 Fluorescence Resonance Ener gy Transfer (FRET) .........................134 7.4.6 Fluorescence Correlation Spectroscop y (FCS)................................134 7.5 Conclusion....................................................................................................134 7.6 Acknowledgments ........................................................................................134 References..............................................................................................................135

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7.1 INTRODUCTION The cell biology of AMPAR synthesis, transport, tar geting and surf ace expression is of crucial importance for correct neuronal function. These processes rely on spatially and temporally coordinated protein-protein interactions to f acilitate and regulate receptor traf ficking. While considerable adv ances ha ve been achie ved in unravelling the comple xity and roles of some of these interactions, the dynamic aspects of AMPAR trafficking are less well-characterized. We have been w orking to visualize AMPAR movement in li ving hippocampal neurons to define the prop erties of AMPAR trafficking under basal and act vated conditions. Here, we re view current techniques and the progress achie ved.

7.1.1 AMPARS For nearly 50 years [1,2], glutamate has been kno wn as one of the most important excitatory neurotransmitters in the mammalian central nerv ous system (CNS). Glutamate receptors (GluRs) can be divided into two functionally distinct categories: metabotropic and ionotropic glutamate receptors. Metabotropic receptors are coupled to G-protein second messenger systems and, upon acti vation, can act to modulate synaptic plasticity. Ionotropic glutamate receptors are ligand-gated ion channels that mediate a lar ge proportion of the f ast e xcitatory transmission at synapses within the CNS. The ionotropic glutamate receptors can be classified into thre distinct subgroups based upon their pharmacology and molecular characteristics namely: α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMP AR), N-methyl-D-aspartate receptors (NMDAR) and kainate (KA) receptors. AMPARs have been an object of intensive investigation for more than a decade. In addition to f ast e xcitatory neurotransmission, the y play fundamental roles in synapse stabilisation and plasticity [3]. Four different AMPAR subunits exist, named GluR1 to GluR4 (or GluRA to GluRD). The GluR1 sub unit was first cloned afte screening an e xpression library [4,5] and subsequently , standard cDN A homology screens revealed the remaining GluR2, GluR3 and GluR4 homologues [5–8]. Each subunit has a molecular weight between 105 to 116 kD [9] and contains three membrane-spanning domains, one membrane-associated h ydrophobic domain, extracellular N-terminal and intracellular C-terminal domains ( Figure 7.1a) [10]. The mature AMPAR is a tetrameric complex [11–17]. Current opinion suggests that the proximal e xtracellular N-terminal domain mediates initial sub unit associations into dimers [18,19] b ut other re gions, such as the membrane re gion and S2 portion, are also necessary for formation of the mature tetrameric receptor [20]. The subunit genes could undergo alternative splicing in two distinct regions, resulting in flip or flop ariants in an e xtracellular domain and also sub units that ha ve either long or short intracellular C-termini [21]. GluR2 and GluR4 e xhibit both long and short forms; the short form is homologous to the C-terminus of GluR3, the long isoform homologous to that of GluR1. In vivo , over 90% of GluR2 is e xpressed as the short alternative splice variant [22]. Different isoforms of GluR4 are e xpressed preferentially in differing areas of the brain; for e xample, GluR4short in cerebellar

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NTD NH2 L1

L2

Glu

Signal peptide EGFP

NH2

Linker

M4

M3

M1

M2

Out

In

Posttranslational modiﬁcation

COOH

COOH

(a)

(b)

FIGURE 7.1 (a) General structure of AMPAR subunits. There are four hydrophobic domains (M1 through M4) of which three are transmembrane (M1, M3, M4) and M2 forms a channel pore-lining loop without spanning the membrane. L1 and L2 are ligand-binding domain lobes. The function of the N-terminal domain (NTD) appears to be connected to the subunit specifi assembly of AMPARs. (b) To construct N-terminal XFP-AMP AR chimeras, the fluorescen protein sequence w as inserted into the receptor cDN A after the signal peptide and further post-translational modification yielded mature N-terminal-labeled receptors

granule cells and Bergmann glial cells [23]. To date, GluR1 has only been found as the long-form splice v ariant in vivo and GluR3 as the short form.

7.1.2 TRANSPORT AND TARGETING OF AMPARS WITHIN NEURONS Neurons possess thousands of synapses, each containing multiple AMPARs with potentially differing subunit compositions. Interestingly, up to 50 to 70% ofAMPARs are not surf ace-expressed in neurons, with a significant proportion being localize within dendrites [24,25]. Routes by which receptors can be traf fic ed in neurons include long-distance dif fusion in the plasma membrane or transport of mRN As within the cell and local synthesis of proteins close to their destination [26]. Ho wever, intracellular vesicular transport to the vicinity of their target site (synapse), followed by excoytosis [27] and then lateral dif fusion in the lipid bilayer and anchoring at their target destination [28], is perhaps the most attracti ve model for most receptor trafficking. Vesicles are driven by the molecular motor proteins kinesin and dynein, which are responsible for mo vements to ward opposite ends of the microtub ules. Thus, polarized v esicle trafficking is li ely to require specific interactions betwee domain-specific motors and esicle cargo. Inhibition of dynein or kinesin by monoclonal antibodies substantially reduces synapticAMPAR- but not NMDAR-mediated responses [29]. Furthermore, AMPARs under go car go-selective polarized v esicle transport via dendrite-specific kinesin motors [30]. Nonetheless, la ge questions remain, for example, targeting of proteins to specific sites is li ely to require either address motifs in the cargo proteins themselves or some kind of a recognition domain allowing passing v esicles to be captured and retained at rele vant sites. The nature and identities of these motifs and the molecular interactions in volved have not yet been identified

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7.1.3 DYNAMIC REGULATION OF FUNCTIONAL SYNAPTIC AMPARS As for AMPAR transport, the re gulation of AMPAR surface expression is undoubtedly a complex process that requires multiple protein interactions. Se veral proteins have been identified that interact selectvely with intracellular C-termini of individual AMPAR subunits and the best, b ut still incompletely, characterized protein interactors include the PDZ domain-containing proteins GRIP and PICK1 for GluR2 subunit and SAP97 for the GluR1 subunit. These interactors appear to be particularly important for acti vity-dependent changes in AMPAR synaptic e xpression [31–37]. These observ ations ha ve stimulated attempts to in vestigate the routes by which AMPA receptors are traffic ed. A useful approach to study any sort of translocation of receptor molecules is to visualize the dynamics of their mo vement. This can be achieved by tagging the receptor molecules with a fluorescent label, a fluoresce protein that is translated in frame with the receptor sub unit and expressed in situ by gene transfer into cells.

7.2 FLUORESCENT PROTEIN TOOLS 7.2.1 GREEN FLUORESCENT PROTEINS Green fluorescent protein (GFP) as originally identified in the bioluminescen jellyfish Aequorea victoria , which produces light when ener gy is transferred from the Ca 2+-activated photoprotein aequorin to GFP [38–40]. The wild-type GFP (wtGFP) gene w as cloned 30 years later [41,42] and w as established as a no vel genetic reporter tool after its e xpression in heterologous systems [42–46]. The total length of the amino-acid polypeptide forming GFP is 238, of which 222 aminoacids are necessary for the matured protein fluorescence despite only a short tripep tide formed upon cyclization of the residues 65-S-dehydroY-G-67, for wtGFP, acting as the chromophore [47–49]. Thus, most of the GFP polypeptide pro vides a milleu for the chromophore to fluoresce by formation of a ery stable protecti ve “barrel” (Figure 7.2a) [50–52]. Development of the protect ive complex and oxidation of the chromphore is a multistep protein maturation process that tak es time and requires oxygen. The wtGFP maximum peaks of absorbance lie in areas of UV and blue light (main, 395 nm; minor , 470 nm), whereas the emission peak has a green wavelength of 509 nm [40]. Because of its potential as a mark er in li ving cells, GFP has attracted close attention that has resulted in modifications to the original molecule [45,53]. S veral silent mutations were introduced to create an open reading frame encoding almost entirely preferred human codons. Other mutations ha ve been introduced to signifi cantly increase expression of GFP in mammalian cells. Perhaps the most important mutations, however, have been those that ha ve altered the fluorescent properties o GFP. Among these are substitutions in the amino-acid sequence resulting in increased brightness and impro ved chromophore formation (F64L and S65T — EGFP) [54–56]. In addition, mutations that shift the e xcitation and emission peaks ha ve significantly xpanded possibilities for simultaneous multi-fluorophore labeling Currently excitation/emission maxima of most used GFP v ariants span from EBFP

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2

3

11

10

7

8

9

4

5

123

6

0.8 0.6 0.4 0.2 0.0 350

HcRed1

0.8 0.6 0.4 0.2 0.0 450

450 550 Wavelength (nm)

DsRed2

1.0

EGFP EYFP

ECFP

HcRed1

DsRed2

Emission

1.0

Normalized emission intensity

Normalized excitation intensity

Excitation

EGFP EYFP

ECFP

(a)

550

650

Wavelength (nm)

(b)

FIGURE 7.2 (Color figure fol ow page 176 .) (a) Topology of the GFP folding pattern [51]. α-helices and connecting loops distrib ute 11 anti-parallel β-sheet strands (arro ws) and form the sides of the barrel that surrounds the GFP chromophore. (b) Excitation and emission spectra of some fluorescent proteins: Aequorea victoria GFP variants, dsRed and hcRed.

(380 to 440 nm) through ECFP (433 to 475 nm), EGFP (489 to 509 nm) to EYFP (513 to 527 nm) (Figure 7.2b) [55,57]. This broad range of colors has been extended further (558 to 583 nm) with the disco very of no vel “GFP-lik e” proteins from nonbiolumuniscent representatives of the class Anthozoa (dsRed) [58,59], increasing the f amily size to 30 dif ferent members. Most of the widely used forms of these fluorescent proteins are n w commercially available from BD Biosciences/Clontech.

7.2.2 CONSTRUCTION PROTEINS

AND

USE

OF

FLUORESCENTLY LABELED

GFP and its deri vatives (collecti vely kno wn as XFPs) are no w routinely used to monitor multiple biological processes. F or e xample, the y are commonly used as markers for gene transfer and also as a visual label fused to a protein of interest. In the first case, fluorescent protein (usually GFP) is translated independently of t recombinant protein of interest and the GFP acts only to identify which cells are transduced. The expressed XFP freely diffuses and fills the entire cell, including th nucleus, making it a convenient marker. It is also possible to monitor cell morphology using free XFP. The most reliable method for independent e xpression of free XFP together with a protein of interest is to use a v ector that contains the recombinant

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protein sequence, follo wed by internal ribosome entry site (IRES) and then by fluorescent protein cD A [60,61]. This bicistronic arrangement allo ws the tw o proteins to be synthesised separately from a single construct with initial translation of the unlabeled protein of interest follo wed by subsequent e xpression of the XFP (which is comparati vely small and has a high translation ef ficien y). While it is optimal, that sequences for XFP and the protein of interest are encoded on the same vector is not a requirement. Cells can be co-transfected with separate plasmids. F or example, this approach has been used to study the ef fect of AMPAR subunit GluR2 overexpression on cultured neuron spine size and density [62]. Ho wever, because cells often do not tak e up multiple viruses, when using viral deli very systems (cell transduction), incorporating the cDN As for the recombinant proteins into a bicistronic vector is often adv antageous. The direct tagging of recombinant proteins by fusion of the cDN As encoding XFP and the protein of interest is now commonplace. Nonetheless, special attention must be paid to the polylinker between the XFP and the host protein. That the linker chain is short b ut fl xible enough to allo w proper folding and functioning of both molecules is preferable. A general practice in construction of fusion proteins is to utilize glycine, the smallest and most fl xible amino acid with serine inserts to improve solubility of poly-Gly chain. In recent years, such GFP-based fluorescen probes, including labeled glutamate receptors, ha ve been successfully applied to neuroscience to visualize synaptic structures and e vents in li ving neurons [63–70]. Initially, XFP fused at the C-terminal GluR sub units w as often found to result in chimeras that lose their ability to interact with other proteins. To circumv ent this problem, most researchers w orking with glutamate receptors no w use XFP-N-ter minal chimeras constructed by inserting the XFP sequence in GluR cDN A immediately after the signal peptide sequence ( Figure 7.1b). This process is technically more involved and usually requires site-directed mutagenesis by o verlap extension polymerase chain reaction (PCR) techniques to introduce necessary restriction sites inside cDNA of GluRs.

7.2.3 IMAGING HARDWARE In parallel with the rapid progress achie ved in fluorophore design, correspondin advances have also been seen in optical detection methods. F oremost among these is the continued e xpansion and refinement of confocal microsco y techniques that have opened up ne w possibilities for GFP imaging. F or e xample, spinning disc microscopes are a vailable that allo w high-speed acquisition of images while minimizing photobleaching of the fluorophore. These machines are ideally suited for tracking movement of GFP-tagged proteins or organelles in cells. Multicolor imaging systems are also widely available that permit simultaneous detection of two or more proteins tagged with dif ferent spectral v ariants of GFP . Total internal reflectio fluorescence (TIRF) microsco y is a new development that is ideally suited to study membrane fusion e vents as it specifically monitors nea -membrane fluorescence Multiphoton (MP) imaging allows high-resolution imaging of small structures such as synapses.

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7.2.4 VIRAL TRANSDUCTION In our laboratory, we have used two different kinds of virus, namely adenovirus and Sindbis. Each system has its adv antages. For example, adenovirus infection allows long-term expression of the protein of interest with minimal c ytotoxicity or immunoresponse making it useful for in vivo experiments. We also ha ve tetrac yclineinducible versions of the virus to allo w the levels and timing of protein production to be controlled. Ho wever, adenovirus is not neuronally selecti ve and is relati vely difficult to ma e. Sindbis, on the other hand, is relati vely straightforward to mak e and usually preferentially transduces neurons o ver glia. Sindbis has been used by several groups to transduce neurons [71]. It is usually cytotoxic in prolonged (3 days after infection) e xperiments; however, a v ersion of Sindbis with a mutation in the nsP2 protease domain has been reported that retains the neurospecificity ut is much less toxic, allowing longer-term expression observations to be undertak en [72]. We have optimized conditions for ef ficient infection of dispersed hippocampa cell cultures and hippocampal slice cultures with bicistronic v ersions of both adenovirus and Sindbis viral v ectors. As discussed abo ve, the bicistronic viruses mak e two separate proteins and we generally e xpress GFP and a peptide or protein of interest. The expression of GFP allo ws us to visually identify which cells e xpress the recombinant polypeptide. If required for biochemical e xperiments, essentially all neurons in a culture or in a slice can be infected [73]. Ho wever, we routinely use relatively low viral titers that achieve sparse neuronal transduction that result in infected neurons adjacent to control, noninfected neurons for side-by-side compar ison. Using these systems, we ha ve inserted se veral fluorophore-tagged GluR sub units, interacting proteins, defined mutants and blocking peptides into one or bot types of virus and the y all display ef ficient neuronal xpression.

7.2.5 GENERAL APPLICATIONS MICROSCOPY

OF

XFPS

IN

CONFOCAL

Direct observation of fluorescence distri ution is the first o vious use of XFP-labeled chimeras. Ho wever, to define the dynamics of receptor m vement, using photobleaching techniques is usually necessary [74–76]. These approaches e xploit the fact that illumination at high intensity photochemically modifies GFP fluoropho to render it irre versibly nonfluorescent without damaging of the cell [55,77,78] Light at 488 nm from an argon ion laser disrupts microtubules at the intensity range of 0.5 to 150 MW/m 2 [79]. Estimation of the ener gy to which the object is e xposed is also important for the pre vention of damage. The total energy of exposure within the range 0.05 to 0.09 MJ/m 2 (10% of the e xposure that can cause breakage of microtubules) is well within the limits of tolerance. Furthermore, by decreasing the time of the exposure, safely using a laser beam at peak intensity of up to 2.7 MW/m2 without cell damage is possible [80]. Nonetheless, calculating the intensity of the light and its duration for each particular imaging system is always necessary. Several photobleaching techniques are widely used in XFP-labeled protein traf ficking an interactions, of which FRAP (fluorescence rec very after photobleaching) and FLIP (fluorescence loss in photobleaching) are among the most used

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FRAP is based on the measurement of fluorescence rec very in a photobleached area. After fluorescent molecules are irr versibly photobleached, a dif fusion of nonbleached molecules into the area occurs that can be monitored under nonbleaching (low-energy laser) conditions. This approach allows measurement of the mobility of XFP-tagged proteins in particular compartments of li ving cells [81–85]. FLIP measures fluorescence loss outside of a repeatedly bleached area due to di fusion of bleached XFP molecules. This technique can be used for estimation of XFP exchange between different cell compartments as well as for the removal of excessive fluorescence from areas to r veal structures hidden by e xcessive fluorescence o overexpressed XFP proteins. When using FRAP and FLIP in neurons, specificity o the cell shape must be considered. F or e xample, normalizing the initial le vels of fluorescence in the dendritic shaft is important as their diameter might not be constan along the length of monitored segment. This could lead to confusion, as fluorescenc in thicker segments will appear to reco ver faster and be lost slo wer, but this does not necessarily correspond to the actual molecule’ s migration. Thus, in dendritic shafts, the initial le vels of fluorescence must be analyzed using a coe ficient [63 that relates the fluorescence l vel to the shaft diameter at the point of measurement (F1 = kF2, where k = D12/D22; F1 and F2 are fluorescence l vels and D1 and D2 are the diameters of the dendritic shaft at the measurement point) [66]. To exclude lateral diffusion of bleached molecules, dendritic shafts were bleached through the full depth of the structure.

7.3 APPLICATION OF XFP IMAGING TO AMPARS 7.3.1 VISUALIZING INTRACELLULAR MOVEMENT AND GLUR2

OF

GFP-GLUR1

As discussed briefly ab ve, all aspects of AMPAR synthesis, post-translational modifications, folding, assembly and tra ficking to d gradation are tightly regulated. Immunocytochemical analysis of fi ed, permeabilized neurons ha ve re vealed that unlike NMDARs, AMPARs are dif fusely distributed in dendrites at early stages of neuronal development [86,87]. Furthermore, GluR2/3 containing vesicles (50 to 300 nm in diameter) co-fractionate with some kno wn AMPAR binding proteins such as GRIP and PICK1 b ut do not contain NMD A receptors [88]. The unique morphological structure of neurons requires the prompt and timely delivery of newly synthesized proteins to remote parts of the dendritic tree. Despite the f act that acti vity-dependent local synthesis of AMPARs in dendrites has been shown [89], it is not likely to be sufficiently rapid to participate in short-term plasti changes. Therefore, the rapid supply of receptors synthesized in the cell body (and possibly in other parts of the dendritic tree) to the vicinity of the synapse undergoing change lik ely requires tar geted intracellular traf ficking of AMPARs by using the microtubular system and actin filaments [29,90]. In our laborator , we have focused on two basic questions about AMPAR trafficking: H w are AMPARs directed to the correct locations in the neurone at the right time? and What are the temporal and spatial characteristics of AMPAR surface expression?

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We have directly visualized the intracellular transport of AMPARs using Sindbis virus to transduce GFP-GluR1 into dispersed cultures of living hippocampal neurons [66,91]. In young neurons, we observed that the distribution and behavior of EGFPGluR1 and EYFP-GluR2 were dif ferent, with EGFP-GluR1 mainly distrib uted in an even, diffuse manner. In contrast, EYFP-GluR2 e xhibited more granulated fluo rescence and seems to be less mobile. In older neurons (21DIV) EYFP-GluR2 fluorescence formed distinct puncta in perimembrane areas while the center of th dendritic shaft remained relati vely diffuse. These data are broadly consistent with previous immunoc ytochemical data for nati ve GluR1 from our cell cultures [25] and, importantly , GFP-GluR1-containing receptors are surf ace-expressed with a synaptic distribution indistinguishable from nati ve AMPARs. To determine the rates of EGFP-GluR1 and EYFP-GluR2 traf ficking in th dendrites, we performed a series of e xperiments in which a dendrite w as subjected to repeated bleaching at a defined point and FLIP as monitored in a location distal to the bleached s egment (Figure 7.3b) [66,91]. The delay in fluorescence loss is function of a distance between the tw o points and allows the speed of movement to be calculated. The measured rate of EGFP-GluR1 translocation v aried from 0.03 to 0.53 µm/sec and up to 2.2 µm/sec, b ut the v ast majority of the bleached sub units traffic ed with speeds in the range of 0.03 to 0.2 µm/sec. In FRAP e xperiments (Figure 7.3a), the initial rate of fluorescence rec very was rapid (70% reco very in 1 to 2 min for EGFP-GluR1 b ut less than 10% for EYFP-GluR2). Subsequent recovery to the prebleached fluorescence took considerably longer for both recom binant receptors and can be e xplained by the e xistence of their slo w e xchange clusters, especially the in case of EYFP-GluR2, where most of the receptors are relatively immobile. Nonetheless, a small proportion of YFP-GluR2 was traffic ed in a similar manner to EGFP-GluR1, showing a diffuse fluorescence pattern and ast FRAP. Li ve neurons labeled with anti-GFP antibodies re vealed about 40% colocalization of intracellular YFP-GluR2 clusters with membrane areas where the receptors were surface expressed. This result is consistent with significant amount of GluR2 sub units accumulated at intracellular sites in the proximity of synaptic sites. Gi ven the wealth of e vidence no w a vailable, the y are lik ely retained in the clusters by the PDZ-interacting protein GRIP , which does not interact with GluR1 [34,35,37,92,93]. For both EGFP-GluR1 and EYFP-GluR2 fluorescence, rec very w as bidirectional with a predominantly proximal to distal direction. The speed of translocation of the f ast population of AMPARs is in line with the rates for other proteins and vesicles in cultured neurons [94–98]. Ov er the time course of our e xperiments (20 to 30 min), AMPAR transport in dendrites was not altered by activation or inhibition of ionotropic glutamate receptors or by the addition of glycine, a protocol kno wn to induce LTP in dispersed cultures [99]. Importantly , even complete inhibition of synaptic transmission did not alter FRAP in dendritic spines or shafts.Taken together, these data indicate that acute changes in the number of surf ace-expressed AMPARs do not immediately af fect receptor deli very and that constituti ve transport or local stores of AMPARs can accommodate f ast ph ysiological changes in the le vels of surface e xpression. We propose that the intracellular transport of AMPARs is not activity regulated but rather v arious subunit combinations of the receptor comple x

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X

Y

Fluorescence Intensity

X

Y

X

100 50

Y

Time (a) X

Fluorescence Intensity

X

Y

100 50

Time (b)

Y

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FIGURE 7.3 (Color figure fol ow page 176 .) (a) An example of FRAP of EGFP-GluR1 in dendritic spines of cultured hippocampal neurons. The area of dendrite between points X (proximal) and Y (distal) w as photobleached and subsequent fluorescence rec very w as measured at 5-sec interv als. The chart represents typical result for quantification of th fluorescent intensity measurements. (b) An e xample of FLIP of EGFP-GluR1 in dendritic spines of cultured neurons. Fluorescence at point Y was measured after repeated photobleaching at point X. The chart sho ws typical changes in the fluorescence intensity at the point o measurement (Y). These measurements allo w estimation of the speed of traf ficking of th bleached molecules along the dendritic shaft [66].

are lik ely to be translocated widely throughout the soma and dendrites. Synapses may then “capture” these passing receptors as and when required. In hippocampal slice cultures, tetanic synaptic stimulation has been reported to cause the rapid NMDAR-dependent delivery of GFP-GluR1 into dendritic spines in hippocampal slice preparations [100]. GFP-labeled subunits have been visualized in combination with electroph ysiological tagging, where the channel rectificatio properties of recombinant AMPARs comprising specific su units are altered by point mutations, e.g., GluR2(R586Q)-GFP . A dif fuse distrib ution w as observ ed throughout the dendritic tree b ut with no comparable le vels of fluorescence i dendritic spines. However, LTP induction with high-frequenc y synaptic stimulation induced the translocation of GFP-GluR1 to spines and dro ve recombinant receptors to the surf ace of dendritic shafts. This redistribution of GFP-GluR1 w as similar to that caused by the activation of NMDA receptors [69]. Immunocytochemical studies in dissociated cultured neurons demonstrated abundant GluR1 expression at synapses [101] and in live, non-stimulated, cultured neurons, EGFP-GluR1 fluorescence as detected in both dendritic shafts and spines at the similar le vel [66,91]. Using these approaches, dif ferential tar geting mechanisms for AMPARs ha ve been proposed comprising either GluR1/GluR2 or GluR2/GluR3 sub unit assemblies. More specifically, the hypothesis is that GluR1/GluR2 receptors are added to synapses predominantly during plasticity via a process that requires interactions between GluR1 and group I PDZ domain proteins [102]. In this model, CaMKII and L TP drive synaptic expression of GluR1-containing AMPARs. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors in a constitutive manner dependent on interactions by GluR2 with NSF and group II PDZ domain proteins ( Figure 7.4) [100]. We used simultaneous bleaching of spines and adjoined areas of the dendritic shaft to visualize real-time transport of recombinant protein into spines in dispersed hippocampal cultures under non-stimulated conditions. In our hands, both the shaft and spines reco vered fluorescence at similar rates, suggesting free access of th recombinant sub units to the dendritic spines. This condition w as not the case for EYFP-GluR2, where FRAP in spines took a much longer time in comparison to FRAP in adjoined areas of the dendritic shaft. Since EYFP-GluR2 shows a clustered and perimembrane distribution in dendritic shafts, we attribute this difference to the fact that GluR2 receptors are relati vely constrained at synapses, possibly as a result of localized recycling between the spine lumen and the membrane [103]. In addition, a freely mobile population of EYFP-GluR2 receptors e xist that are constituti vely delivered into the spines.

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I LTP

LTD II - LTP

III Activity dependent (LTP and LTD)

Constitutive and LTD

GluR1 GluR2L GluR4

GluR2 GluR3 GluR4c

Non-Silent Synapse

Silent Synapse GluR1 GluR2 GluR3

(a)

(b)

FIGURE 7.4 Synaptic AMPARs recycling in LTP and LTD. (a) Deli very to the synapse of AMPARs containing subunits with long C-terminal domains (GluR1, GluR2L and GluR4) is proposed to require L TP whereas receptors containing sub units GluR2, GluR3 and GluR4c (short C-terminal domain) rec ycle constituti vely. (b) L TP-induced dynamic changes of AMPAR expression in non-silent and silent synapses [63]. Under basal conditions, equilibrium exists between surf ace-expressed and intracellular receptors (I). F ollowing LTP induction, new GluR1-containing receptors are inserted into both non-silent and silent synapses (II) and, later, a ne w equilibrium is established with the possibility of e xchanging GluR1-containing with GluR3-containing receptors (III). See te xt for details.

7.3.2

PH-SENSITIVE

SURFACE

GFP (PHLUORIN) TO MONITOR AMPAR EXPRESSION AND DIFFUSION

Despite providing insight into AMPAR transport inside neurons, GFP-tagged receptors are limited as a tool to study real-time changes in surf ace e xpression of the receptors. The amount of intracellular GFP-AMP AR e xceeds the number of AMPARs expressed at the surf ace and the signals cannot be dif ferentiated in realtime. Innovative single-particle and single-molecule tracking approaches ha ve been used to show that AMPARs lose their high mobility in the e xtrasynaptic membrane after entering a synaptic region [28,104,105] but receptors can only be followed for a relatively brief time. Thrombin cleavage assay and antibody labeling has been used to estimate the rate of e xocytosis and location of surf ace-expressed AMPARs subunits [27]. These workers tracked AMPARs in cultured neurons and suggested the direct insertion of GluR2 receptors into the synaptic membrane b ut that GluR2 subunits are accumulated initially at e xtrasynaptic sites. Ho wever, a significan drawback of the technique is that it has comparati vely slow temporal resolution. An alternative strategy is to use a fluorescent tag that changes its fluorescen if exposed outside the cell membrane. Such a label became a vailable from studies investigating random mutations of wild-type GFP , which led to the disco very of pHluorins. The fluorescence intensity of these proteins can be manipulated b

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Ι

I

ΙΙ

ΙΙΙ

0.1 NMDA

II

∆F/F0

0 0.1 Punctate Diﬀuse

0.2 −50 II

Intracellular

(a)

250 550 Time(s)

(b)

FIGURE 7.5 (Color figure fol ows page 176 .) (a) Ecliptic pHluorin protein is fused to the N-terminal domain of anAMPAR subunit and is positioned inside the acidic lumen of transport vesicles in the secretory pathway. Fluorescence is quenched until e xocytosis and exposure of the pHluorin to the neutral e xtracellular environment, where a rapid increase in fluorescenc occurs. This fluorescence is lost when pHluorin-tagged receptors are internalized into aci endocytotic vesicles. (b) NMD A-induced endocytosis of AMPARs in cultured hippocampal neurons [67]. I: Fluorescence under basal conditions from synaptic (red) and e xtrasynaptic (blue) pHluorin-GluR2; II: Rapid endocytosis of extrasynaptic pHluorin-GluR2 after NMDA receptors activation; III: Subsequent slo w removal of synaptic pHluorin-GluR2, presumably by lateral dif fusion out of the synaptic area. See te xt for details.

changes of pH. In particular, ecliptic pHluorin (EP) is almost in visible at pH values less than 6.0 b ut the fluorescence intensity increases with pH up to a maximum o 8.5 [106,107]. Thus, a chimera of pHluorin fused to the N-terminus of an AMPAR sub unit (pHluorin-GluR2) acts as a marker for a surface-expressed receptor. This characteristic is due to the transfer of the N-terminal domain of GluR2 containing the pHluorin to a neutral extracellular pH from the relatively acidic internal vesicles compartments (Figure 7.5a) [107]. An endocytosed plasma membrane also forms v esicles that are rapidly acidified [108], so fluorescence from internalized pHluorin-GluR2 is al quenched. We used adenovirus to express pHluorin-GluR2 in cultured hippocampal neurons to distinguish the spatio-temporal properties of surf ace expressed synaptic, surface expressed extrasynaptic and intracellular receptors, in near real-time [67]. The pHluorin-GluR2 forms functional receptors, is targeted to the plasma membrane and concentrates in dendritic spines opposed to pre-synaptic terminals. As e xpected, inside neurons pHluorin-GluR2 e xhibited relatively low fluores cence, whereas surf ace-expressed subunits resulted in bright areas at the edges of dendritic shafts. This fluorescence as due to the surface-expressed pHluorin-GluR2 because it co-localized with anti-GFP antibody immunoreacti vity in li ving nonpermeabilized cells. Punctuate dendritic fluorescence as reversibly eliminated by a brief acidic w ash that does not af fect intracellular pH, as measured on cells fille with fluorescent pH indicator BCECF-AM ( ′,7′-bis-(Carboxyethyl)-5(6′)-carboxyfluorescein Acetoxymethyl Ester). Re versible ele vation of intracellular pH with

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NH4Cl w as used to estimate proportion of surf ace e xpressed pHluorin-GluR2. In neurons e xpressing pHluorin-GluR2, surf ace fluorescence as decreased by brief washing of the cells with acidic (pH 4.5) buffer. Subsequent change to 5 0mM NH4Cl in neutral pH buffer, which caused an elevation of total fluorescence to its maximu due to restoring surface fluorescence back to initial l vels and simultaneously revealing intracellular pHluorin-GluR2. We determined that around 45% of pHluorinGluR2 is surf ace-expressed under the resting conditions in dendritic re gions. Surface-expressed pHluorin-GluR2 fluorescence sh wed bright punctate synaptic areas (confirmed by immunolabeling with the synaptic mar er synaptoph ysin and FM4-64) and lo wer diffuse e xtrasynaptic distrib utions. This made comparing activity-dependent internalization of synaptic and e xtrasynaptic pHluorin-GluR2 [67] initiated by application of NM DA to the cultures possible ( Figure 7.5b ) [109–111]. During, and for a fe w minutes following NMDAR stimulation, synaptic pHluorin-GluR2 fluorescence as stable b ut then gradually declined. In mark ed contrast, e xtrasynaptic pHluorin-GluR2 fluorescence rapidly decreased as soon a NMDA was applied and returned to basal le vels 2 to 3 min after NMD A removal. These results sho w that internalization occurs mainly outside the post-synaptic region. We interpret our data to suggest that synapticAMPARs are initially protected from rapid internalization, whereas AMPARs at e xtrasynaptic sites are rapidly endocytosed. This dif ferential re gulation of synaptic and e xtrasynaptic AMPARs suggests that synaptic receptors laterally dif fuse a way from the PSD to sites of endocytosis in the e xtrasynaptic membrane [67]. Most recently, we ha ve used an enhanced v ersion of pHluorin termed super ecliptic pHluorin (SEP) [112] to specifically visualize AMPARs on the surf ace of neurons. This pH-sensitive variant of GFP e xhibits much brighter fluorescence an an increased signal-to-noise ratio compared to pHluorin. When expressed in hippocampal neurons and visualized by laser scanning confocal microscop y, SEP-GluR1 and SEP-GluR2 fluorescence h ve distributions characteristic of the plasma membrane and are particularly enriched in the heads of dendritic spines. Our current experiments are focused on determining the parameters for surface diffusion of SEPGluRs.

7.4 FUTURE IMAGING POSSIBILITIES 7.4.1

PH

SENSORS

Perhaps the most rapid developments will occur with new generations of pH sensors. For example, new dual-emission, pH-sensitive GFP variants allow ratiometric analysis of pH using single excitation wavelength [113]. Multicolor imaging would also be extremely useful: pH sensors with different colors would allow the simultaneous characterization of e xocytosis/endocytosis of tw o or more membrane proteins. F or example, multimeric proteins such as AMPARs might undergo internalization, subunit rearrangement leading to the loss of particular sub units, reassembly and reinsertion at the synapse. This possibility could be directly tested using subunits each tagged with a dif ferent colored pH-sensitive fluorophore

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7.4.2 PHOTOACTIVATABLE GFP We have focused on tracking protein mo vement using photobleaching protocols. However, photoactivatable GFP (PA-GFP) variants could make these types of experiments more direct [84]. P A-GFP displays little fluorescence until act vated by a pulse of blue light [114]. The photoactivated proteins can then be follo wed as they move. This approach will potentially be more sensiti ve because the acti vated fluo rescence is monitored ag ainst a dark background.

7.4.3 BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS (BIFC) Several emergent approaches based on fluorescent protein technology promise sig nificant ad antages b ut ha ve ha ve not yet been applied to the study of AMPARs. For example, the ability of partial GFP polypeptides to form a fluorescent molecul has been used to in vestigate interactions among bZIP and Rel f amily transcription factors [115]. GFP has a β-barrel-like structure with a central α-helix surrounding the chromophore. The GFP was divided into two fragments within loops at the ends of β-barrel and the fragments fused to C-terminal ends of bZIP domains of c-F os and c-Jun that form the heterodimeric c-Fos-c-Jun transcription factor. This process, known as bimolecular fluorescence complementation analysis (BiFC) can be use for direct localization of protein interactions in living cells. The method was further developed with the use of BiFC analysis for complementation between fragments of GFP-f amily proteins with dif ferent spectral characteristics [116], pro viding the possibility to visualize multiple simultaneous protein interactions in cells. A modification to this approach has been reported where t o interacting proteins (calmodulin and M13) were genetically fused with N- and C-terminal halv es of split EGFP that were flan ed by a domain for protein self-splicing (intein) deri ved from Saccharomyces cerevisiae [117,118]. In the cell, self-catalyzed e xcision of intein links the EGFP halves back by a peptide bond. While some technical issues remain to be resolved, this approach appears to hold tremendous potential, for e xample, imaging dynamic interactions between AMPAR subunits and interacting proteins.

7.4.4 FLUORESCENT TIMER Changes in protein e xpression can be monitored using a destabilized GFP v ariant that is de graded more rapidly than standard GFPs, resulting in a lo w resting fluo rescence level. This property allo ws detection of an increase in the rate of protein synthesis. The best example is fluorescent timer [119], a mutant of the coral protei dsRed that changes from green to red, according to the age of the protein. Changes in the relative amounts of green and red fluorescence can be analyzed to determin the balance between protein synthesis and de gradation. This kind of tool has man y possibilities for the investigation of the life-cycle of AMPARs and their component subunits.

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7.4.5 FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Fluorescence resonance energy transfer (FRET) exploits the phenomenon of energy transfer from donor fluorophore to acceptor fluorophore/chromophore via dipol dipole coupling and allo ws the resolution of molecular interactions and conformational changes in the 1 to 10 nm range [120,121]. This technique is no w relatively well-established and FRET -based imaging has been used to monitor numerous protein interactions, including F-actin/G-actin equilibrium in dendritic spines [122]. In that study, actin tagged with c yan (CFP) and yello w (YFP) fluorescent protein allowed real-time observ ation of the equilibrium between its glob ular (G-) and filamentous (F-) forms. The energy transfer from the donor (CFP) to the acceptor (YFP) was detected when tagged molecules of actin monomers formed filamentou structures with 55 Å of distance between them. As for BiFC, FRET analysis has the potential for investigating the dynamics and locations of interacting protein binding to AMPAR.

7.4.6 FLUORESCENCE CORRELATION SPECTROSCOPY (FCS) Fluorescence correlation spectroscopy (FCS) of fluorescent protein tags is a poten tially useful technique for studying protein transport in li ving neuronal synapses [123]. FCS can be used to calculate rates of dif fusion and acti ve transport of fluorescent molecules by measuring spontaneous fluctuations in their fluorescen This data can be used to define their position, concentration or chemical kinetics For e xample, ECFP-tub ulin has been used to study tub ulin molecular dynamics [124].

7.5 CONCLUSION Given the rapid advances in fluorophore and microscope technolog , live cell imaging of the traf ficking of AMPARs and other proteins will continue to pro vide ever deeper insight into the molecular and cellular mechanisms underlying these vital processes. Furthermore, not only will we g ain more understanding at the le vel of individual cells, an increasing number of researchers are turning their attention to using imaging techniques to monitor more comple x neuronal netw ork acti vity in cultured cells and in vivo.

7.6 ACKNOWLEDGMENTS We are grateful to the Medical Research Council, Wellcome Trust and the European Union for funding. We also thank Dr . Michael Ashby, Dr. Atsushi Nishimune and Dr. Kyoko Ibaraki for access to their data and helpful advice.

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8

Neurotransmitter Dynamics Sabine Lévi and Antoine Triller

CONTENTS 8.1 8.2

Introduction ..................................................................................................143 Receptor Traffickin .....................................................................................144 8.2.1 Extrasynaptic Pool of Receptors...................................................... 144 8.2.2 Intracellular Pool of Receptors ........................................................145 8.3 Receptor Dynamics ......................................................................................146 8.3.1 Importance of the Dif fusion-Trap Mechanism in Synaptic Receptor Aggregation.......................................................................146 8.3.2 Use of Single-P article Tracking Experiments to Access Diffusion in the Synapse ..................................................................147 8.3.3 Diffusion Properties of Neurotransmitter Receptors: What Is Common and What Is Different? ......................................147 8.3.3.1 Extrasynaptic Receptors ...................................................147 8.3.3.2 Synaptic Receptors ...........................................................149 8.3.4 Receptor Dynamics and Synaptic Plasticity ....................................149 8.3.4.1 Excitatory Glutamate Receptors .......................................149 8.3.4.2 Inhibitory Glycine Receptors ...........................................150 References..............................................................................................................151

8.1 INTRODUCTION Most e xcitatory and inhibitory neurotransmitter receptors are concentrated at the post-synaptic density (PSD) facing pre-synaptic terminals containing the corresponding neurotransmitter [1–3]. The preferential accumulation of receptors at synapses is achieved by their specific interactions with a molecular sca fold that links them to the underlying c ytoskeleton [4,5]. This scaffold forms the so-called PSD, which acts as a molecular machine and locally controls some aspects of synapse formation, maintenance, plasticity and function. In recent years, the use of single-molecule and real-time imaging has revealed that neurotransmitter receptors are in constant, rapid movement at the neuronal surface and are transiently trapped at PSDs so as to modify the number and composition of receptors that are a vailable to respond to released

143

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neurotransmitter [6–8]. Such is the case for the glycine receptor (GlyR), the GABAA receptor (GABAAR), the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-meth yl-D-aspartate (NMDA) receptors, indicating that the phenomenon can be generalized.This dynamic behavior has profoundly modifie our view of the synapse. The aim of this chapter is to re view some aspects of our knowledge on receptor dynamics.

8.2 RECEPTOR TRAFFICKING Receptor trafficking i volves the intracellular mo vement of receptors from sites of synthesis to the plasma membrane, to ward synapses and then to sites of de gradation. The receptors are inserted into and remo ved from the plasma membrane by exocytosis and clathrin-mediated endocytosis, respectively [9–12]. At the neuron surf ace, receptors are first inserted into the xtrasynaptic plasma membrane and then diffuse in and out synapses (Figure 8.1) and, in some instances, that they could be directly inserted into the plasma membrane at synaptic sites has been hypothesized [13,14].

8.2.1 EXTRASYNAPTIC POOL

OF

RECEPTORS

Extrasynaptic receptors were initially detected using electroph ysiological e xperiments [15]. A newly developed technique combining freeze fracture and immunocytochemistry now allows us to visualize and quantify the densities of extrasynaptic and synaptic receptors, which were found to be 19 ± 2 receptors/µm 2 and 910 ± 36 receptors/µm2, respectively, for the AMPARs [16]. The ratio of synaptic over extrasynaptic receptor densities is lik ely to range between 10 and 50, depending on receptor types. Therefore, despite the difference in densities, because the e xtrasynaptic membrane occupies 98% of the total membrane, e xtrasynaptic receptors account for a much larger pool of receptors than synaptic ones [17]. In some cases, extrasynaptic and synaptic receptors are preferentially composed of dif ferent subunits. In the adult hippocampus, the NR2A sub unit is at extrasynaptic and synaptic membranes, whereas the NR2B sub unit is only at e xtrasynaptic membranes [15]. In the cerebellum, inhibitory GAB AARs containing the δ subunit are preferentially associated with extrasynaptic membrane [18]. GAB AAR subcellular localization is correlated with function: tonic inhibition could be mediated mainly by extrasynaptic δ-containing GAB AARs whereas phasic inhibition relied on synaptic GAB AARs devoided of δ subunits. That e xtrasynaptic receptors are acti vated by neurotransmitter spillover from the synaptic junction follo wing massive synaptic transmision has been proposed. Our e xperiments [19] and those by Choquet and colleagues [20,21] have shown that e xtrasynaptic receptors can enter synapses by lateral diffusion, revealing that the “dif fusion-trap” mechanism is a relati vely general mechanism for synaptic receptor aggregation. Now postulated is that extrasynaptic receptors form a reserv e pool, which can be rapidly deli vered by lateral dif fusion at the synapse upon request [8].

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4

4

2 3

5

1

6

Scaﬀold Receptor

FIGURE 8.1 Receptor trafficking and synaptic plasticit . Neurotransmitter receptors are synthesized in the cell body and transported to the periphery (1), inserted by e xocytosis in the plasma membrane directly at the post-synaptic density (PSD, 2) or at a distance (3) where they dif fuse laterally (4). Extrasynaptic receptors can be single or in microclusters and associated or not with scaffolding proteins. Extrasynaptic receptors access the PSD (localized on spines at e xcitatory synapses or on shafts at inhibitory synapses) by lateral dif fusion and are trapped by the submembraneous scaffold linked to the cytoskeleton. Receptors are released and diffuse away from the PSD follo wing a decrease in the af finity of sca fold-receptors or cytoskeleton-scaffold complexes. Receptors no w join the e xtrasynaptic pool where the y can be endoc ytosed follo wing capture by chlatrin-coated pits (5), further rec ycled to the cell surface (2) or sent to lysosomes for de gradation (6) depending on the nature of the internalization-triggering signal. Synapses beha ve as receptor donor/acceptor modules. The amount of receptors at synapses is determined by an equilibrium between e xtrasynaptic and synaptic pools of receptors. Synaptic plasticity controls both surface levels of AMPARs and their lateral diffusion in and out synapses. F or instance, LTP increases the number of surf ace AMPARs (black broken arrows) by changing the rate of exocytosis without an effect on lateral receptor diffusion. In contrast, the L TD-mediated increase in AMPARs endoc ytosis (gray brok en arrows) is due to an increase in synapticAMPARs lateral diffusion. Receptors therefore rapidly exit the PSD and become a vailable for internalization at the periphery .

8.2.2 INTRACELLULAR POOL

OF

RECEPTORS

Large intracellular pools of receptors ha ve been detected for GAB AARs and AMPARs at the light and ultrastructural level [22–24]. Intracellular pool of receptors close to synapses can form follo wing transport from the soma [25], local synthesis [14,26] or endocytosis [23,24]. Endocytosis, which also constitutes the intracellular pool of receptors, does not occur at the PSD b ut rather takes place at the periphery or at a distance from the PSD [27–29]. Therefore, receptors ha ve to dif fuse out of

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the synapse for internalization. Later , internalized receptors can be directed to lysosomes for de gradation or rec ycled at the cell surf ace, depending on the nature of the internalization-triggering signal [30]. If not de graded, intracellular receptors can also form a reserv e pool ready to be inserted in the plasma membrane and recruited at the synapse in response to a specific synaptic act vity pattern.

8.3 RECEPTOR DYNAMICS 8.3.1 IMPORTANCE OF THE DIFFUSION-TRAP MECHANISM SYNAPTIC RECEPTOR AGGREGATION

IN

A pioneer study used time-lapse imaging of thrombin-clea vable extracellular tags to follow GlyR dif fusion at the dendritic surf ace of spinal cord neurons [31]. The quantitative analysis re vealed that GlyR mo ved along the plasma membrane of dendritic processes at a speed of 1 to 2 µm/min. Meanwhile, Meier et al. [32] measured real-time surface GlyR dynamics using latex beads coated with antibodies directed against the extracellular region of transfected tagged GlyR α1 subunit. The mean diffusion coefficient of xtrasynaptic receptors was 0.029 ± 0.005 µm2/sec (D ± S.E.M.), a value in the same range than that found with the clea vable tag experiment. The extrasynaptic surface GlyR displayed free Brownian movements. Brownian movements depend on thermal agitation of molecules (in this case, lipids shaking on proteins) and are characterized by a linear relationship between the surf ace explored versus time, measured by the mean square displacement (MSD) function. In these e xperiments, GlyR slo wed do wn when encountering scaf folding GFPtagged geph yrin clusters and w as confined within gep yrin cluster boundaries. Actually, confined m vements are characterized by a spatial limitation of dif fusion due to obstacles, such as transmembrane proteins or membrane domains, or to transient binding to scaf fold proteins. GlyRs were transiently confined (tens o seconds) within geph yrin scaf fold and were dif fusing between aggre gates. This kinetic behavior with transitions between f ast Brownian and slo w confined m vements w as the first direct demonstration of the “di fusion-trap” model initially proposed to explain the formation of AchR hotspots at the neuromuscular junction [33]. Rapid Bro wnian mo vements in the e xtrasynaptic membrane were observ ed for all neurotransmitter receptors analyzed so far: the metabotropic glutamate receptors [34], the AMPA- and NMD A-types of glutamate receptors [20,21,35] and for the inhibitory GlyR [19,32]. Receptors-scaf folding molecule interactions resulted in confined m vements for GlyR and geph yrin [19,32], GAB AAR and geph yrin [36], metabotropic glutamate receptors and homer [34], AMPAR and PSD-95 [37]. Other obstacles, such as corrals and pick ets formed by the submembraneous c ytoskeleton and transmembrane proteins, respectively, could also participate in confin ing the mo vements of receptors. Although this has not yet been demonstrated, transient association of receptors with lipid rafts [38–40] could also contrib ute to confinement of receptors at synapses

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8.3.2 USE OF SINGLE-PARTICLE TRACKING EXPERIMENTS TO ACCESS DIFFUSION IN THE SYNAPSE The initial tracking e xperiments used beads whose size (500 nm) precluded the analysis of receptor diffusion in the synapse. Receptors were then tracked with much smaller probes using single-fluorescent molecule imaging [19–21]. These e xperiments confirmed what could be xpected from basic laws of diffusion: the behavior and dif fusion properties of receptors in the e xtrasynaptic plasma membrane were not dependent on the size of the probe used. Ho wever, with these small probes, tracking receptors in narrower compartments such as synapses was possible. In these experiments, receptors were labeled with antibodies directed against an extracellular epitope and coupled to organic dyes (e.g., Cy3, Cy5 and rhodamine) or the recently developed semiconductor nanocrystal also named quantum dot (QD). Or ganic dyes are smaller (1 nm) than QDs (5 to 10 nm) b ut they photobleach rapidly (t < 10s) whereas QDs do not. QDs can be used to track receptors for unprecendented duration (greater than 20 min) [19]. Another advantage of QDs is that the y can be used at the same time at the optical and the ultrastructural le vel. Single organic fluorescen molecules are identified by their one-step photobleaching. In contrast, ind vidual QDs do not photobleach but are identified as single entities by their blinking propert, i.e., their random intermittency of their fluorescence emission [41,42]. Whatever the probe used, individual molecule point sources form a halo of defined diameter an intensity that can be imaged with a char ged-cooled CCD camera. Their localization can be determined in a nanometer-scale and for the QDs, due to the excellent signalto-noise ratio, the diffusion coefficient measured with precision (1 4 to 105 µm2/sec). Unfortunately, whatever the probe used, tracking indi vidual fluorescent molecule does not necessarily mean following individual receptors because the labeled receptor could be part of a dif fusing cluster.

8.3.3 DIFFUSION PROPERTIES OF NEUROTRANSMITTER RECEPTORS: WHAT IS COMMON AND WHAT IS DIFFERENT? To date, lateral dif fusion has been analyzed for the inhibitory GlyR and GAB AAR and the e xcitatory AMPAR and NMD AR in primary cultures of spinal and hippocampal neurons, respectively [19–21,32,36]. Although it is a rough estimate, diffusing molecules can be arbitrarily di vided in two groups: the highly mobile (D > 7 × 103 µm2/sec) and the slo w (D < 7 × 103 µm2/sec). 8.3.3.1 Extrasynaptic Receptors All receptors shared a common beha vior in the e xtrasynaptic plasma membrane. GlyR α1 subunit, GABAAR α2 subunit, AMPAR GluR2 subunit and NMDAR NR1 subunit display Bro wnian-type movements and a close proportion (40 to 50%) of highly mobile molecules (Figure 8.2). This proportion is smaller than that of another transmembrane molecule such as N-Cam (95% of mobile molecules). Actually, the expression of N-Cam molecules with short intracellular tails predominates in maturating neurons, suggesting that the slo wer lateral dif fusion of e xtrasynaptic

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A1

A2

A3

A4

b2 b1 b3 B

C

0.4

MSD (µm2)

MSD (µm2)

0.5 0.3 0.2 0.1

D 0.05

***

0.02 0.01 0.2 0.4 0.6 0.8 1 Time (s) F

75 50 25

100 Mobile receptors (%)

100 Mobile receptors (%)

*

0.03

0.2 0.4 0.6 0.8 1 Time (s) E

**

0.04

75 50

GlyR GABAAR AMPAR NMDAR N-Cam

25

FIGURE 8.2 (Color figure fol ows page 176 .) Characteristics of neurotransmitter receptor lateral dif fusion. (a) Example of GlyR motion o ver the neuritic surf ace of spinal cultured neurons. Images were e xtracted from a sequence of 850 frames (acquisition time, 75 msec). a1 to a4 correspond to frames 6, 118, 150 and 629, respectively. QD fluorescence spots (green and FM4-64-labeled synaptic boutons (red). One QD (arro w), first located at bouton b1 diffuses in the extrasynaptic membrane (a1 to a3) and associates with bouton b2 (a7).Another QD (arrowhead) remains associated with synaptic boutons b3 and blinks at image a4. Scale bar, 2 µm. (b) MSD v ersus time, calculated for a continuous sequence of images between frames 54 and 161, which sho ws the extrasynaptic motion. (c) MSD v ersus time, calculated for a continuous sequence of images between frames 503 and 597, when the QD is located at the periphery of bouton b2. Error bars sho w mean ± S.D. (d) QD-GlyR dif fusion during long recording. Projection of time-lapse recording (1 Hz, 20 min) of QD-GlyR trajectories (green) overlaid with FM4-64 staining (red) and bright-field image. Extrasynaptic QD-Gly (*) e xplored lar ge surf aces of the membrane, and synaptic QD-GlyRs were stable (**) or mobile (***) in a confined domain around the synapse. Scale ba , 5 µm. Fractions of mobile (D > 7 × 103 µm2/sec) molecules at e xtrasynaptic (e) and synaptic (f) sites. Only slo wly mobile synaptic N-Cam molecules were considered. Similar fraction of mobile molecules is found in the e xtrasynaptic membrane for all neurotransmitter receptor types. Note that neurotransmitter receptors are slo wer in the e xtrasynaptic membrane than the transmembrane adhesion molecule, N-Cam. Note the smaller fraction of mobile inhibitory receptors compared to excitatory receptors at synapses.

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neurotransmitter receptors is due to an interaction of their c ytoplasmic re gions (which range from 1 to 10 nm) with the submembraneous elements acting as fences. In agreement, pharmacological depolymerization of microtub ules greatly increased the speed of e xtrasynaptic GlyR movements and the proportion of rapid molecules [42]. Receptor-fence interactions could be direct or indirect in volving a scaffolding protein associated with the receptor as it dif fuses laterally on the e xtrasynaptic membrane. This beha vior is supported by the f act that post-synaptic scaf folding proteins such as PSD-95 [43], star gazin [44,45], synapse-associated protein (SAP)102 [46] for NMD AR, AMPAR-binding proteins (GRIP/ABP) [47,48] and gephyrin for GlyR [25] can associate with receptors as the y travel toward or a way from the cell surf ace. In addition, pick ets formed by transmembrane molecules posted in the membrane also contrib ute to the limitation of receptor mo vement in the plasma membrane. 8.3.3.2 Synaptic Receptors Several parameters of synaptic dif fusion are common to all receptors studied. AMPAR, NMD AR, GlyR and GAB AAR enter and e xit synapses through lateral diffusion [19–21,36]. This behavior implies that movements of extrasynaptic receptors are likely to regulate synaptic receptor numbers and that pools of e xtrasynaptic and synaptic receptors are in a dynamic equilibrium. A change in this equilibrium (effect on k on and k off kinetics or lateral dif fusion characteristics) will alter the amount of receptors present at the synapse (see below). Furthermore, receptors travel from one synapse to another by mean of lateral dif fusion. Receptors mo ving in or out of synapses al ways transited through a perisynaptic area (400 nm) where the y remained for a longer period than e xpected from their lateral dif fusion coefficien in the extrasynaptic plasma membrane. Activity can alter the proportion of receptors in the peri-synaptic re gion [20], reinforcing the notion that this area is not simply a re gion for entering and e xiting synapses b ut could constitute a reserv e pool of receptors rapidly a vailable at synapses upon request. In contrast to what w as seen at extrasynaptic membranes, diffusion within synapses is confined. Both mobile an immobile receptors are detected at the synapse. Mobile synaptic receptors ( Figure 8.2) at variable proportion depending on receptor types could reflect d fferent properties of scaf fold-receptor interaction and dif fusion kinetics. This transient binding property sets specific on and k off v alues for the receptors to PSD interactions. More precisely, about 30% of GlyR, 45% of GAB AAR, 65% of AMPAR and 60% of NMDAR were highly mobile. Mobile synaptic receptors were confined withi about 0.1 µm 2 [20] corresponding to the e xpected surface area of the PSD.

8.3.4 RECEPTOR DYNAMICS

AND

SYNAPTIC PLASTICITY

8.3.4.1 Excitatory Glutamate Receptors The implication of lateral diffusion in the rapid regulation of receptor number at the synapse during synaptic function and plasticity w as recently demonstrated. KClinduced depolarization of hippocampal neurons increased AMPAR diffusion [21]. Inversely, blocking neuronal activity for 48 hours with tetrodotoxin (TTX) decreased

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extrasynaptic AMPAR diffusion. In agreement with previous electrophysiology and cell biology experiments supporting the notion that synaptic AMPAR, in contrast to NMDAR, are highly dynamic [12,50], none of these treatments af fected NMDAR lateral diffusion in the e xtrasynaptic and synaptic membranes. Therefore, separate mechanisms control the trafficking ofAMPAR and NMDAR. Only following protein kinase C (PKC) acti vation is lateral NMD AR diffusion increased in e xtrasynaptic and synaptic membranes [21]. This condition is consistent with the implication of lateral diffusion in the dispersal of synaptic NMD AR clusters in an uniform membrane distribution [51]. Synaptic plasticity in volves both re gulated exocytosis and endocytosis of AMPARs at e xtrasynaptic sites. AMPARs are internalized during NMDAR-dependent LTD and inserted into the membrane during NMD AR-dependent LTP [50]. The use of a pharmacological approach (bath application of strychnine and bicucculine to block inhibition and glycine to potentiate NMD ARs in hippocampal cultured neurons) known to increase the number of AMPARs through mechanisms similar to NMDAR-dependent LTP [13,52] increased AMPAR lateral diffusion at synapses, b ut only transiently [20]. The increase in AMPAR number in the PSD is consistent with an increase in the rate of e xocytosis without changing the equilibrium between extrasynaptic and synaptic pools of receptors. In contrast, bath application of glutamate to decrease the surf ace number of AMPARs [53,54] in a mechanism similar to NMDAR-dependent LTD [55,56] increased the percentage of peri-synaptic receptors and the dif fusion of synaptic receptors without changing dynamics of receptors in the extrasynaptic membrane [20]. LTD is known to increase AMPAR endocytosis and internalized AMPAR are immobile [20]. Therefore, LTDmediated increase in AMPAR endocytosis is due to receptors rapidly diffusing from synaptic to extrasynaptic sites where the y are then internalized in the e xtrasynaptic membrane. In this model, synaptic plasticity alters the equilibrium between e xtrasynaptic and synaptic pools of receptors. The increase in synaptic AMPAR diffusion is now thought to result from a complex set of events involving receptor-scaffolding protein unbinding, untethering of receptors from the c yoskeleton following depolymerization or a change in transmembrane adhesion molecules that w ould regulate the number of corrals and pick ets [57]. 8.3.4.2 Inhibitory Glycine Receptors The lateral diffusion of GlyR is also controlled by synaptic acti vity. Acute blockade of excitatory but not inhibitory activity increased GlyR diffusion in the extrasynaptic and synaptic membrane [43]. This effect is more pronounced in the e xtrasynaptic membrane. This increase in diffusion is mimicked by buffering intracellular calcium with B APTA-AM or blocking post-synaptic, L-type, v oltage-dependent calcium channels. Indeed, b uffering intracellular calcium also increased AMPAR diffusion [35]. Elevation of intracellular calcium acti vates a signaling cascade, which could induce man y ef fects including c ytoskeletal rearrangements at the PSD [58]. In agreement, depolymerizing microtub ules and actin c ytoskeleton increased GlyR mobility [43]. Because intracellular calcium in dendrites depends on the acti vation of excitatory receptors, these results suggest an homeostatic regulation of the balance between excitatory and inhibitory transmission.

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Altogether, these data indicate a role for extrasynaptic receptors in the regulation of synaptic transmission. Receptors are c ycling between extrasynaptic and synaptic compartment. At rest, extrasynaptic and synaptic receptors are at an equilibrium that could be displaced during synaptic plasticity to reach a ne w equilibrium set point to change receptor number at synapses.

REFERENCES 1. Triller, A. et al., Distrib ution of glycine receptors at central synapses: An immunoelectron microscopy study, J. Cell Biol ., 101, 683, 1985. 2. Baude, A. et al., High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus,Neuroscience, 69, 1031, 1995. 3. Takumi, Y. et al., The arrangement of glutamate receptors in e xcitatory synapses, Ann. NY Acad. Sci ., 868, 474, 1999. 4. Kneussel, M. and Betz, H., Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: The membrane activation model, Trends Neurosci., 23, 429, 2000. 5. Kim, E. and Sheng, M., PDZ domain proteins of synapses, Nat. Rev. Neurosci., 5, 771, 2004. 6. Choquet, D. and Triller, A., The role of receptor dif fusion in the or ganization of the postsynaptic membrane, Nat. Rev. Neurosci., 4, 251, 2003. 7. Triller, A. and Choquet, D., Synaptic structure and dif fusion dynamics of synaptic receptors, Biol. Cell ., 95, 465, 2003. 8. Triller, A. and Choquet, D., Surf ace traf ficking of receptors between synaptic an extrasynaptic membranes: And yet the y do mo ve! Trends Neurosci., 28, 133, 2005. 9. Carroll, R.C. et al., Role of AMPA receptor endoc ytosis in synaptic plasticity , Nat. Rev. Neurosci., 2, 315, 2001. 10. Malinow, R. and Malenka, R.C., AMPA receptor traf ficking and synaptic plasticit , Ann. Rev. Neurosci., 25, 103, 2002. 11. Sheng, M. and Kim, M.J., Postsynaptic signaling and plasticity mechanisms, Science, 298, 776, 2002. 12. Bredt, D.S. and Nicoll, R.A., AMPA receptor traf ficking at xcitatory synapses, Neuron, 40, 361, 2003. 13. Passafaro, M., Piech, V., and Sheng, M., Subunit-specific temporal and spatial pattern of AMPA receptor exocytosis in hippocampal neurons, Nat. Neurosci., 4, 917, 2001. 14. Gardiol, A., Racca, C., and Triller, A., Dendritic and postsynaptic protein synthetic machinery, J. Neurosci., 19, 168, 1999. 15. Tovar, K.R. and Westbrook, G.L., The incorporation of NMD A receptors with a distinct subunit composition at nascent hippocampal synapses in vitro, J. Neurosci., 19, 4180, 1999. 16. Tanaka, J. et al., Number and density of AMPA receptors in single synapses in immature cerebellum, J. Neurosci., 25, 799, 2005. 17. Rusakov, D.A. et al., Synapses in hippocampus occupy only 1–2% of cell membranes and are spaced less than half-micron apart:A quantitative ultrastructural analysis with discussion of ph ysiological implications, Neuropharmacology, 37, 513, 1998. 18. Nusser, Z. et al., Se gregation of dif ferent GABAA receptors to synaptic and e xtrasynaptic membranes of cerebellar granule cells, J. Neurosci., 18, 1693, 1998.

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19. Dahan, M. et al., Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking, Science, 302, 442, 2003. 20. Tardin, C. et al., Direct imaging of lateral mo vements of AMPA receptors inside synapses, EMBO J., 22, 4656, 2003. 21. Groc, L. et al., Dif ferential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors, Nat. Neurosci., 7, 695, 2004. 22. Petralia, R.S. and Wenthold, R.J., Light and electron immunoc ytochemical localization of AMPA-selective glutamate receptors in the rat brain, J. Comp. Neurol., 318, 329, 1992. 23. Connolly, C.N. et al., Cell surf ace stability of g amma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition, J. Biol. Chem., 274, 36565, 1999. 24. Connolly, C.N. et al., Subcellular localization and endocytosis of homomeric gamma2 subunit splice v ariants of g amma-aminobutyric acid type A receptors, Mol. Cell. Neurosci., 13, 259, 1999. 25. Hanus, C. et al., Intracellular association of glycine receptor with geph yrin increases its plasma membrane accumulation rate, J. Neurosci., 24, 1119, 2004. 26. Ju, W. et al., Activity-dependent regulation of dendritic synthesis and traf ficking o AMPA receptors, Nat. Neurosci., 7, 244, 2004. 27. Blanpied, T.A., Scott, D.B., and Ehlers, M.D., Dynamics and re gulation of clathrin coats at specialized endoc ytic zones of dendrites and spines, Neuron, 36, 435, 2002. 28. Petralia, R.S. et al., Internalization at glutamater gic synapses during de velopment, Eur. J. Neurosci., 18, 3207, 2003. 29. Racz, B. et al., Lateral or ganization of endocytic machinery in dendritic spines, Nat. Neurosci., 7, 917, 2004. 30. Ehlers, M. D., Reinsertion or degradation of AMPA receptors determined by activitydependent endocytic sorting, Neuron, 28, 511, 2000. 31. Rosenberg, M. et al., Dynamics of glycine receptor insertion in the neuronal plasma membrane, J. Neurosci., 21, 5036, 2001. 32. Meier, J. et al., Fast and reversible trapping of surface glycine receptors by gephyrin, Nat. Neurosci., 4, 253, 2001. 33. Young, S.H. and Poo, M.M., Rapid lateral dif fusion of extrajunctional acetylcholine receptors in the de veloping muscle membrane of Xenopus tadpole, J. Neurosci., 3, 225, 1983. 34. Serge, A. et al., Receptor acti vation and homer dif ferentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane,J. Neurosci., 22, 3910, 2002. 35. Borgdorff, A.J. and Choquet, D., Re gulation of AMPA receptor lateral mo vements, Nature, 417, 649, 2002. 36. Schweizer, C. et al., Lateral diffusion of GABAA receptors studied by single particle tracking, presented at the French Society for Neuroscience, Lille, France, May 17–20, 2005 (unpublished). 37. Bats, C. and Choquet, D., Star gazin regulates AMPA Receptors diffusion within the neuronal plasma membrane, presented at the Society for Neuroscience, Washington, DC, USA, No vember 12–16, 2005 (unpublished). 38. Suzuki, T. et al., Biochemical e vidence for localization of AMPA-type glutamate receptor subunits in the dendritic raft, Brain Res. Mol. Br ain Res., 89, 20, 2001. 39. Bruses, J.L., Chauv et, N., and Rutishauser , U., Membrane lipid rafts are necessary for the maintenance of the (alpha)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons, J. Neurosci., 21, 504, 2001.

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40. Becher, A., White, J.H., and McIlhinney, R.A., The gamma-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum, J. Neurochem., 79, 787, 2001. 41. Nirmal, M. et al., Fluorescence intermittenc y in single cadmium selenide nanocrystals, Nature, 383, 802, 1996. 42. Empedocles, S.A., Norris, D.J., and Bawendi, M.G., Photoluminescence spectroscopy of single CdSe nanocrystallite quantum dots, Phys. Rev. Lett., 77, 3873, 1996. 43. Levi, S. et al., Control of inhibitory receptor lateral dif fusion by e xcitatory input, presented at Society for Neuroscience, San Die go, California, USA, October 23–27, 2004. 44. El-Husseini Ael, D. et al., Synaptic strength re gulated by palmitate c ycling on PSD95, Cell, 108, 849, 2002. 45. Schnell, E. et al., Direct interactions between PSD-95 and star gazin control synaptic AMPA receptor number, Proc. Natl. Acad. Sci. USA , 99, 13902, 2002. 46. Chen, L. et al., Star gazin re gulates synaptic tar geting of AMPA receptors by tw o distinct mechanisms, Nature, 408, 936, 2000. 47. Sans, N. et al., NMD A receptor traf ficking through an interaction between PD proteins and the e xocyst complex, Nat. Cell Biol ., 5, 520, 2003. 48. Dong, H. et al., GRIP: A synaptic PDZ domain-containing protein that interacts with AMPA receptors, Nature, 386, 279, 1997. 49. DeSouza, S. et al., Dif ferential palmito ylation directs the AMPA receptor -binding protein ABP to spines or to intracellular clusters, J. Neurosci., 22, 3493, 2002. 50. Collingridge, G.L., Isaac, J.T ., and Wang, Y.T., Receptor traf ficking and synapti plasticity, Nat. Rev. Neurosci., 5, 952, 2004. 51. Fong, D.K. et al., Rapid synaptic remodeling by protein kinase C: Reciprocal translocation of NMDA receptors and calcium/calmodulin-dependent kinase II, J. Neurosci., 22, 2153, 2002. 52. Lu, W. et al., Activation of synaptic NMD A receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons, Neuron, 29, 243, 2001. 53. Carroll, R.C. et al., Dynamin-dependent endoc ytosis of ionotropic glutamate receptors, Proc. Natl. Acad. Sci. USA ., 96, 14112, 1999. 54. Beattie, E.C. et al., Re gulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD, Nat. Neurosci., 3, 1291, 2000. 55. Carroll, R.C. et al., Rapid redistrib ution of glutamate receptors contrib utes to longterm depression in hippocampal cultures, Nat. Neurosci., 2, 454, 1999. 56. Man, H.Y. et al., Re gulation of AMPA receptor–mediated synaptic transmission by clathrin-dependent receptor internalization, Neuron, 25, 649, 2000. 57. Murase, S. and Schuman, E.M., The role of cell adhesion molecules in synaptic plasticity and memory, Curr. Opin. Cell. Biol ., 11, 549, 1999. 58. Lamprecht, R. and LeDoux, J., Structural plasticity and memory, Nat. Rev. Neurosci., 5, 45, 2004.

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9

Receptor Dynamics at the Cell Surface Studied Using Functional Tagging Philip Thomas and Trevor G. Smart

CONTENTS 9.1 9.2 9.3 9.4 9.5

Introduction ..................................................................................................155 Perspective on Nonfunctional Receptor Tagging Techniques.....................156 Early Studies Using Functional Tagging.....................................................158 The Nature of the Epitope: Criteria for Selecting a Functional Tag ..........160 Advantages of the Functional Tag in the Study of Mobile Receptors .........................................................................................161 9.6 AMPA Receptor Rectification and Synaptic AMPAfication ...................162 9.7 MK801 Channel Block Exposes the Transient Nature of Synaptic NMDA Receptors .........................................................................165 9.8 Synaptic Inhibition: The Mobility of Extrasynaptic GABAA Receptors........................................................................................167 9.9 Photoreceptive Potassium Channels as Switches of Neuronal Excitability ...................................................................................169 9.10 Disadvantages and Limitations of the Functional Tag................................170 9.11 Conclusion....................................................................................................172 References..............................................................................................................172

9.1 INTRODUCTION Heisenberg’s Uncertainty Principle, originating from quantum mechanics, can also be appropriately applied to the study of receptor dynamics in cell membranes. Essentially, the “uncertainty” is introduced because to measure or track receptor movements in cell membranes, we really need to tag the proteins, which in itself could alter receptor mobility. The nature of the tag might involve a sequence of amino acids inserted into the protein (structural modification), or simply the addition of a antibody to a nati ve epitope on the receptor . In either e xample, the accurac y of measurements of mobility will innately have a degree of uncertainty associated with them. The key objective of an y method emplo yed to measure receptor mo vements

155

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is to minimize this uncertainty while maximizing the resolution fidelity for a appropriate population of receptors. To date, the principal and most technically accessible methods for monitoring the dynamic movement of receptors within the membrane of neuronal or other cells most commonly in volves some form of biochemical or optical procedure. Optical approaches have one critical advantage in that they provide reasonably high temporal resolution, especially when images of membrane receptors or clusters of receptors are obtained from live cells that are monitored using organic fluorophores or quantu dots, in concert with the rapid acquisition capabilities of confocal microscop y. However, both biochemical and optical techniques require certain assumptions to be made before the y can impart information about the mo vements of functional receptors in the cell membrane. Such techniques will, to some e xtent, ine vitably include receptors from se veral pools (e.g., cell surf ace membrane and intracellular pools), some of which might be nonfunctional, or ha ve limited in volvement in maintaining the appropriate excitable state of the neurone.The functional approaches described in this chapter use a dif ferent strate gy based on electroph ysiological methods. This provides a measure of the current which results only from the binding of activating lig ands to their respecti ve lig and-gated ion channels (LGIC) thereby resolving only those receptors that actually contrib ute to the e xcitable state of the neuron. This can include both synaptic and e xtrasynaptic pools of functional receptors but, importantly, it will discount submembranous receptors. As such, the influ ence these tagged receptors contrib ute to the metaplastic changes (potentiation or depression) of the synaptic response can, in principle, be readily monitored. As we will see, this method of functional tagging requires that particular receptor isoforms carry either a bioph ysical or pharmacological reporter , or tag, that can be readily studied in live cells, and that such reporters provide distinctive functional signatures. This could be manifest by the activation, modulation or ablation of receptor function, depending on the tag and its lig and as described in the proceeding sections.

9.2 PERSPECTIVE ON NONFUNCTIONAL RECEPTOR TAGGING TECHNIQUES Why should we seek alternati ve receptor tracking strate gies to the man y optical, biochemical and other nonfunctional methods that follow the life-cycle of membrane receptors? The simplest answer to this question is that certain procedures are more suitable to address specific questions with r gard to the traf ficking of membran proteins and, importantly, not all these methods selectively monitor the same parameters. In man y cases, several of these techniques are complementary . The application of a functional tag to a receptor (also referred to as “electrophysiological tagging”) permits e xperiments that monitor the stability , biophysical properties and pharmacological profile of a functionally competent population o receptors, which are resident in the cell membrane, beha ving functionally as the y would in vivo . The functional tag therefore e xcludes an y direct contrib utions that might be made to the o verall monitoring of receptor mo vement by immature, dysfunctional or submembranous receptors. Moreo ver, by applying tags to certain

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specific populations of receptors, or by enabling a tag to become visible only durin particular functional states of the receptor, the relative trafficking of synaptic recep tors, as distinct from e xtrasynaptic receptors, can be deduced. This is an important difference between functional tagging and nonfunctional biochemical or optical methods. The adv ent of or ganic dyes such as FM4-64 [1,2] and related products, which can be uptak en into synaptic v esicles, has attempted to rectify this to some extent by permitting only the identification of act ve synapses, although this fluo rescent dye does not identify the mo vement of functionally acti ve receptors [3]. Calcium imaging of dendritic spines of fers another optical approach to the measure of functional receptor acti vity, although this can ha ve spatio-temporal resolution limitations and still represents a second-order response following receptor activation. It has yet to be fully e xploited to monitor receptor mo vements in and around the cell membrane. As receptor tracking e xperiments are often required to monitor mo vements in the membrane o ver relati vely short time periods, the temporal resolution of biochemical and fi ed staining procedures can be a limitation. The practicalities of conducting pulse-chase experiments (either with antibodies conjugated to radioactive tracers, avidin or specific o ganic fluorophores) is generally not conduc ve to following the f ast movements of membrane-delimited proteins, which occur minuteby-minute under conditions of both basal and stimulated neuronal acti vity. As far as immunocytochemistry is concerned, temporal issues ha ve been resolved to some extent through no vel protein modifications, such as the introduction of cle vable epitopes such as thrombin and the haemagglutinin tag [4]; fusions with GFP; pHsensitive GFPs (pHlourins) [5,6]; c ysteine-biarsenicals [7–9]; and the introduction of high-affinit , irreversible binding sites for high potenc y toxins, e.g., α-bungarotoxin [10]. Man y of these epitopes or tags can be used in li ve staining procedures. Perhaps the most useful of these are the pHluorins, particularly ecliptic pHluorin, which uniquely loses fluorescence at one xcitation wavelength when placed in an acidic pH en vironment that is normally only e xperienced by internalized receptors rather than their cell surface counterparts. Thus, this pHluorin should faithfully report the mo vements of principally cell surf ace receptors, although certain trans-Golgi compartments, from which synthesized receptors originate, have sufficiently neutra pH to permit fluorescence.Thus, with few exceptions, fluorescently labeled receptor that are internalized or are being traf fic ed to the cell surf ace are usually dif ficul to unequivocally distinguish from those that only reside at the cell surf ace. Antibody labeling techniques ha ve further limitations. Antibodies are man y orders of magnitude lar ger than the receptor the y are labeling. Generally , this disparity in size is exacerbated by the requirement for a secondary antibody to be linked to an organic fluorophore for visualization and, indeed, this disparity is xacerbated further with quantum dot coupling. In addition, the primary antibody needs to be raised to e xternal receptor epitopes to distinguish only cell surf ace receptors and avoiding the need to membrane permeabilize to allo w the antibody to g ain access to intracellular epitopes, which w ould mak e intracellular and membrane receptor pools indistinguishable. Furthermore, dual labeling of receptors in concert with specific synaptic mar er proteins only indicates the general “proximity,” not absolute apposition, of a receptor to a synaptic site. One further ca veat also suggests that by

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no means are all synaptically labeled receptors functionally acti ve [3]. Yet further ambiguity is raised because synaptic and e xtrasynaptic receptor classification at th optical le vel is usually ascribed on the basis of immunofluorescent puncta size although what part indi vidual receptor-antibody-fluorophore compl xes represents in a single punctum and ho w this reflects on the potential number of receptors pe punctum is unknown. Further, under conditions of prolonged e xposure to excitable wavelengths of laser light, such as occurs with repetiti ve line scanning of the same region of interest (a common technique used to optimize an image and to perform time-series studies), certain organic fluorophores are susceptible to xtensive bleaching. As such, unintentional fluorescence loss in photobleaching (FLIP) or fluoure cence reco very after photobleaching (FRAP) measurements can gi ve the f alse impression of receptor mobility .

9.3 EARLY STUDIES USING FUNCTIONAL TAGGING Relatively few examples are found of the use of functional tags to study the membrane mo vements of neuroph ysiologically important proteins. Because of technological limitations, many early examples were unable to introduce a tag into a precise part of the receptor structure. As such, early experiments, although often ingenious, were some what unrefined as, for xample, in e xperiments designed to assess the mobility of voltage-gated potassium and sodium channels within the plasma membrane of frog muscle fibers. Here, a patch pipette as used to measure membrane currents and then also to shine UV light onto the membrane patch to cause photodestruction of the ion channels (and, presumably , other surf ace proteins also) [11]. By subsequently monitoring the f aster reco very of membrane potassium currents relative to sodium currents, potassium channels were concluded to possess greater mobility than their sodium channel counterparts. An earlier yet some what more sophisticated approach relied on the use of an inherent pharmacological reporter within the muscle nicotinic acetylcholine receptor (AChR), which is irre versibly inhibited by α-bungarotoxin with nanomolar af finit . The density of AChR on the myotomal muscle cell surf ace of Xenopus tadpoles was monitored by the re gular iontophoretic application of ACh, which caused membrane depolarizations [12]. Local inactivation of these functional AChRs by α-bungarotoxin and monitoring the reco very of the depolarizations ( Figure 9.1a and Figure 9.1b) permitted a determination of the diffusion coefficient D) for these receptors. The value of D (1.5 × 109 to 4.0 × 109 cm2/sec) implied that simple diffusion-based redistribution is the likeliest mechanism for receptor mo vement during synaptogenesis. An approximate estimate of ho w long ( t) it takes a particle to dif fuse a given distance (d) knowing its diffusion coefficient D) can be found from: t1/2 – ≈ (d1/2)2/D. Thus, for a diffusion coefficient of 2.5 × 109 cm2/sec, to mo ve a quarter of the w ay around a muscle fiber of diameter 30 µm will ta e approximately 37 min. F or Young and Poo’s study [12], half recovery following α-bungarotoxin was reached for similar sized fiber in approximately 21 min. Therefore, diffusion alone w as sufficient to xplain the rate of recovery observed.

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V α-BTX

ACh

(a) α-BTX

(b)

Con-A

(c)

FIGURE 9.1 Early form of receptor tagging. (a) Intracellular recording (V) from a Xenopus tadpole myotomal muscle cell showing repetitive localized application of acetylcholine (ACh) in the absence or presence of focally-applied α-bungarotoxin (BTX). (b) The amplitude of the ACh-induced membrane depolarizations are irre versibly reduced by approximately 70% by the local application of α-BTX (solid bar). This recovers to approximately 50% of the full amplitude following removal of the toxin, representing lateral transition of unblock ed receptors into the area of membrane acti vated by ACh. (c) The same preparation no w pretreated with concanavalin-A, applied over the muscle fibers (for 18 min) to immobilize the receptors is unable to show any recovery after α-BTX treatment. Adapted, idealized and redrawn from reference [12]. The calibration bars represent 2.5 mV (vertical bar) and 1 min (horizontal bar).

The technique successfully e xploited another specific pharmacological reporte of the muscle AChR, concanavalin-A, which w as used to immobilize AChRs and thus block dif fusion (Figure 9.1c). This agent pre vented the dif fusion of toxinblocked and unblocked receptors affecting the dynamics of recovery. The mechanism of this reco very represented the first electrop ysiological demonstration that functional receptors were able to diffuse freely within the membrane. From these studies, the suggestion of a “dif fusion trap” model for protein mo vement within the membrane originated. This entailed the re gion of innerv ation on a post-synaptic membrane serving as a sink, or trap, for functional receptors that are readily dif fusing in the membrane, thus leading to their initial concentration during synaptogenesis and subsequent maintenance at mature synapses. Recently, this concept has again found favor in explaining the movements of neurotransmitter receptors in cell membranes [13].

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9.4 THE NATURE OF THE EPITOPE: CRITERIA FOR SELECTING A FUNCTIONAL TAG Trying and achieving several of the following criteria is desirable when selecting or adopting a functional tag to monitor the mo vement of surf ace receptors. The main objective is to monitor the receptors without unduly af fecting their beha vior in the membrane by the inclusion of a functional tag. Importantly, the epitope could be an innate part of the nati ve receptor, which is functionally susceptible to the binding of a ligand, for example, or it could be part of the receptor that can be con veniently engineered by mutagenesis and reintroduced into a cell to become susceptible to another ligand that might not normally associate with that protein. Although either approach is useful, other criteria are w orthy of consideration. Tag size . This attribute should be k ept to a small size; lar ge tags, particularly those that increase the size of the receptor in terms of b ulk or mass, are more lik ely to have an impact on receptor mo vement and speed of dif fusion in the membrane. Silent ta g. The incorporation of a tag alone should not af fect the mobility , distribution or the function of the receptor . Thus, the tag should act in a passi ve capacity. Binding a ligand to the tag might then specifically alter the function of th receptor, which can be used as a monitor of receptor mo vement. Essentially, the tag must therefore remain silent until it is activated or becomes bound to another moiety. Irreversible tag activation. To accurately follow the movements of tagged receptor proteins, tag acti vation should be irre versible or , at best, only v ery slo wly reversible. This criterion will ensure that a tagged receptor that becomes functionally altered follo wing lig and binding can be track ed f aithfully until it is endoc ytosed without any confounding observ ations resulting from the lig and dissociating from the tag and the receptor function altering as a result. Receptor assembly . The inclusion of the tag must not interfere with sub unitsubunit assembly for hetero- or homo-oligomeric receptors. Innate ta g. Where possible, to guarantee the characteristics listed abo ve, it is better to use an innate tag, i.e., an epitope or binding site naturally present on the receptor. This use will ensure the least disruption to normal receptor function and trafficking. As is often the case, innate tags, apart from being sensiti ve to selecti ve antisera, can be quite unsuitable for tracking receptor mo vement due to the inappropriateness of the binding lig and for such a function (i.e., the ef fect of the lig and might not be easy to monitor). State-dependent tag. To gain maximum use of a functional tag, only re vealing the epitope to a lig and during particular conformational states of the receptor is helpful. For example, if the tag is present in the ion channel, then only re vealing this tag when the ion channel is acti vated is useful, thus rendering acti ve channels susceptible to tagging by a suitable lig and but not their closed counterparts. Rapid functional r eporter. To pro vide immediate feedback on receptor mo vement, the binding of a lig and to an epitope and its subsequent ef fect on receptor function should be rapid or at least se veral orders of magnitude f aster than the movement of receptors that are being track ed.

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9.5 ADVANTAGES OF THE FUNCTIONAL TAG IN THE STUDY OF MOBILE RECEPTORS The binding of a lig and to a receptor possessing an inte gral ion channel in volves a characteristic signal transduction e vent involving the opening of the ion channel and fl w of ions. This ionic current represents the normal ph ysiological function of the receptor but, as such, it can also be used to act as a reporter for the receptor’ s movement in real-time. The ability to measure and monitor currents through functional receptors is the principal property that mak es the functional tagging approach a more ph ysiological measurement than corresponding biochemical or optical techniques. Generally, the phasic release of neurotransmitter from pre-synaptic specializations (in mature primary cultures, acute tissue slices, or ganotypic cultures or e ven primary and secondary cell line co-cultures) acti vates only post-synaptic receptors that are located directly apposed to the transmitter release sites.These phasic synaptic currents are transient in nature and their profile depends on the receptor subtyp (subunit composition) present at synapses, their bioph ysical characteristics (i.e., activation, deactivation and desensitization rates, de gree of rectification and so on) their state of modulation (e.g., phosphorylation) and the speed of remo val of the transmitter by transporters or inacti vating enzymes. These post-synaptic receptors can often be distinct in terms of composition and pharmacology from their perisynaptic and e xtrasynaptic equivalents that populate the remainder of the neuronal membrane. Extrasynaptic receptors that are persistently acti vated by neurotransmitter spillover from synapses or basal le vels of transmitter from other sources (e.g., astrocytes) are responsible for tonic currents, which partly set the threshold at which action potentials are generated, thereby contrib uting to “signal inte gration” in the neuron [14–16]. For a particular receptor family or a receptor subtype within a single family, the presence of a functional tag allo ws the current fl w through specified receptors t be manipulated using specific pharmacological agents, often inhibitors. or the agent to be maximally useful, its binding site can only be e xposed once the channel is activated, thus making the use-dependent channel blocking agent a popular tool for functional tagging studies. The ideal, irre versible (co valent) binding reaction of a ligand to the epitope ensures that an y recovery of function can only be attrib utable to the introduction (by rec ycling, exocytosis or lateral translocation) of unblock ed receptors into the cell membrane. The advantage of this type of block of receptors in a neuron is that the spontaneous and miniature synaptic currents, due to neurotransmitter release only at the synapse, can be studied in isolation from the e xtrasynaptic receptor pool, i.e., only the activated receptors at the synapse are susceptible to block. Once the functionally irreversible current “knockdown” is complete, any recovery of synaptic activity will be the result of unblock ed receptors mo ving in from either e xtrasynaptic sites or from exocytotic pathways to displace these blocked synaptic receptors. By the nature of the a vailable lig ands, some receptors possess a nati ve, inherent functional tag, e.g., α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors and N-methyl-D-aspartate (NMDA) receptors, b ut others require to ha ve such tags engineered into the protein, as is the case for the GABAA receptors (see section 9.8).

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9.6 AMPA RECEPTOR RECTIFICATION AND SYNAPTIC “AMPAFICATION” Early measurements of protein mobilities, revealed that integral membrane proteins such as rhodopsin ha ve restricted mo vement in the plane of the cell membrane compared to being able to dif fuse freely in the lipid bilayer [17]. Furthermore, measurements of AChR mobility at the neuromuscular junction revealed such a slow diffusion coefficient D < 1014 cm2/sec) that monitoring movement was difficult wit any accuracy [18,19]. Indeed, given such a small diffusion coefficient, the calculation in section 9.3 regarding the movement of AChRs around one-quarter of a muscle fiber ould now take approximately 17.6 years. Thus, such receptors could be considered as static entities.

Now well-established is the f act that this stability of the receptors in the postsynaptic membrane is essential for ef fective intercellular communication, as the entrapment of mobile receptors permits the apposition of pre- and post-synaptic structures at neurotransmitter release sites. The slowness of some receptors to diffuse in the membrane has been unequi vocally established as due to specific interaction between anchoring proteins and post-synaptic receptors in the post-synaptic density (PSD) [20–22]. Although anchored at the synapse, these receptors are able to e xhibit mobility as observed during activity-dependent changes to the ef fica y of synaptic transmission that underlie plasticity changes such as long-term potentiation (LTP) and longterm depression (L TD). The molecular basis for these changes could stem from changes to the channel conductance [23], alterations to the phosphorylation state of the receptor [24–27], as well as increased deli very of receptors to the synapse [28]. The first studies to correlate the e fects of neuronal acti vity on the traf ficking o AMPA receptors at individual synapses coupled observations of cell surface epitopetagged AMPA receptors and electroph ysiological recordings of mEPSC e vents [29–31]. Subsequent functional tagging studies [32] clarified vidence from optical and biochemical data that the pool of membrane receptor proteins, rather than being rigidly corralled and retained as part of the PSD, is v ery dynamic, traf ficking an targeting specific receptors into established synapses. As a consequence of this synaptic tuning, detectable changes to the functional signature of synaptic receptors could be recorded in response to stimulation. Recording from cerebellar stellate cells, Lui and Cull-Candy [32] isolated AMPA-mediated excitatory post-synaptic currents (EPSCs) by minimally e voking the parallel fiber input in the presence of appropriate receptor bloc ers. The functional tag in this case exploited the susceptibility of AMPA receptor-derived EPSCs (which, importantly , lack the GluR2 sub unit) to v oltage-dependent block by the intracellular application of the polyamine, spermine. The net result is that while nonGluR2 subunit-containing AMPA receptors exhibit inward rectification due to thei calcium permeability, inclusion of GluR2 confers relati ve calcium impermeability and no inw ard rectification [33–36]. This characteristic deri ves from the e xclusive editing of the GluR2 sub unit in the pore-lining re gion in volving a Q/R mutation

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[37]. Interestingly, the GluR2 sub unit is also the k ey to the susceptibility of AMPA receptors to other pharmacological agents. F or instance, Joro spider toxin, pentobarbital and thioc yanate ions all dif ferentially block AMPA-derived EPSCs depending on the presence of GluR2. By measuring the de gree of rectification for voked EPSCs in the presence of spermine, any changes taking place to the functional properties of synaptic glutamate receptors in response to high-frequenc y stimulation could be monitored [32]. Synaptic currents, measured between 15 to 30 min after cessation of the stimulation, were seen to change from being predominantly inwardly-rectifying to linear (Figure 9.2). These results reflected a change in th AMPA receptor phenotype at the synapse due to an increased presence of the GluR2 sub unit and emphasized the highly dynamic nature of the receptors in the PSD. However, the cellular origin of this new phenotype of synaptic receptor was not investigated but might have been as a result of increased protein e xpression, exocytotic delivery into the membrane from intracellular pools or in the membrane lateral translocation from e xtrasynaptic sources. The time frame for these changes suggested limited in volvement of de novo protein expression. Thus, high-frequency stimulation can trigger the appearance of calciumimpermeable AMPA receptors at cerebellar stellate cell synapses that can be monitored as a functional change. Importantly, the same effect was not observed for lowfrequency stimulation b ut could be emulated by bath application of glutamate or kainate. Thus, this is the first significant study to xploit an innate electroph ysiological tag (i.e., the edited GluR2 sub unit) to monitor the dynamics of a functional receptor. As an aside, this study also highlighted the importance of calcium entry through the AMPA receptor in inducing this sub unit composition change, enabling a form of plasticity that is self-re gulating because the e xpressed replacing synaptic receptors have much lower permeability to calcium. Mediation of f ast e xcitatory synaptic transmission in all parts of the central nervous system (CNS) is undertaken by AMPA receptors, whereas NMDA receptors are more crucial to a diverse range of developmental, physiological and pathological processes. Despite such disparate neurophysiological roles, these two receptor families are mutually dependent. This relationship is demonstrated early in development by the awakening of “silent” glutamater gic synapses whereby neuronal stimulation promotes the deli very of AMPA receptors (probably from e xtrasynaptic compartments, as well as intracellular pools) into the quiescent NMDA R-containing synapse causing their functional “a wakening.” Ev en at later de velopmental stages, immunogold labeling techniques re veal that although NMD A receptors can be found in most mature synapses of the CA1 hippocampus, the same cannot be said of AMPA receptors [28,38,39]. Ho wever, changes in the plasticity of such mature pathw ays induced by stimulation are also underpinned by introducing functionalAMPA receptors at synapses (AMP Afication) through the act vation of NMD A receptors [40]. Thus, se veral parallels between synaptic maturation and L TP e xist, principally involving the activity-driven delivery of AMPA receptors following NMDA receptor activation. The dynamics of “silent” synapse acti vation and LTP have been addressed in a complementary study to that of Lui and Cull-Candy [32] using the same rectificatio property of GluR2-deficient receptors as a functional reporte . Hayashi et al. [41]

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Pre-stimulation

Post-stimulation

40 −40

20 pA

−60

2 ms (a)

−60

80

pA

pA

80

40

−20 −40

20 mV

−60

60

40

−20 −40

−80

20 mV

60

−80

(b)

FIGURE 9.2 Mobile AMPA receptors at glutamater gic synapses. (a) After high-frequenc y stimulation (300 stimuli at 50 Hz), cerebellar stellate-cell EPSC amplitudes were reduced at –60 mV b ut increased at +40 mV holding potentials. (b) The inw ardly-rectifying currentvoltage relationship before stimulation (open circles) became linear after 50 Hz stimulation (filled circles). Adapted from [32].

over-expressed the GFP-tagged GluR1 AMPA receptor subunit in hippocampal slice neurons. Importantly, this caused the accumulation of lar gely homomeric GluR1 receptors in extrasynaptic regions of the dendrite, thus maintaining the linear rectification properties of endogenous GluR2-containing synaptic responses. Activation of CaMKII (which would occur following NMDA receptor activation and associated Ca2+ influx) caused the insertion of homomeric GluR1 into synapses, as detected b inward rectification of the EPSCs. Notabl , pairwise recordings from GluR1-infected or noninfected cells in the absence of activated CaMKII failed to generate this switch. Further, an LTP induction protocol caused similar changes to the rectification of th EPSCs due to the import of GluR1 homomeric receptors into the synapse, which occurred 30 min after a stable potentiation period. Thus, phosphorylation e vents mediated by CaMKII appear to re gulate the deli very of dif ferent phenotypes of synaptic AMPA receptors from extrasynaptic compartments into the synapse, changing the functional character of the synaptic response.

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9.7 MK801 CHANNEL BLOCK EXPOSES THE TRANSIENT NATURE OF SYNAPTIC NMDA RECEPTORS The NMDA receptor, unlike their AMPA-sensitive counterparts, were until recently considered to be relati vely immobile elements of the PSD, being tightly anchored in the post-synaptic membrane [42]. Their resistance to deter gent extraction from PSDs is testament to this [43,44], although, surprisingly , the half-life of NMD A receptors in cultured cerebellar granule cells is approximately only 1 day and not too dissimilar to that for AMPA receptors [45]. NMD A receptors are present at the synapse early in development and, until the activity-dependent importation of AMPA receptors into the synapse, constitute the “silent” synapse. The signal transduction cascades, mediated by Ca 2+ influx through the NM A receptor, are responsible not only for synapse formation b ut also for their modification and elimination [46,47] Much of the plasticity of glutamatergic synapses has been attributed to the profound mobility of the AMPA receptor f amily but more recent e vidence has underscored the contribution made by NMD A receptor turno ver and trafficking [48–50] The NMDA receptor open channel blocker, MK801, has proved a useful tool in the limited number of ph ysiological studies of NMD A receptor dynamics in neurones. Whole-cell responses to NMDA are completely blocked by MK801 and show little recovery thereafter [51], suggesting limited replenishment of surface receptors. Interestingly, exposure of neurones to a constituti vely active form of protein kinase C increased the e xocytosis of unblock ed, functional receptors to the membrane by nearly four-fold, thus highlighting the ability of these receptors to respond to stimuli and readily modify their numbers at the cell surf ace. A subsequent e xploitation of MK801 was undertaken by Tovar and Westbrook [52] who, for the first time in physiological study, were able to suggest that NMD A receptors mo ve quite freely within the plane of the lipid bilayer between synaptic and e xtrasynaptic sites. Using cultured hippocampal neurones forming autapses, all synaptic and e xtrasynaptic NMD A receptors were completely block ed by co-applying NMD A and MK801 to the whole cell. No reco very of NMDA receptor function w as evident 30 min after MK801 washout, suggesting a very slow basal exocytosis rate for NMDA receptors (Figure 9.3a). However, when only synaptic receptors (measured as stimulated EPSCs) were block ed by MK801 (applied during the e voked EPSCs), a maximal recovery of about 40% in the synaptic response w as seen within 20 min (Figure 9.3b). Because recovery from MK801 blockage was less than complete, only a limited reserve of NMDA receptors seems to be quickly mobilized into the synapse. This recovery of synaptic receptors could be accounted for by a number of scenarios, including dissociation of MK801 or an increase in the number of synaptic NMD A receptors. The latter could occur either at e xisting synapses, following new synapse formation or by an increase in the size of e xisting synapses. MK801 unbinding was discounted because exposing MK801 blocked synaptic receptors to AP5 (to prevent channel opening and MK801 unbinding) during w ashout sa w the same le vel of current reco very. Moreo ver, insertion of ne w receptors w as clearly not the route for recovery since the whole-cell responses did not reco ver. Latrunculin, which arrests dendritic spine mobility, also discounted active zone or pre-synaptic terminal

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Agonist-evoked block

Synaptic block

MK-801 EPSC amplitude (pA)

EPSC amplitude (pA)

NMDA / MK-801 500 400 300 200 100 0

0

20 10 Time(min)

(a)

30

400 300 200 100 0

-5

0

5 10 15 20 Time(min)

(b)

FIGURE 9.3 (Color figure fol ows page 176.) Lateral mobility of NM DA receptors. (a) Agonist-evoked block of synaptic receptors. Whole-cell application (top panel and arro ws in lower panel, for 1 sec) of NMD A in the presence of MK801 resulted in complete and irreversible block of the NMD A receptor -mediated EPSC. (b) Selecti ve block of e voked synaptic NMDA receptors (top panel) by MK801 application (solid bar , lo wer panel) also completely blocked the EPSC. Ho wever, following removal of MK801, the EPSC sho wed a 30–40% recovery over the course of se veral minutes. Filled circles indicate MK801 application. In both top panels, acti vated receptors are red, inacti ve are blue and the shaded area represents that o ver which the agonist is dispersed. Adapted and redra wn from [52].

migration as another possibility . Thus, the strongest candidate mechanism for synaptic receptor recovery was the lateral movement of unblocked extrasynaptic receptors within the membrane. Further e xperiments undertak en with another NMD A channel blocker, ketamine, revealed the true dynamic nature of these receptors, in that about 25% of the receptors on the entire cell surf ace entered a synapse within 5 min. This reinforces the dif fusional rate constants seen for other lig and-gated ion channels in neuronal membranes [13,53–55] and also the high density of acti ve synapses present on in vitro neurons, which could only be a fraction of the in vivo scenario. A further observ ation from these studies w as that the number NMD A receptors moving into and out of synapses o ver several minutes appeared to be at a steady-state, thus not changing the o verall size of synapses. Synaptic NMD A receptors preferentially contain the NR2A sub unit, whereas NR1/NR2B assemblies lar gely form e xtrasynaptic receptors [56] and should be sensitive to the NR2B-specific antagonist, ifenprodil [57]. With these criteria in mind, post-MK801 reco vering EPSCs might be assumed to become sensiti ve to ifenprodil due to the import of e xtrasynaptic NR1/NR2B receptors. Ho wever, this did not occur, implying that synaptic receptors might be just as mobile as e xtrasynaptic receptors in that reco very of synaptic receptors could actually be occurring

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because of the import of other synaptic receptors as well as, or instead of, e xtrasynaptic receptors. This study by Tovar and Westbrook [52] emphasized how the synaptic complement of NMD A receptors are v ery much more dynamic than pre viously realized. This result raises the possibility that the trafficking and ta geting of functional forms of the NMD A receptor are realistic mechanisms for modulating synaptic strength. Clearly, a change in the number and type of synaptic NMD A receptor w ould alter the receptor mediated Ca 2+ concentration in spines, with important implications for the activity of kinases and phosphatases mediating changes in synaptic strength.

9.8 SYNAPTIC INHIBITION: THE MOBILITY OF EXTRASYNAPTIC GABAA RECEPTORS If the general or ganization of the inhibitory synapse is not dissimilar to that of the excitatory synapse, one might expect a similar degree of regulation of receptor traffic Molecular scaf fold proteins (such as geph yrin and other associated proteins lik e GABARAP, Raft, Collibistin and Plic1 [58,59]) are stabilized by the c ytoskeleton, an association that ensures the sub-synaptic localization of glycine and GAB AA receptors. Ho wever, much recent e vidence from confocal microscop y studies has revealed that inhibitory synapses are also subject to rapid structural modification [13]. In fact, the application of single particle and quantum dot tracking of metabotropic glutamate receptors, AMPA receptors, NMDA receptors and glycine receptors, within and outside the synapse, has established lateral mobility of these receptors as an increasingly important mechanism in the or ganization of the post-synaptic membrane [53,54,60,61]. We have recently studied the dynamics of functional GAB AA receptors at synaptic and extrasynaptic loci in hippocampal neurons, specifically to establish whethe these receptors are as mobile as other LGIC members. Unfortunately , unlike many of the other LGICs, the GABAA receptor does not have the benefit of an irr versible blocker of receptor function in its pharmacopoeia (such as MK-801 for the NMD A receptor or α-bungarotoxin for the nACh receptor). Nor does any evidence exist that the bioph ysical profile of a GA AA receptor can be suf ficiently changed by th import or export of specific receptor subtypes into or out of the synapse in respons to stimulation. Thus, functional studies of this nature ha ve been hampered by the lack of an inherent electroph ysiological tag. To overcome this condition, we engineered a silent mutation into the channel lining transmembrane domain of the α1 subunit of the receptor , at a location pre viously re vealed by systematic c ysteine scanning mutagenesis [62]. By mutating a h ydrophobic residue in the ion channel region to a c ysteine at the 2 ′ position (V257C), the application of the c ysteinemodifying reagent MTSES to mutant recombinantα1V257Cβ1γ2 receptors was largely ineffective in the absence of GAB A. However, after receptor acti vation by GABA, and presumably full exposure of the cysteine residue to MTSES, an irreversible 75% reduction in current amplitude was observed (Figure 9.4a) [62]. This difference in sensitivity to MTSES between open and closed GAB A channels and its predictable use-dependence has formed the basis for a functional tag of α1-containing GABAA

α1V257C-containing neurons

N275C R274C A273C S272C I271C S270C L269C T268C T267C M266C T265C L264C V263C T262C T261C V260C G259C F258C V257C T256C R255C A254C P253C V252C S251C E250C WT −75 −50 −25

Recovery 120

100

80

60 +MTSES 40

20

−75 −50 −25

0 25 50

Eﬀect (%)

Pre MTSES MTSES

0

0

25 50

WT

Eﬀect (%) (b)

FIGURE 9.4 Tracking synaptic GABAA receptor movements. (a) Application of 10-mM MTSES to Xenopus oocytes injected with the α1V257Cβ1γ2 subunits of the GABAA receptor show irreversible current blockade in the presence (left panel) b ut not absence (right panel) of GAB A. The residues indicated a re those of the α1 subunit TM2 region (extracellular at the top) that were individually and systematically mutated to a cysteine. Bars indicate normalized current responses to whole-cell GABA application. Def ections to the left indicate inhibition of the GAB A current. (b) Introduction of the same mutation ( α1V257C) into hippocampal neurones causes a reduction in the amplitude of mIPSC e vents. A control period of mIPSC acti vity (left panel, top trace) sho ws reduced amplitudes during the perfusion of MTSES (10 mM, middle trace). Approximately 10 min after the remo val of MTSES, mIPSC amplitudes sho w partial reco very to near control le vels (left panel, bottom trace). Scale bars represent 10 pA (vertical) and 2 sec (horizontal). Right panel: mean data (± S.E.M.) demonstrating mIPSC amplitude changes follo wing MTSES perfusion on wild-type hippocampal neurons (WT) and those transfe cted with the α1V257C subunit. Recovery of amplitudes, due to translocation of unblock ed receptor into the synapse, occurs 10 min after remo val of MTSES. P anel (a) is tak en with permission from [62].

© 2006 by Taylor & Francis Group, LLC

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(a)

α1V257C

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receptors in hippocampal neurons [63]. Transfection of neurons with the mutated subunit cDNA allowed the correct assembly and transport of the mutant sub unit to appropriate locations frequented by wild-type α1 sub units. When these mutant receptors are located at synaptic sites, thus becoming subject to spontaneouslyreleased GABA, they will also be susceptible to block by MTSES, whereas those extrasynaptic receptors not “seeing” released GABA would remain resistant. Under these conditions, the normal function of synaptic receptors (monitored as mIPSCs) is inhibited by up to 50% foll owing MTSES exposure (Figure 9.4b). The reaction of MTSES with the channel cysteine is covalent and thus irreversible; therefore, any recovery of receptor function will come from either direct insertion into the synapse of unblock ed receptor or from lateral dif fusion of e xtrasynaptic receptors. Both scenarios are possible because MTSES is not membrane permeant and the absence of GABA means that intracellular pools of receptor will be unaf fected by MTSES, and e xtrasynaptic receptors ha ve not been e xposed to the same concentrations of GABA as those at the synapse and thus remain unblock ed. We can largely discount tonic le vels of GAB A because the e xperiments were performed on lo w-density cultures and only mIPSC events were recorded, so spillover of neurotransmitter was kept to a minimum. With this e xperimental paradigm, we were able to observ e the very rapid recovery of blocked synaptic receptors within 10 to 20 min of the maximal MTSES ef fect (Figure 9.4b). The unblock ed receptors appeared to mo ve into the synapse, replacing blocked receptors by lateral diffusion because inhibitors of vesicular, exocytotic delivery (i.e., botulinum toxin B and N-ethylmaleimide) were unable to affect the basal deli very of GAB AA receptors to the membrane from inside the cell during the timescale of the recovery. Moreover, blockade of all surface receptors in the neurone (synaptic plus extrasynaptic) by MTSES resulted in no recovery over a longer timescale, suggesting that surf ace receptor mobility is k ey to the reco very process. Taken overall, these observations indicate a v ery slow turnover rate of GAB AA receptors by endo- and exocytotic processes, and would thus largely discount receptor deli very from within the cell as the reco very mechanism. The more lik ely explanation is that reco very of synaptic GAB AA receptors results from lateral diffusion of unblock ed receptors into the synapse, restoring the ef fica y of synaptic transmission. This represents the first study using an electrop ysiological tag to establish the dynamics of GAB AA receptors in neurons.

9.9 PHOTORECEPTIVE POTASSIUM CHANNELS AS SWITCHES OF NEURONAL EXCITABILITY The ion-channel pore belonging to f ast-acting, ligand-gated receptors has pro ved a useful locus for functional receptor tags. This approach could also be applied to voltage-gated ion channels. An inno vative v ariation on this theme has recently evolved for the Shak er K + channel, which, lik e other members of this f amily, is susceptible to re versible channel block by quaternary ammonium ions such as tetraethylammonium (TEA). This channel has been tagged by b uilding a no vel “chemical gate” that controls the activation/inactivation of the channel re gulated by

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δ light of particular w avelengths. The elegant generation of this so-called SP ARK (synthetic photoisomerizable azobenzene-re gulated K +, Figure 9.5a) channel involves the tethering of a synthetic maleimide-azobenzene deri vative to an introduced cysteine residue at a known location in the S5 and S6 pore unit of the Shak er potassium channel [64,65]. This cysteine is sufficiently close to the ion channel tha TEA, attached to one end of the tether , can bind to its site in the ion channel and block ion fl w. In the case of the photoisomerizable azobenzene group, the w avelength of light determines whether the azo moiety adopts a cis or trans conformation consequently shortening or lengthening the tether and so either removing or allowing block of the ion channel by TEA (Figure 9.5b). Exposure to UV light (380 nm) induces the cis conformation that shortens the link er and effectively pulls the TEA moiety at its extremity away from the channel δ-preventing pore block (Figure 9.5b), whereas visible light (460 to 500 nm) allo ws revision of the molecule to the ther modynamically δ-favored trans conformation, which is longer by 7Å, re-establishing channel block. The tethered lig and thus acts as a light-acti vated chemical g ate. Switching between these tw o states of block and unblock is achie ved very rapidly. The protein modification required to permit attachment of the synthetic molecul introduces a cysteine into the protein to which a maleimide molecule at one end of the tether attaches in an irre versible manner. This mutation is functionally silent. Once attached, under visible light the tether passively blocks the K+ channel, ablating almost 1 nA of current in ooc ytes (Figure 9.5c). Following appropriate mutation of the SPARK channel to increase the neuronal resting conductance for K+ (by eliminating rapid inactivation) and subsequent expression in hippocampal neurons, spontaneous action potential acti vity w as mark edly reduced. The activation of the SP ARK channels via the tethered TEA resulted in a general increase in neuronal acti vity within seconds of e xposure to UV light (due to channel block, Figure 9.5d). Clearly , this technique could ha ve a number of applications, not least in the programmable silencing of other functional ion channels or LGICs, whose membrane dynamics could subsequently be track ed. The convenient nature of the channel block during episodes of normal light (i.e., due to the favored trans configuration) ould allow functional receptors to be e xposed selectively under UV light.

9.10 DISADVANTAGES AND LIMITATIONS OF THE FUNCTIONAL TAG Many of the limitations of the functional tag assay are also applicable to biochemical or optical methods, though any modification of a protein that might a fect its normal function (i.e., current flux) should be thoroughly assessed in the isolation of recombinant system prior to its use in a nati ve environment. In particular the tag must be functionally silent until acti vated or bound by a lig and. Thus, one needs to check ion-channel g ating in response to agonist, whether an y alteration to agonist potency is found, or if unusual rectification properties are present after recepto activation. In addition, and ag ain common to optical and biochemical tracking methods, ensuring that the use of a functional tag does not introduce an epitope that

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MAL-AZO-QA

O NH

N

O NH

N O

N O HN

500nm

O

O

~10 Å

NH

N

N

~17 Å

N

O

380nm N N

O

171

(a) NH S

N H

Out

+

+ 380nm 380nm

K+

500nm 500nm

In (b)

Current (pA)

1000 Vis UV

800 600 400

20 mV

200

500 390 nm nm

0 0

50

10s

100 150 200 250 300

Time (s)

(c)

(d)

FIGURE 9.5 Engineering membrane channel inactiv ation with light. (a) Synthetic maleimide-

azobenzene-TEA (MAL-AZO-QA) tether used in the modif cation of the Shaker K + channel. The maliemide moiety attaches to a c ysteine residue; the azobenzene moiety changes the length of the tether through the transformation of a cis-trans bond in response to light; and the quaternary ammonium group blocks the functional K + channel. (b) Introduction of this tether into the K + channel generates a SPARK channel (see text) whose function can be switched “on” or “of f” in the presence or absence of UV light, respectiv ely, by means of the contracted or e xtended tether releasing or blocking the ion channel. (c) Excised inside-out patch from Xenopus oocytes injected with SP ARK reveal the almost complete ablation and subsequent restoration of a 1 nA current follo wing removal of, or e xposure to, UV light, respectiv ely. (d) Introduction of multiple mutations to the SP ARK channel caused silencing of spontaneous neuronal acti vity, which was re versed upon treatment with MAL-AZO-QA and UV light. All panels are provided with permission from [65].

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could inadvertently trigger endocytosis or affect interactions with accessory proteins is necessary, for example, by disrupting a receptor’s phosphorylation state, anchoring or trafficking. All these aspects can be check ed in control e xperiments. F or these reasons, if a receptor has an inherent functional tag, then this should be e xploited in preference to engineered tags by mutagenesis. Clearly , if an epitope must be introduced, the af fects of e xpressing a protein in a neurone must then also be considered. Many transfection protocols, especially those using viral v ectors, have become very efficient ven in primary cell cultures. The consequence of this ef fi ciency is that the cell could be overwhelmed with exogenous DNA products, causing the basal equilibrium of protein manuf acture to be upset. Under these conditions, certain LGICs might be inappropriately tar geted or traf fic ed, or others might be under-represented in the cell membrane. All these considerations must be taken into account when interpreting the data.

9.11 CONCLUSION Functional tagging of receptor proteins of fers a unique method of tracking receptor movement. It has one overriding advantage in that, unequivocally, one is measuring the movement of functional surf ace receptors by virtue of the f act that the y can be activated and pass current. The method does not need to rely on co-localization with other proteins to assume importance at synapses, nor is inferring that the y are functional receptors necessary. In the future, use of more inno vative silent tags will allow the resolution of receptor translocation in the surf ace membrane to become even more accurate, and with more specific inhibitors of xo- and endoc ytotic processes, we will g ain a better understanding of the life-c ycle of important neurotransmitter receptors in the central nerv ous system.

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RNAi and Applications in Neurobiology Tija C. Jacob

CONTENTS 10.1 Introduction to RNAi ...................................................................................178 10.1.1 miRNAs............................................................................................178 10.1.2 Chapter Overview ............................................................................179 10.2 RNAi Mechanism.........................................................................................180 10.2.1 Early Days—Historical Perspective ................................................180 10.2.2 dsRNA Cleavage by Dicer ...............................................................181 10.2.3 RISC Complex Assembly and Activity ...........................................181 10.2.4 Organism-Specific Variations in the RN Ai Mechanism ..................182 10.3 Generating RNAi — Types of RNAi ..........................................................183 10.3.1 siRNAs .............................................................................................183 10.3.2 Hairpin dsRNAs and shRNAs .........................................................183 10.3.3 Inducible Systems ............................................................................184 10.4 RNAi Delivery — Specific Methods for Empl ying RNAi in Various Experimental Systems ....................................................................185 10.4.1 C. elegans .........................................................................................185 10.4.2 D. melanogaster ...............................................................................186 10.4.3 Mammalian Systems ........................................................................186 10.4.3.1 Mammalian Cell Culture and Primary Culture ................186 10.4.3.2 Oocytes, Pre- and Post-Implantation Embryos, and Post-Natal Animals ....................................................188 10.4.3.3 Adult Mice ........................................................................189 10.4.3.4 Stable Inheritable Genetic Knockdo wn — Transgenic Mice and Rats ................................................190 10.5 RNAi Experimental Design Strate gy...........................................................191 10.5.1 siRNA/shRNA Design .....................................................................191 10.5.2 Specificity Concerns: O f-Target Effects and the Interferon Response .........................................................................192 10.5.3 Controls ............................................................................................193 10.6 Conclusion....................................................................................................193 References..............................................................................................................194

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10.1 INTRODUCTION TO RNAi RNA interference (RN Ai) describes a conserv ed biological response to doublestranded RNA (dsRNA) resulting in the degradation of homologous messenger RNA. In the last fe w years, this process of sequence-specific, post-transcriptional gen silencing has become a key technique for rapidly assessing gene function in species ranging from plants to mammals. Fire et al. pro vided the first insight into the R Ai mechanism by identifying dsRNA as the trigger of RNAi in Caenorhabditis elegans in 1998 [1]. However, a similar gene-silencing phenomenon w as reported in earlier studies in both plants and Neurospora [2,3]. The basic RN Ai response starts with long dsRNA being processed into small interfering RN As (siRNAs) by a ribonuclease (RNase) III enzyme, Dicer . Next, the siRN A is incorporated into the RN Ainduced silencing complex (RISC). For target RNA recognition to occur, the siRNA duplex must be unwound, allowing binding of one siRNA strand to the target mRNA. This is follo wed by RISC clea vage of the homologous mRN A. Recent w ork has shown that the RN Ai machinery is also in volved in anti viral responses, transposon silencing, development and heterochromatin formation [4].

10.1.1

MIRNAS

The involvement of RNAi components in basic cellular processes is partly explained by the discovery of endogenously encoded small RN A molecules, known as microRNAs (miRNAs), that are encoded in the genomes of humans, mice, w orms, fruit flies and plants. Initially transcribed as long R A, miRNAs are processed in the nucleus by the RNase III enzyme Drosha into a pre-miRNA of about 70 nucleotides (nt) [5,6]. The pre-miRNA, which forms an imperfect hairpin structure, is bound by exportin-5 and e xported into the c ytoplasm [7,8] where Dicer further processes it into a mature, single-stranded miRN A [6,9,10]. miRN As from animals appear to regulate gene expression by binding to partially complementary sequences in the 3 ′ untranslated regions (UTRs) of tar get mRNAs and suppressing translation [11]. In plants, however, miRNAs primarily direct tar get mRNA cleavage. The key to this difference appears to lie in the le vel of homology, as outside of the plant kingdom, no miRNAs have been identified that sh w full identity to any mRNA. Furthermore, animal miRNAs are capable of directing mRN A cleavage if supplied with a fully complementary mRNA [12,13]. Similarly, siRNAs are able to suppress translation: Doench et al. sho wed that a siRN A could suppress translation of a mRN A where the 3 ′ UTR had been engineered to contain multiple binding sites (all containing a central mismatch to stop RISC-dependent mRN A clea vage) [14]. Ho wever, the requirement for se veral binding sites, with apparent cooperati ve activity, decreases the lik elihood of siRN As producing miRN A-like ef fects on mRN As with partial homology. The number of cloned miRN As is growing rapidly, with man y showing cell and developmental specific xpression patterns. Of particular interest to neurobiologists is the identification of numerous neuronal specific mi As isolated from both mammalian embryonic neurons and the adult murine brain [15–18]. miRN As identified from rat cortical cultures all co-fractionated with polyribosomes, impli cating miRNA regulation of translation in man y aspects of neuronal function [18].

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10.1.2 CHAPTER OVERVIEW This chapter be gins with an o verview on the RN Ai pathway [19–21]. To provide a brief historical perspective on some of the initial steps of an e xponentially growing research area, a fe w key early e xperiments into the mechanism of RN Ai are listed in the first paragraph. This is follo wed by a thorough description of the RN Ai mechanism including Dicer processing and RISC comple x assembly and acti vity. Next, specific methods to generate (Figure 10.1) and del ver RNAi in various experimental systems are detailed ( Figure 10.2). In the section c overing RNAi delivery, applications in the field of neurobiology are g ven, with a focus on murine e xperimental systems. Finally , general RN Ai e xperiment design strate gy is discussed, including siRNA/shRNA design, RNAi specificity concerns and important controls A

B

C

dsRNA

pre-miRNA

shRNA

Cleavage by Dicer

siRNA or miRNA

D

5′-p 3′-OH

OH-3′ p-5′

RISC assembly

miRNP assembly Incomplete pairing

Exact pairing 3′-OH 7 mG Target mRNA cleavage

p-5′ AAAA

3′-OH 7 mG

p-5′ AAAA

Translational repression

FIGURE 10.1 Sources for RN A-induced gene silencing include (a) dsRN As, (b) miRN As, (c) shRNAs and (d) chemically synthesized siRN As. Dicer processes dsRN As, pre-miRNAs and shRNAs into siRNA/miRNA duplexes containing 5 ′ monophosphate groups and 2-nt 3 ′ overhangs. Next, these duple xes are unw ound and incorporated into the appropriate ef fector complex (RISC/miRNP). The degree of pairing between the siRN A/miRNA and the tar get mRNA appears to determine whether silencing occurs via mRNA degradation or translational repression. 7 mG, 7-meth yl guanine; AAAA, poly-adenosine tail.

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RNAi delivery method

RNAi source

C. elegans

injection into adults bacterial feeding of dsRNA soaking worms transgenic worm strains expressing hairpin dsRNA

dsRNA

D. melanogaster

injection into embryos and adults soaking cells cells plated on dsRNA microarray transgenic flies expressing hairpin dsRNA

dsRNA

Mammalian

transfection transduction by infection nucleofection soaking/addition to media cells plated over RNAi microarray

siRNA, shRNA viral shRNA shRNA V-siRNA siRNA, shRNA

oocytes & preimplantation embryos

dsRNA microinjection, electroporation, and in vivo electroporation

dsRNA

later embryonic stages

maternal delivery via tail vein injection, in vivo electroporation, and in utero electroporation

siRNA, shRNA

in utero electroporation, in vivo electroporation, and injection transduction by viral injection “systemic delivery’’ by intravenous injection

siRNA, shRNA

postnatal & adult stages

viral shRNA chol-siRNA

microinjection into pronuclear stage fertilized eggs

shRNA

ES cell electroporation followed by chimera formation or tetraploid aggreagation lentiviral delivery to ES cells or 1 cell embryos

shRNA

cell culture & primary culture

Mammalian (murine)

transgenic methods

viral shRNA

FIGURE 10.2 A description of RN Ai approaches including model system, RN Ai delivery method and RN Ai source. RN Ai sources include dsRN A (double stranded RN A), siRN A (small interfering RN A), shRNA (short hairpin RN A), V-siRNA (siRNA conjug ated to the vector peptide Penetratin I) and chol-siRN A (siRNA conjugated to cholesterol).

10.2 RNAi MECHANISM 10.2.1 EARLY DAYS—HISTORICAL PERSPECTIVE Research in se veral model systems using dif ferent e xperimental approaches has contributed to our current understanding of RN Ai [21]. In 1999, analysis of posttranscriptional gene silencing (PTGS) in plants led to the detection of 21 to 25 nt antisense RNA complementary to the tar get mRNA, indicating a means to generate specificity for silencing [22]. N xt, in vitro experiments in Drosophila melanogaster embryo e xtracts re vealed that long dsRN A w as clea ved into 21 to 23 nt siRN As [23], and these endogenous siRNAs or chemically synthesized siRNAs [24] mediated target mRNA cleavage. Furthermore, clea vage of the tar get mRNA occurred only within a re gion of identity to the siRN A trigger. At nearly the same time, in vivo cleavage of dsRN As into siRN As was shown in w orms [25] and fly embryos [26] Using a biochemical approach in Drosophila S2 cells, Hammond et al. identified sequence-specific nuclease that co-fractionated with short R As of approximately

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25 nt [27]. Subsequent efforts in the Hannon group led to the cloning of the enzyme Dicer, a member of the RNase III f amily [28].

10.2.2

DSRNA

CLEAVAGE

BY

DICER

Dicer processes dsRNAs into 22 nt siRN As that contain 5 ′ monophosphate groups and two nucleotide 3′ overhangs [23,24,28]. The general structure of RNase III-type endonuclease Dicers includes several distinct domains: an N-terminal DExH/D box RNA helicase domain, a domain of unkno wn function (DUF283), a P AZ domain, two catalytic endonuclease domains (RIII) and a dsRN A-binding domain (dsRBD). For an in depth re view of the biochemical and structural features of RNase III enzymes in gene silencing and models for Dicer clea vage, see Carmell and Hannon [29]. Humans and C. elegans have one Dicer , which is responsible for processing both siRNA and miRNA precursors. Drosophila, on the other hand, has tw o Dicers with DCR-2 being the major siRNA-producing enzyme for RNAi and DCR-1 functioning in miRNA-induced gene silencing [30]. Ho wever, there appears to be some functional overlap between DCR-1 and DCR-2. Endogenous and recombinant DCR2 processing of dsRNAs into siRNAs occurs in an ATP-dependent manner [30–32]. In contrast, dsRN A cleavage by human recombinant Dicer sho ws no ATP requirement [33,34].

10.2.3 RISC COMPLEX ASSEMBLY

AND

ACTIVITY

Once cleavage by Dicer has occurred the mature siRN A or miRNA is released and integrated into the appropriate ef fector comple x, RISC (for siRN As) or miRNP (miRNA containing ef fector complex). RISC and miRNPs ha ve been identified o varying sizes and components but the common features include the siRNA/miRNA, the complementary target mRNA and a member of the Argonaut (Ago) family. Ago family proteins contain two major domains, PAZ and PIWI. Biochemical and crystal structure studies indicate that the P AZ (piwi-ar gonaute-zwille) domain, also conserved in Dicer, is an RNA binding domain (RBD) that can bind to the end of siRNA and miRNA duplexes. Recently, a crystal structure for the P AZ domain of human argonaute eIF2c1 was solved in complex with a 9-mer siRN A-like duplex, with the 2 nt 3 ′ overhang of the duple x held in a conserv ed binding pocket [35]. This result led to the proposal that the Dicer P AZ domain could be used to hold a siRN A and, following clea vage, could pass of f the other siRN A end to the P AZ domain in Argonaute, allowing direct transfer of the siRNA into RISC. The PIWI domain was first proposed to participate in cle vage of target RNA, as structural studies indicated high similarity between the archaebacterial Ago protein and the RNase H f amily that cleaves the RNA strand of DNA/RNA duplexes [36]. As mutation of the homologous cryptic RNase H domain in the mammalian Argonaute 2 (Ago2) inacti vates RISC, and only Ago2-containing RISC is capable of mRNA cleavage, Ago2 appears to provide the “slicing activity” for RISC [37]. The number of Ago family members varies between or ganisms: C. ele gans has o ver 20, Drosophila has fi e and both mouse and humans have eight [38]. These numbers suggest thatAgo proteins provide specificity to RISC/miRN , although most ha ve yet to be characterized.

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Other proteins contrib ute to formation of RISC/miRNP comple xes and mRN A cleavage/translational suppression. For target mRNA recognition to occur, the siRNA duplex must be unwound. Assembly of active RISC has an ATP energy requirement [31,39], lik ely due to the step of unwinding a siRN A or miRN A duple x or other configurational changes in the compl x. Proteins proposed to participate in thisATPdependent step include the DEAD-box RNA helicases. One such candidate inDrosophila is the putative RNA helicase Armitage, as mutants are defective in later RNAi events including forming an active RISC complex [40]. Once formed, RISC cleavage of target RNA shows no ATP requirement [27,31]. As well as the core components of RISC/miRNP complexes, several other proteins have been identified from di ferent model systems. Additional proteins found in Drosophila RISC include Vasa intronic gene product (VIG), the Drosophila homolog of fragile X mental retardation protein (dFXR) and the putative endonuclease Tudor-SN (TSN-1) [41,42]. Using C. elegans extracts and mammalian cells, a complex containing TSN-1, VIG homologs, siRNAs and Argonaute was identified [42]. Drosophila miRNPs have been identifie associated with se veral factors: AGO2, dFXR, the DEAD-box helicase RM62 and ribosomal proteins RPL5 and RPL11 [43]. Biochemical experiments detected human Ago2/eIF2C2 interactions with fragile X mental retardation protein [44]. A RISC/miRNP-like comple x w as purified from HeLa cells that contained huma Ago2/eIF2C2, gemin3 (a DEAD-box helicase), gemin4 (a protein lacking identifie motifs) and miRN As [45]. At present, the role of these v arious proteins in RN Amediated gene silencing is not known. In a recent review, Meister and Tuschl provide a comprehensive list of proteins involved in RNAi or transcriptional gene silencing, complete with domains, functions and references [19].

10.2.4 ORGANISM-SPECIFIC VARIATIONS MECHANISM

IN THE

RNAI

Not surprisingly, differences are found in Dicer clea vage and RISC assembly when comparing model systems. As discussed earlier , Drosophila DCR-2 functions primarily in siRN A processing, whereas DCR-1 processes miRN As. DCR-2 lacks a PAZ domain, so it must first form an initiation compl x with the dsRBD containing R2D2 protein to be able to bind a siRN A and incorporate it into RISC [32]. DCR2 also appears to function do wnstream of long dsRN A clea vage as null mutants cannot be rescued by siRN A injections into Drosophila (which bypass the Dicer cleavage step), and in vitro experiments show that DCR-2 is required for all steps of RISC comple x assembly [39]. This requirement for Dicer in RISC assembly is consistent with data from mammalian cell culture [46]. Although the C. ele gans Dicer contains a PAZ domain, it also interacts with the dsRBD protein RDE-4 [47] and this interaction is required for production of siRN As but not subsequent RN Ai steps [48]. RDE-4 also interacts with the P AZ-domain-containing protein RDE-1 and a conserved DExH box RNA helicase [47]. RDE-4 and RDE-1 are proposed to function together in detection and presentation of dsRN As to Dicer. Regulation of RNA silencing is another area where model systems dif fer. In C. elegans [1], systemic silencing occurs where a small amount of dsRNA is amplifie and spreads throughout the or ganism. Systemic silencing, which also tak es place in

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plants, requires RN A-dependent RNA polymerases (RdRPs). RdRPs are proposed to amplify RNAi effects by replication of trigger dsRNAs, as well as siRNA-primed replication of the tar get mRNA or its clea vage products. Interestingly , RdRP-lik e proteins are not found in the Drosophila or vertebrate genomes, and considerable evidence indicates that an y amplification of R A silencing is not lik ely to occur here [49–51].

10.3 GENERATING RNAi — TYPES OF RNAi 10.3.1

SIRNAS

Long dsRNA, which is processed into siRN As by Dicer , was historically the most commonly used method for RNAi in worms, flies and plants. In mammals, h wever, dsRNA longer than 30 nt induces the interferon anti-viral response, resulting in the sequence-nonspecific shutd wn of gene e xpression. Interferon production acti vates two enzymes, the dsRNA-dependent protein kinase (PKR) and 2 ′-5′-oligoadenylate synthetase (O AS1). O AS1 induction leads to acti vation of RNase L and mRN A degradation, whereas PKR phosphorylation of the translation initiation f actor eIF2 results in the global inhibition of mRN A translation [52]. F ortunately, this nonspecific response can generally be voided by the use of siRN As shorter than 30 nt [53]. Chemically synthesized 20 to 23 nt siRNA duplexes that are structurally similar to endogenous Dicer-processed dsRNAs and contain 2 nt 3 ′ overhangs are efficien mediators of RN Ai [54]. The number of companies pro viding custom synthesized siRNAs has grown dramatically in the last fe w years; a fe w of the main companies and specific production details are listed in a siR A methods paper by Elbashir et al. [55] and on the Tuschl lab website [56]. An alternative method is to use recombinant Dicer to process in vitr o transcribed long dsRN A into siRN As [57,58]. Although this does pro vide greater co verage of the tar get mRN A, this approach cannot be as rigorously controlled and chances of off-target effects, such as miRNAlike responses, are higher [58]. Of f-target effects and the interferon response will be discussed at more length in the later section on RN Ai experimental design.

10.3.2 HAIRPIN

DSRNAS AND SHRNAS

Due to the transient nature of siRN A-mediated RNAi, researchers constructed v arious DNA vectors encoding a long in verted repeat that w ould form a hairpin RN A when transcribed in vivo. Dicer subsequently cleaves the hairpin RNA into siRNAs. These long hairpin RNAs were successfully used in model systems with little to no interferon response, such as mouse ooc ytes and preimplantation embryos [59], nematodes [60] and fruit flies [61]. or mammalian studies that required short hairpin RNAs (shRN As), DN A v ector systems were de veloped that primarily emplo y an RNA polymerase III (pol III) H1 or U6 promoter [62–67]. One dif ference between these pol III promoters is that the U6 promoter requires a guanosine at the +1 position, whereas the H1 promoter does not. Another key feature needed to generate a shRNA are four to fi e thymidines at the 3 ′ end that encode a pol III transcription termination sequence. The general fold-back stem-loop structure of a shRN A is

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encoded as follo ws, starting from the 5 ′ end: an approximately 19 to 29 nt-long sense strand is follo wed by a short 4 to 9 nt loop, then the antisense strand and, finall , the termination sequence. Researchers ha ve de veloped shRN A e xpression systems with man y different stem and loop lengths and compositions. Conflictin reports are also found on the effects of sense and antisense orientation in the hairpin, where one group sho wed strand re versal resulted in diminished RN Ai [65] while the other reported no change [66]. Recent data indicate that 5 ′-end instability is important for incorporation into RISC, suggesting that using the orientation of sense strand followed by antisense strand might be preferable. Another shRNA variation has been to introduce stem mismatches, resulting in a miRNA-like imperfect hairpin duplex [68]. Other less commonly used v ector-based RNAi systems, including the use of tandem pol III promoters to separately dri ve individual siRNA strands [69] and pol II promoters, are discussed in a recent review by Dykxhoorn et al. [20]. The use of pol II promoters has increased recently , as these promoters result in high levels of functional siRNAs in a variety of cell types [70,71] and offer other features, including tissue-specific promoters and inducible transcription

10.3.3 INDUCIBLE SYSTEMS RNAi has g ained additional v ersatility through the introduction of inducible forms of shRNA v ectors. These v ectors are particularly useful where RN Ai phenotypes involve cell gro wth and dif ferentiation or apoptosis pathw ays. Tetracycline- (Tet-) or ecdysone-inducible systems are most commonly used in mammalian cells. Initial efforts involved a U6 pol III-based v ector combined with sequences from the tetracycline operator (TetO) [72]. Use of this inducible system requires cells e xpressing the tetracycline repressor (TetR) protein. Transcription of the shRN A is suppressed by TetR binding to a TetO sequence, and this suppression is remo ved when tetracycline or doxyc ycline is added to the media. This inducible system has also been combined with H1 promoter -driven shRNAs [73]. One apparent problem with Tetinducible systems is “leakiness” or lo w levels of basal transcription in the absence of tetrac ycline in some cell types. Modifications of Tet-inducible pol III-based shRNAs are in development to extend inducible system functionality [74]. Ecdysone systems emplo y the insect hormone ecdysone and tw o receptors, the modifie ecdysone receptor (VpECR), retinoid X receptor (RXR) and an ecdysone response element (ECRE). In the presence of ecdysone, the receptors dimerize and bind to the ECRE, acti vating transcription. Although this system is more elaborate, it generally has tighter regulation and as a lipophilic steroid, ecdysone has greater potential for in vivo applications due to less toxicity and ease of tissue infiltration an metabolism. Gupta et al. designed an ecdysone-inducible system that coordinates with a GAL-4 system to drive a U6 promoted shRNA in mammalian cells [75]. This system results in highly ef fective p53 suppression in a dose- and time-dependent manner. Furthermore, it is also re versible, such that normal cell phenotypes and protein levels return after inducer withdra wal. Additional vector systems are being developed that rely on other induction mechanisms [76]. Recently , Chang et al. developed SIRIUS-CRE, an inheritable and inducible RN Ai system that combines

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the Cre-loxP and Tet-on techniques allowing for inducible tissue-specific xpression of shRNAs in mice [77].

10.4 RNAi DELIVERY — SPECIFIC METHODS FOR EMPLOYING RNAi IN VARIOUS EXPERIMENTAL SYSTEMS Depending on what model system one is emplo ying, the particular area of research, developmental window of interest and experimental time constraints, various RNAi approaches can be used. A thorough description of these methods, including type of RNAi trigger and appropriate mode of delivery, is given in the following section, organized by model system. As the w orm and fly protocols are well-established i the RNAi field, a concise paragraph of methodologies is listed here.This is followed by a more in-depth discussion of RN Ai approaches applied to mammalian model systems, particularly the laboratory mammals used for most neurobiological studies, rats and mice. The segment on the mammalian model systems includes cell culture, primary culture, early de velopmental stages from ooc yte to early postnatal, adult rodents and transgenic animals. By presenting a fe w recently published e xamples of RNAi in the field of neurobiolog , one can more easily choose appropriate RNAi techniques for today’s and tomorrow’s experiments. Moreover, the juxtaposition of different RNAi tools and methods might inspire further no vel RNAi applications in neurobiology.

10.4.1 C.

ELEGANS

dsRNA can be delivered directly to C. elegans by microinjection into the body cavity [1], feeding of bacteria e xpressing dsRN A [78] or soaking of w orms in dsRN A solution [79]. In addition, transgenic worm strains can be made that e xpress hairpin dsRNA [60]. The C. elegans scientific community d velops and supports a wealth of online resources [80] including a database of RN Ai phenotypes [81]. These functional genomics tools, combined with the ease and speed of standard w orm genetic approaches, rapidly adv anced RN Ai applications leading to lar ge-scale projects such as genome-wide RN Ai feeding screens [82]. The library of bacterial strains used in this screen, co vering 86% of the predicted w orm genes, w as made publicly available along with the necessary protocols to conduct other RNAi screens [83]. C. elegans is also a key model system for studying neural and neurodegenerative disorders. One challenge has been the relative resistance of the worm nervous system to RN Ai, complicating neuronal studies [84]. This hurdle w as initially o vercome when transgenic w orms carrying an inducible heat shock promoter dri ven hairpin dsRNA displayed in vivo RNAi susceptibility in some neurons [60]. The identification of t o mutants that are h ypersensitive to RNAi, rrf-3 [85] and eri-1 [86], now allows for greater application of RN Ai to study the nematode nerv ous system [87,88]. These strains are particularly useful in RN Ai screens for v arious neuronal phenotypes. A recent RN Ai screen for re gulators of polyglutamine aggre gation identified 186 genes that led to the premature accumulation of protein aggr gates

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when suppressed [89]. A recent re view by Buckingham et al. lists other e xamples of RNAi in w orm models of neuronal diseases [90].

10.4.2 D.

MELANOGASTER

RNAi in D. melanogaster was first sh wn by injection of dsRNA into embryos [91]. This method of RN Ai delivery is some what limited as RN Ai in fly embryos onl lasts for se veral days. Studies of gene function at later stages of de velopment and for longer time periods are possible with flies carrying a stably int grated hairpin dsRNA transgene that provides heritable genetic knockdown [61]. To improve RNAi effectiveness, particularly in neuronal tissue, genomic cDN A cassettes carrying intronic spacers were made that encoded hairpin dsRN As [92]. Adult fly R Ai can also be achieved by dsRNA injection into the abdomen and results in RNAi throughout the body and central nerv ous system (CNS) [93,94]. In addition, man y RNAi studies are no w done using Drosophila cell culture [95], where RN Ai is easily achieved by addition of dsRN A to the culture media [96]. The next advancement was the application of RNAi to genetic screening in Drosophila cell culture: a highthroughput RNAi screening method was developed with a 384-well plate assay and used to identify genes involved in cell morphogenesis [97]. Recently, this screening technology w as scaled up for a genome-wide RN Ai screen and a dsRN A library was generated to enable screening of 91% of the predicted Drosophila genes for a role in cell growth and viability [98]. To further facilitate the use of large-scale RNAi screens, Harv ard Medical School, in collaboration with P aro’s group (EMBL, Heidelberg, German y) established a Drosophila RNAi screening center as a fl community resource where researchers can perform approved high-throughput RNAi screens [99]. Another significant ad ancement is the RN Ai li ving-cell microarray screening method developed in the Sabatini laboratory, with publicly available methods and protocols [100]. This method utilizes dsRN As printed on glass microarray slides with Drosophila cells cultured directly on top of the arrays. RNAi phenotypes are determined by e xamining the appearance of groups of cells attached o ver a particular dsRNA spot. The microarray RN Ai technology allo ws screening of the complete fly genome on three standard glass slides and can also be used to stud genetic interactions, as tw o genes can be suppressed at once [101].

10.4.3 MAMMALIAN SYSTEMS 10.4.3.1 Mammalian Cell Culture and Primary Culture RNAi experiments in mammalian cells were initiated in 2001 when Elbashir et al. reported successful RN Ai with 21 nt siRN A duple xes transfected using cationic liposomes [53]. In the ensuing years, researchers ha ve used various different RNAi approaches and deli very methods to achie ve RNAi in mammalian cell culture systems, primary cells and whole or ganisms. siRNA experiments are limited to shortterm studies (24 to 72 hours after siRN A deli very) and principally use standard transfection techniques to carry siRN As into cells. siRN As can be transfected in mammalian neurons via lipid reagents [102,103].

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Although transfection works well for most cell culture systems, lower efficien y in primary cultures has generally limited researchers to single-cell studies, hamper ing other e xperiments that require a lar ge transfected population. P articularly in cultured neurons, researchers have tried alternative methods to improve transfection efficien y and a void lipid mediated c ytotoxity with v ariable success. Soaking cultured neurons in “nak ed” siRNA resulted in uptak e into endosomes, no observ able RNAi and a decrease in general metabolic acti vity [104]. F ortunately, the use of chemically modified siR As appears more promising and could also allo w earlier observation of RN Ai effects by eliminating the need to w ait for neuronal reco very post-transfection. Recently, Davidson et al. conjugated siRNAs to the vector peptide Penetratin I (Pen1) and observ ed highly ef ficient del very (99% after a 2-hour incubation) with lo w toxicity and rapid siRN A-mediated protein knockdo wn in hippocampal cultures. Upon entering the reducing en virons of the c ytoplasm, the disulfide bond linking the siR A and Pen1 is clea ved, releasing the siRN A [105]. In comparison to transfections with lipid reagents, these Pen1-modified siR As (VsiRNAs) resulted in improved neuronal survival: the survival rate was 99% in control untreated neurons, 92% with V-siRNA treatment and 58% with Lipofectamine 2000 siRNA transfection. Interestingly, protein knockdown was observed within 6 hours of treatment b ut the initial decrease in protein le vels w as not accompanied by a commensurate drop in mRN A le vels, suggesting a miRN A-like translational suppression mechanism. Another modification that might be applicable to primar neuronal culture is a chemically stabilized siRN A with cholesterol, discussed later with regard to systemic deli very in adult mice [106]. Biotech companies ha ve also developed specific siR A transfection reagents, some of which ha ve been successfully used in primary cortical neurons, cerebellar neurons, dorsal root g anglion neurons and Schw ann cells [107–109]. For long-term studies, se veral groups de veloped shRN A v ectors with RN A polymerase III H1 or U6 promoters to pro vide more persistent RNAi in cell culture systems [62–67]. shRNA vectors can be delivered into cells by standard transfection means including lipid reagents, calcium phosphate and electroporation. These methods can also be applied to primary neuronal culture; for example, cerebellar granule cultures from P6 animals ha ve been successfully transfected with shRN As by a modified calcium phosphate method [110,111]. More ver, in the case of shRN A vectors, the issues of limited transfection ef ficien y and lipid toxicity can be o vercome by nucleofection, a modified electroporation technology (Amaxa, Gaithers burg, MD), which is used both in standard cell culture systems and primary cultures. By increasing the percentage of transfected neurons, this procedure opens the door to biochemical e xperiments, cell surf ace biotin ylation assays, analysis of mRN A levels and studies on neuronal netw orks. F or e xample, primary cortical neurons nucleofected with shRN A vectors showed protein knockdo wn by Western analysis and immunofluorescence at 2 to 3 DIV [112]. Nucleofection of plasmids in ra hippocampal cultures results in transfection efficiencies of approximately 40 to 50 [113]. shRNA vectors are nucleofected into rat primary hippocampal cultures with similar efficien y, allowing long-term RNAi up to 21 DIV (Jacob TC, unpublished results). One limitation of this method of shRNA delivery is that nucleofection must be done at plating. Finally, with the goal of RNAi-based therapeutics, shRNA vectors

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have also been incorporated into retroviral vectors and show stable reduction of gene expression in mammalian cell lines [114–117] and primary cells [118–121]. Although more labor-intensive than transfection or nucleofection, viral vectors could be used as another method for deli vering shRN As to primary neuronal culture. Finally, great potential for RN Ai-based screens is also e xpected as high-throughput approaches based on methods de veloped in the Sabatini laboratory [122] ha ve recently been applied to mammalian cell culture, using microarrays of siRN As or shRNAs spotted with a lipid transfection agent [123]. 10.4.3.2 Oocytes, Pre- and Post-Implantation Embryos, and Post-Natal Animals The first xamples of RNAi in mammals were in mouse oocytes and pre-implantation embryos, using longer dsRNA molecules of several hundred base pairs [59,124,125]. Longer dsRN As do not appear to initiate the interferon response in these early developmental stages. For these studies, dsRNAs were microinjected, although electroporation appears to be a new optimal method for dsRNA delivery [126]. To study later embryonic de velopment, ho wever, siRN As and shRN As are used to a void nonspecific suppression of translation initiated by the interferon response. A more complex technique w as required for deli very of endoribonuclease-prepared siRN A (esiRNA) to defined r gions of the de veloping CNS of embryonic day 10 (E10) mouse embryos; this involved exo utero surgery combined with simultaneous injection into the neural tube and oriented electroporation [127]. In this paper , after 1 day in whole embryo culture, a β-galoctosidase ( β-gal) esiRN A w as sho wn to suppress a coelectroporated β-gal reporter plasmid without affecting a co-electroporated eGFP reporter plasmid expression in neuroepithelial cells. Moreover, an eGFP esiRNA was able to suppress endogenously e xpressed eGFP (in a knock-in mouse line expressing eGFP in neuroepithelial cells). As whole embryo mouse cultures are made during E7 to E12 and can be cultured for only approximately 2 days [127], this somewhat limits the application of in vivo electroporation for post-implantation embryonic studies. However, longer-term studies and greater fl xibility is possible through the application of other techniques, such as maternal deli very via tail v ein injection [128] or in utero electroporation [129,130]. After a v ector encoding both a shRN A for bone morphogenetic protein 4 (Bmp-4) and a dsRed reporter w as injected into the tail v ein of an E6.5 pre gnant dam, embryos showed DsRed expression 24 hours later and Bmp-4 loss of function phenotypes such as defects in neural fold closure and cardiac morphogenesis appeared subsequently [128]. Immuno-histochemical analysis showed a decrease in Bmp-4 protein levels and a four - to eight-fold drop in BMP-4 mRN A compared to control embryos, whereas other mRN A le vels were not af fected. An e xample of RNAi through in utero electroporation is seen in w ork from Bai et al., where the y targeted doublecortin (DCX) e xpression [131]. shRN As were deli vered to E14 rat embryos and dissociated cortical cultures and slices were e xamined 24 hours to 4 days later. These experiments revealed that DCX is required for radial migration in the developing rat neocortex. Furthermore, the observed migration defects in RNAiexpressing cells also disrupted the migration of neurons e xpressing normal le vels

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of DCX, indicating an additional, non-cell autonomous phenotype due to the cooperative nature of radial migration. Mutations in the human dcx gene result in an Xlinked dominant disorder characterized by cerebral neocortex malformations, mental retardation and epilepsy; ho wever; dcx knock out mice ha ve no observ able neocortical defects [132]. The DCX RN Ai phenotype closely resembles the defects seen in humans, indicating the value of RNAi experiments, which can bypass the masking of phenotypes due to both molecular compensatory mechanisms and possible species-specific di ferences. RNAi delivered by in utero electroporation can also be e xamined at later, postnatal time points. One of the key advantages of RNAi is the ability to quickly assess gene function without the time required to mak e knockout mice, and the option of assessing gene function in a rat. A recent paper by Nguyen et al. highlights this reverse-genetic RNAi approach, where the y e xplored the role of Nudel, a protein expressed highly in the developing, post-natal and adult CNS [112]. Although Nudel is part of the neuronal perinuclear microtub ule netw ork in volved in the nuclear translocation step of neuronal migration [112], its later function in maturation and maintenance of neurons is unkno wn. By e xamining the ef fects of Nudel RN Ai in primary neuronal culture and in post-natal mouse brain ( in utero electroporated at E15/E17), they reveal that Nudel is a k ey protein in neurofilament (NF) assembl , transport and general neuronal inte grity. In vivo electroporation can be applied to post-natal rats or mice, further xtending e the stage at which one can use shRNAs to produce RNAi. Furthermore, by combining in vivo and in vitro RNAi studies, researchers are able to access the adv antages of these dif ferent systems. F or e xample, K onishi et al. emplo yed the follo wing approaches: shRNAs in primary cerebellar neuron culture, overlay of RNAi expressing cerebellar neurons on or ganotypic cellular slices or a myelin substrate and in vivo RNAi in the cerebellum of P6/8 rat pups [111]. RN Ai of Cdh1-APC in P6 rat cerebellar granule neuronal culture sho wed that Cdh1 inhibits axon elong ation and inappropriate gro wth o ver myelin, whereas the slice assay and in vivo studies revealed a role for Cdh1 in controlling layer -specific axonal outgr wth and parallel fiber o ganization in the cerebellum [111]. 10.4.3.3 Adult Mice RNAi studies are also possible in adult mice. Both siRN A and shRNA vectors can be delivered to adult mice by injection, in some cases with the addition of cationic lipid formulations [133–136]. Injection of adeno viral and lenti viral v ectors also results in successful transfer of shRNAs to adult mouse brain [70,137]. shRNA from a lentiviral vector delivered by stereotactic injection of adult mouse brain w as able to persistently suppress EGFP e xpression [137]. In this series of reported e xperiments, RN Ai injections were performed in the right striatum, and eGFP/control injections occurred in the contralateral hemisphere. Knockdo wn of eGFP w as observed at 1 week, still persisted at 6 months and could also be achie ved by injection of lenti viral shRN A v ector 1 week after injection of eGFP had occurred. Viralmediated RNAi has also been used to knock do wn endogenous genes. Injection of an adenoviral shRNA targeting tyrosine hydroxylase blocked dopamine synthesis in

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mid-brain neurons of adult mice and resulted in decreased motor performance [138]. Dopamine staining w as reduced on the tyrosine h ydroxylase shRNA side, whereas the contralateral control side (scrambled shRN A injected) sho wed no change. This RNAi-targeted knockdown of dopamine could become a useful model of Parkinson’s disease in addition to e xisting toxin-induced models. Recently, RNAi was tested as a potential therapy in a mouse model of the human neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) [71]. SCA1 is a dominant genetic disorder caused by polyglutamine e xpansion in the ataxin-1 protein. Transgenic mice expressing a mutant human SCA1 transcript in Purkinje cells exhibit defects typical of human SCA1, including uncoordinated movement, Purkinje cell de generation and thinning of cerebellar molecular layers. After identifying functional shRNAs targeting the mutant ataxin-1 in HEK 293 cells, an adeno viral shRNA was tested in the SCA1 mice by intracerebellar injection. shRN A treatment not only resulted in decreased ataxin-1 e xpression but, more importantly, the mice showed improved motor performance. The improved motor function correlated with rescued neuropathology: ataxin shRNA mice had cerebellar molecular widths equal to wild-type mice and control LacZ shRNA mice exhibited the characteristic reduction in cerebellar molecular layer width. A complete block of Purkinje cell intranuclear inclusion formation in ataxin shRN A-treated mice was also seen compared to control LacZ shRNA-treated SCA1 mice. Using a dif ferent RNAi approach, recent attempts to identify a clinically v alid means of deli vering therapeutic siRN A led to the de velopment of a chemically stabilized siRNA with cholesterol conjugated to the 3′ end of the sense strand (cholsiRNA). Chol-siRNA was shown to function ef ficiently in cell culture and, impo tantly, adult mice that recei ved intravenous injection of chol-siRNA targeting apoB had decreased plasma le vels of apoB protein (sho wn to result from mRN A degradation) and reduced total cholesterol [106]. In comparison, intra venous delivery of nonmodified siR As produced le vels of siRNA that were belo w detection resulted in no biological acti vity. Clearly , the use of such chemically modified siR As increases the potential applications of RN Ai in disease treatment. Additionally, this could dramatically improve RNAi effectiveness in primary culture and other systems that ha ve been less tractable to the standard RN Ai deli very mechanisms outlined above. In summary, the growing consensus is that initial results present an encour aging picture for the therapeutic use of RN Ai, although much remains to be done to improve RNAi delivery mechanisms and specificity [90,139,140] 10.4.3.4 Stable Inheritable Genetic Knockdown — Transgenic Mice and Rats Finally, the goal of creating knockdo wn animals with stable, inheritable RN Ai has also been attained by various means. Mammalian transgenic RNAi was first achi ved by the ef forts of Hasuw a et al. via shRN A microinjection into pronuclear -stage fertilized rat and mice e ggs [141]. Shortly afterw ards, transgenic knockdo wn mice were made by electroporation of shRN As into embryonic stem (ES) cells follo wed by either chimera formation [142] or tetraploid aggregation [143]. At the same time, an alternative successful approach w as published, emplo ying lentiviral delivery of

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shRNAs to embryos [144,145]. As lenti viral v ectors are inte grated into the host chromosome (unlik e adeno viral v ectors), the shRN A cassette can be transmitted through to subsequent generations. This technique, lik e microinjection, could be particularly valuable as it can be used to mak e transgenic rats as well as potentially other transgenic animals. The ef ficien y of transgenic RN Ai can be suf ficient to result in undetectabl levels of the targeted gene product and produce a complete loss of function phenotype equivalent to a null mutant [143]. On the other hand, transgenic RN Ai can result in differing degrees of knockdown (with the same shRNA construct) seen in approaches using mice derived from pronuclear injection of fertilized eggs, ES cells or lentiviral transduction [141–144,146]. The opportunity for varying degrees of suppression can be v ery useful, particularly where a complete knockdo wn might well result in lethality. One explanation for this phenotypic “spectrum” is the phenomenon known as positional v ariegation, where the le vel of transgene e xpression is determined by the relative quiescence or activity of the chromosomal region where it has integrated. An additional issue here is the possibility that the function of an endogenous gene could be compromised by transgene insertion. By phenotypic e xamination of multiple founder animals, these concerns can be eliminated. Also, the relati ve number of integrated RNAi transgenes can affect the degree of RNAi knockdown. The second way of generating a spectrum of RN Ai phenotypes is by tar geting different regions of the same mRN A. Hemann et al. generated shRN As to dif ferent re gions of the tumor suppressor p53 and determined the RN Ai ef ficien y in tissue culture cells [147]. When these constructs were used to generate transgenic RNAi mice lines, the observed phenotypic se verity matched the le vel of RNAi knockdown in tissue culture. A final consideration is that some situations could xist where in utero electroporation or maternal delivery via injection can provide advantages over transgenic RNAi, where genes can be tar geted that are in volved in growth/maintenance of ES cells or expressed in structures not deri ved from ES cells, i.e., yolk sac and trophoblast.

10.5 RNAi EXPERIMENTAL DESIGN STRATEGY 10.5.1

SIRNA/SHRNA

DESIGN

One of the most important issues in RN Ai experimental design is the appropriate choice of target mRNA sequence. Picking several regions (three to four) for siRN A targeting is generally recommended, as there is wide v ariation in RN Ai efficien y. Next, if multiple model systems will be used, sequence identity should be conserved between all relevant organisms. Of equal importance is the issue of sequence specificity in terms of possible of-target RNAi effects resulting from sequence homology within a protein family or common domain. F or both strands of the siRN A, regions showing sequence homology for 15 or more contiguous base pairs with other genes should not be used. With the discovery of miRNA-dependent suppression of translation, this issue is e ven more critical and a voiding regions sharing even 11 contiguous nucleotides might be advisable. F or shRNA vectors with a pol III promoter , avoiding regions of poly (T) or poly (A) is important, as this w ould lead to early

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termination of transcription of the hairpin RN A. Although most siRN As are made against coding sequence, man y labs ha ve recently tar geted re gions in 3 ′ UTRs to successfully produce RN Ai. The Hannon laboratory pro vides a particularly useful website for RNAi experiment design using siRNAs, shRNAs and miRNA-like hairpins [148]. The Whitehead Institute of Biomedical Research at the Massachusetts Institute of Technology (MIT) freely pro vides online siRN A design tools to the research community [149]. siRNA biotech companies such as Ambion also provide both free online siRN A design tools for researchers who w ant to choose their o wn siRNAs and custom services with the use of proprietary algorithms [150]. In vitro experiments show that the final cle vage-competent “holo” RISC complex contains unwound siRNAs [39] and active RISC retains a single siRN A strand [151]. Moreover, addition of single-stranded antisense RNAs to HeLa extracts reconstitutes RISC comple x formation and transfected single-stranded antisense RN As have similar silencing acti vity to siRN A duplexes [151]. This apparent preference for antisense strands (AS) rather than sense strands (SS) in RISC complex assembly is explained by recent w ork showing that functional miRN As and siRNA duplexes have lower free energy/stability at the 5′ AS end, resulting from a high A/U content in the first t o to fi e nucleotides [152,153]. The current model is that lower stability at the 5 ′ AS terminus biases helicase acti vity to opening the siRN A duple x from this end, resulting in the observ ed strand selectivity. However, sense strands can be incorporated into RISC and tar get mRNA degradation, thereby increasing the lik elihood of of f-target effects. This and other data has led to v arious new recommendations for siRN A/shRNA sequence design: high A/U content at the 5 ′ end of AS; G/C at the 5 ′ end of the SS; lo w G/C content o verall; and the absence of in verted repeats [152–154]. Together, these criteria should improve efficien y and specificit of siRNA through the preferential incorporation of the optimized AS over the SS. These recent guidelines have been incorporated into the publically a vailable siRNA design site siDirect [155,156]. The RNA company Dharmacon also provides asymmetric siRN As where a proprietary chemical modification inhibits sense strand incorporation into RISC [157].

10.5.2 SPECIFICITY CONCERNS: OFF-TARGET EFFECTS INTERFERON RESPONSE

AND THE

The use of proper controls should be considered as part of an y siRNA experimental design, as some reports have shown significant vidence of nonspecific ut sequencedependent effects, so-called “off-target effects,” and interferon response-related side effects from RN Ai. Several microarray-based studies of fer conflicting conclusion on siRN A specificity and the potential for o f-target mRN A clea vage [158–160]. However, clear proof is also found that a single base-pair dif ference in a siRN A duplex can block RN Ai [54], and man y reports show that siRNA activity is highly sequence specific. Recentl , other groups ha ve shown off-target effects that occur specifically through an miR A-like suppression of protein translation rather than by mRNA degradation [161,162]. Although 21 nt siRNAs are generally believed to not induce the interferon response, more recent findings h ve brought this issue back to the forefront. Transfection of some 21 nt siRNAs [163] or lentiviral shRNA vectors

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[164] resulted in up-re gulation of components of the interferon system. In Bridge et al. [164], one shRN A vector induced OAS1 whereas the production of a synthesized siRN A corresponding to that shRN A produced no interferon response and reduced target mRNA levels. In contrast, all six siRN As used by Sledz et al. [163] resulted in Stat1 up-re gulation. Careful shRN A v ector design is also crucial, as a recent paper sho wed that alteration of the original C/G sequence at the –1/+1 positions in a U6-dri ven shRNA vector resulted in interferon induction [165]. Specificall , the presence of a BstB I restriction enzyme-site (TTCGAA) or an AA dinucleotide immediately before the transcription start site induced O AS1. Whether the up-regulation of interferon response components observ ed in these reports produces cellular alterations w as not shown. In conclusion, the e vidence indicates that some shRN A v ectors and siRN As result in up-re gulation of interferon pathw ay components, whereas others do not.

10.5.3 CONTROLS The resulting lack of consensus mak es difficult the determination of the true xtent of non-specific R Ai effects. To address this issue, recommended controls for RNAi experiments were outlined in an editorial by Nature Cell Biolo gy [166] and are summarized here. In addition to sho wing an RNAi phenotype, showing the specifi decrease in tar get mRNA and protein le vels for RISC-mediated RN Ai, or proteinlevel knockdown for miRNA-like suppression of translation, is critical. Appropriate techniques for assessing mRNA levels include gel-based RT-PCR, Northern analysis, nuclease protection assays and real-time PCR. Real-time PCR is the optimal method as it allo ws truly quantitati ve measurements of changes in mRN A levels. Changes in protein levels should be determined by a technique such as quantitati ve Western blotting. Titration of the siRNA is also encouraged, as reducing the siRN A concentration decreases side effects in addition to showing a graded RNAi effect. Specificit is further supported if tw o or more siRN As/shRNAs tar geting the same mRN A produce a similar phenotypic outcome. Non-specific e fects should be check ed by assaying le vels of unrelated proteins. The use of a scrambled siRN A/shRNA is considered to be of little benefit [166]. Perhaps a more useful comparison can b made between the effects of a siRNA/shRNA targeting a non-endogenous gene (such as eGFP or lacZ) to one directed at the gene of interest. Finally , the “ultimate” functional control is to rescue the RN Ai phenotype by e xpressing a form of the target gene that is resistant to RN Ai. This condition can be achieved by introducing multiple silent mutations within the siRN A/shRNA tar get re gion of the gene, although this is not f ail-safe, as miRN A-induced suppression of translation could still persist. As this last recommended control is not al ways feasible, performing a majority of the abo ve controls is suf ficient

10.6 CONCLUSION Ongoing studies on RNA silencing will continue to improve both our understanding of the RN Ai mechanism and the potential applications of RN Ai in research and disease treatment. Although specificit , appropriate controls and deli very

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mechanisms remain important issues, man y solutions ha ve already been pro vided due to the rapid e xpansion of research in the RN Ai field. R Ai approaches are economical and pro vide a tremendous amount of fl xibility by allo wing time- and tissue-specific knockd wn of genes or specific splice forms, in addition to voiding the common problem of embryonic lethality from gene knock out. Furthermore, by targeting a domain shared in a protein f amily, knockdown of multiple mRN As can bypass the problem of genetic redundanc y. Post-transcriptional silencing by RN Ai is also less lik ely to result in compensatory transcription often caused by gene deletion. The ability to produce dose-dependent and graded phenotypic outcomes is another valuable feature of RNAi. Like other research areas, neurobiology stands to benefit greatly from R Ai techniques. P articularly for neurobiology researchers using a rat experimental system, RNAi has dramatically increased the genetic toolkit at hand. A particularly e xciting prospect is the application of functional genomics in neuronal culture through the use of shRN A/siRNA microarray high-throughput screens [123,167].

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154. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S. and Khvorova, A., Rational siRNA design for RN A interference, Nat. Biotech., 22(3), 326–330, 2004. 155. http://design.rnai.jp/. 156. Naito, Y., Yamada, T., Ui-T ei, K., Morishita, S. and Saigo, K., siDirect: highly effective, target-specific siR A design softw are for mammalian RN A interference, Nucl. Acids Res., 32 (Web Server issue), W124–W129, 2004. 157. http://www.dharmacon.com. 158. Chi, J.T ., Chang, H.Y ., Wang, N.N., Chang, D.S., Dunph y, N. and Bro wn, P.O., Genomewide view of gene silencing by small interfering RN As, Proc. Natl. Acad. Sci. USA, 100(11), 6343–6346, 2003. 159. Jackson, A.L., Bartz, S.R., Schelter , J., K obayashi, S.V., Burchard, J., Mao, M., Li, B., Cavet, G. and Linsley, P.S., Expression profiling r veals off-target gene regulation by RNAi, Nat. Biotech., 21(6), 635–637, 2003. 160. Semizarov, D., Frost, L., Sarth y, A., Kroe ger, P., Halbert, D.N. and Fesik, S.W ., Specificity of short interfering R A determined through gene e xpression signatures, Proc. Natl. Acad. Sci. USA , 100(11), 6347–6352, 2003. 161. Scacheri, P.C., Rozenblatt-Rosen, O., Caplen, N.J., Wolfsberg, T.G., Umayam, L., Lee, J.C., Hughes, C.M., Shanmug am, K.S., Bhattacharjee, A., Me yerson, M. and Collins, F.S., Short interfering RN As can induce une xpected and di vergent changes in the levels of untargeted proteins in mammalian cells, Proc. Natl. Acad. Sci. USA , 101(7), 1892–1897, 2004. 162. Saxena, S., Jonsson, Z.O. and Dutta, A., Small RN As with imperfect match to endogenous mRNA repress translation. Implications for of f-target activity of small inhibitory RNA in mammalian cells, J. Biol. Chem., 278(45), 44312–44319, 2003. 163. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. and Williams, B.R., Activation of the interferon system by short-interfering RN As, Nat. Cell Biol ., 5(9), 834–839, 2003. 164. Bridge, A.J., Pebernard, S., Ducraux, A., Nicoulaz, A.L. and Iggo, R., Induction of an interferon response by RN Ai v ectors in mammalian cells, Nat. Genet ., 34(3), 263–264, 2003. 165. Pebernard, S. and Iggo, R.D., Determinants of interferon-stimulated gene induction by RNAi vectors, Differentiation, 72(2-3), 103–111, 2004. 166. Whither RNAi? Nat. Cell Biol ., 5(6), 489–490, 2003. 167. Wu, R.Z., Bailey, S.N. and Sabatini, D.M., Cell-biological applications of transfectedcell microarrays, Trends Cell Biol ., 12(10), 485–488, 2002.

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11

Transfecting and Transducing Neurons with Synthetic Nucleic Acids and Biologically Active Macromolecules Josef T. Kittler, Jonathan G. Hanley, and John T. R. Isaac

CONTENTS 11.1 11.2 11.3

11.4 11.5 11.6

11.7

Introduction ...............................................................................................206 Methods for Neuronal Gene Transfer.......................................................207 Viral Approaches .......................................................................................207 11.3.1 The Sindbis Virus System ............................................................207 11.3.1.1 Transfecting Cultured Neurons with Sindbis Viruses...............................................................208 11.3.1.2 Viral Infection of Neurons in Intact Tissues: The Acute Slice Method ................................................208 11.3.2 Other Viral Systems .....................................................................209 Chemical and Ph ysical Transfection Approaches.....................................211 11.4.1 Microinjection and Biolistics .......................................................211 11.4.2 Chemical Approaches...................................................................212 Electrical Approaches: Electroporation and Nucleofection .....................213 Transducing Neurons with Biologically Active Peptides, Proteins and Antibodies ............................................................................215 11.6.1 Function Blocking and Protein–Protein Interaction Blocking Peptides, Proteins and Antibodies................................................215 11.6.2 Identification of Protein–Protein Binding Domains and Design of Protein–Protein Interaction Blocking Peptides........... 218 Functional Studies Using Function Blocking and Protein–Protein Interaction-Blocking Peptides, Proteins and Antibodies .........................221 11.7.1 Introducing Peptides and Proteins into Neurons via the Electrophysiological Recording Pipette .......................................221 205

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11.7.1.1 Some Considerations .....................................................223 Transducing Neurons with Membrane-Permeant Peptides and Proteins ................................................................................224 11.9 Conclusion.................................................................................................225 11.10 Acknowledgments .....................................................................................228 References..............................................................................................................229 11.8

11.1 INTRODUCTION A major challenge in neurobiology is to better understand the ph ysiological role of the protein–protein interactions and post-translational modifications important fo regulating the acti vity and plasticity of synapses. A significant e fort has been undertaken over the past 10 years to identify the protein components of the postsynaptic domain and the protein machinery important for re gulating the activity of the neurotransmitter receptors that mediate the electroph ysiological responses responsible for synaptic acti vity. Of significant contri ution to this ef fort has been the systematic identification of the protein constituents and protein–protein interac tions of the synapse, using proteomic and yeast tw o-hybrid approaches and the identification and characterization of the signalling path ays, enzymes and synaptic substrates of particular receptor post-translational modifications (such as phospho rylation, palmitoylation, ubiquitination and nitrosylation) important in the regulation of neurotransmitter receptor acti vity and synaptic plasticity [1–5]. Essential to the functional characterization of the molecular machinery important for re gulating synapse de velopment, synaptic transmission and synaptic plasticity has been the development of methods for altering the constituents of a neuron either genetically or by changing the activity of a protein of interest. Although homologous recombination in embryonic stem (ES) cells is now a standard method to mutate the germ line of mice and hence interfere with the activity of a particular gene of interest [6], this approach still has se veral limitations. These include the high costs of producing and maintaining transgenic animals, complications due to embryonic lethality and the potential consequences on nerv ous-system development of introducing into the animal a particular mutation. Other potential limitations include genetic compensation, spatial and temporal specificity and the restriction of orking with mouse tissues. As a consequence, a significant e fort has been directed to ward developing and impro ving methods for introducing biological agents (including DNA, proteins, antibodies and biologically acti ve or protein–protein interactionblocking peptides) into either single or lar ge populations of neurons. Here, we consider some techniques that have emerged for transfecting and transducing neurons with DNA and biologically acti ve macromolecules. We focus, in particular , on the use of these approaches for studying the molecular mechanisms important for re gulating the acti vity and plasticity of synapses and the functional re gulation of the key ionotropic glutamate receptors (NMDA receptors, AMPA receptors and kainate receptors) and inhibitory ligand-gated ion channels (GABAA and glycine receptors) at excitatory and inhibitory synapses, respecti vely [1,7–10].

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11.2 METHODS FOR NEURONAL GENE TRANSFER The ability to introduce synthetic nucleic acids (recombinant DN A, RNA and oligonucleotides) into neuronal cells and thus manipulate gene e xpression or protein function has become an essential approach for research in the neurosciences. Neuronal gene transfer can be used to introduce epitope-tagged proteins into neurons, including wild-type or mutant neurotransmitter receptors or synaptic scaf fold, signalling or trafficking proteins. Gene transfer into neurons has also been used to allw the knockdown of a particular gene of interest using antisense oligonucleotides or RNA interference (RNAi) approaches. Transfected neurons can then be analyzed by biochemical, imaging or electroph ysiological approaches to analyze the functional consequences of these manipulations on aspects of neurotransmitter receptor function and synaptic activity. However, post-mitotic neurons are comparatively difficul to transfect and, furthermore, a wide v ariety of neuronal preparations are used in the study of ionotropic receptor modulation and synapse ph ysiology (cultured dissociated neurons, or ganotypic and acute brain slices and whole animal in vivo studies). This has dri ven the de velopment of a wide v ariety of techniques for the introduction of nucleic acids into these preparations [11,12].

11.3 VIRAL APPROACHES Recombinant viral v ectors have emerged as a k ey tool for neuronal gene transfer . Several types of viral vector, including herpes simplex virus, Sindbis virus, Semliki Forest virus, adeno virus, adeno-associated virus, v accinia virus and lenti virus have been applied to neuronal transfection [12–17]. Viral vectors differ with respect to several parameters, such as ef ficien y of infection, time tak en to e xpress a desired protein, expression levels attained and cellular toxicity [12,18,19]. Hence, the use of a particular viral system depends to a certain e xtent on the specific xperimental application [12].

11.3.1 THE SINDBIS VIRUS SYSTEM The use of Sindbis viral v ectors has become particularly popular with neuroph ysiologists [18,20,21]. These vectors have several advantages: the virus is neurotropic (a high infecti vity for neurons), rapidly produces/e xpresses the protein of interest, is relatively easy to produce, has a defined wind w of non-toxicity and is commer cially available (e.g., pSinRep5, Invitrogen). For a detailed description of the Sindbis system and production of viral particles, see Chapter 12 by Marie and Malenka (see also In vitrogen Sindbis e xpression system manual) [18,21]. Furthermore, Sindbis virus vectors have been engineered to ha ve lower toxicity and allo w simultaneous expression of two proteins (for example, a protein of interest and a reporter protein such as GFP), allo wing significantly greater f xibility in the types of e xperiment that can be performed [22,23]. As a result, the Sindbis system is now routinely used by many laboratories to transfect neurons in se veral types of preparation including cultured dissociated neurons [21,24–27], or ganotypic brain slices [18,21,28,29], acute brain slices [30,31] and neurons in vivo [32,33]. Here, we focus on the use of

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Sindbis viruses to transfect neurons in dissociated primary culture and in acute brain slices. 11.3.1.1 Transfecting Cultured Neurons with Sindbis Viruses For transfection of dissociated cultured neurons, plated cells can be incubated in conditioned neuronal culture media containing Sindbis virus particles (either purifie or unpurified infect ve supernatant solution) for 1 hour , a period long enough to allow neuronal infection, follo wed by return to viral-free neuronal culture medium [24]. Alternatively, viral particles can simply be added to the culture medium. The virus is then allo wed to e xpress for 24 to 48 hours before analysis (detectable expression can be seen within as little as 6 hours, Figure 11.1a) [21,24,25]. Using Sindbis virus, no ob vious ef fects on morphology of neurons infected for up to 3 days has been reported [21,24], although longer infection periods result in cells showing clear signs of toxicity [21]. Depending on the titre of virus used, either relatively lo w le vels of neuronal transfection (5 to 10%) up to high transfection (close to 100%) ef ficiencies of infection can be achi ved allowing both single-cell based approaches (electrophysiology or imaging) or biochemical analyses (for example, biotinylation/co-immunoprecipitation) to be used to analyze transfected neurons [24–27,31]. Several groups have elegantly used the Sindbis system to study v arious aspects of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor synaptic targeting, trafficking and assembly [24–27,31] and their roles in synapti transmission and plasticity. For example, researchers have used Sindbis virus–based infection of cultured hippocampal neurons to e xpress epitope-tagged wild-type and mutant v ersions of AMPA-type glutamate receptor sub units or AMPA receptor associated proteins (such as GRIP/ABP or PICK proteins) to study the molecular mechanisms important for various aspects of AMPA receptor assembly and synaptic targeting in synaptogenesis and synaptic plasticity . The high e xpression levels that can be achieved using the Sindbis system has also allowed live-cell imaging of GFPtagged AMPA receptor sub units [34,35] or AMPA receptor -associated proteins in cultured neurons [36] f acilitating studies of real-time traf ficking of these proteins 11.3.1.2 Viral Infection of Neurons in Intact Tissues: The Acute Slice Method Experiments in intact brain tissues, such as acute and or gantoypic brain slices and neurons in vivo, hold many advantages for studying the activity of synapses because to varying degrees, these preparations maintain basic neuronal connectivity. Neurons transfected in intact tissues can then be analyzed using electrophysiological, imaging or biochemical techniques. A major adv antage of electroph ysiological experiments from transfected neurons in intact tissues is that it allows researchers to make wholecell, patch-clamp recordings from visually identified infected neurons duall expressing a reporter construct such as GFP and another construct of interest while simultaneously recording from neighboring noninfected control neurons from the same slice. Under conditions where dual recordings are carried out simultaneously for both the infected and uninfected neurons, this allo ws the direct comparison of

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the electroph ysiological properties of both e xperimental and control neurons activated by the same stimulus position and intensity , providing a useful control. The use of viral v ector systems such as Sindbis virus has greatly simplified the routin and efficient infection of neurons in intact brain tissues. In combination with t ophoton and confocal microscop y, the efficient xpression of proteins in brain slices achievable using Sindbis v ectors has also f acilitated GFP reporter -based li ve-cell imaging of subcellular structures (e.g., spines), GFP-tagged synaptic proteins or receptors of interest in intact tissues [21,29,37,38]. Se veral groups ha ve elegantly used the Sindbis system in combination with or ganotypic brain slice cultures or in vivo [29,38–41] to gain significant insights into the molecular mechanisms ofAMPA receptor trafficking underlying xcitatory synaptic plasticity. See [21] andChapter 12 by Marie and Malenka for a detailed description of the use of Sindbis v ectors to infect organotypic slice cultures and neurons in vivo , respectively. Recently, the rapid transgene expression rates of Sindbis virus have been applied to the transfection of neurons in “acute slices. ” In this approach, acute slices are produced using standard techniques: 400-µm slices are prepared using a microslicer at 4°C in modified xtracellular solution (low sodium, 1 mM Ca 2+, 5 mM Mg 2+, 0.5 mM ascorbate). Slices are then allo wed to recover for 30 to 60 min at 27°C before being placed in standard sterile culture medium (containing fetal calf serum) and then immediately transfected with Sindbis virus. To increase infection of neurons deeper within the slice, the virus particles can be pressure ejected into a re gion of interest (for e xample, the CA1 p yramidal cell layer of the hippocampus) using a picospritzer (WPI) or Eppendorf injector (Eppendorf). Slices can then be analyzed using electroph ysiological or biochemical techniques within 20 to 40 hours of transfection (Figure 11.1b). Importantly, this type of procedure appears to maintain slice health because noninfected neurons from in-slice controls of infected slices have similar properties to neurons in uninfected slices [31]. This type of approach has several of the advantages of using acute slices, such as the preservation of acute slice connectivity and ph ysiology, and a voids some of the potential disadv antages that can occur in organotypic slices including cell death, abnormal synaptic rewiring and epileptogenic activity. This approach can be used to infect neurons with viruses that can bicistronically express a protein/peptide of interest together with free EGFP, allowing visualization and electroph ysiological analysis of transfected neurons in an acute slice preparation. For example, this approach has been used to o verexpress PICK1 in acute slices and study the role of this protein inAMPA receptor traffickin and excitatory synaptic plasticity [31].

11.3.2 OTHER VIRAL SYSTEMS Although the Sindbis virus expression system has proved to be a very powerful tool for transfecting neurons in intact brain tissues, Sindbis v ectors do have some disadvantages, including neurotoxicity during prolonged infection times, precluding stable long term neuronal transfection with this approach. In addition, other viruses are better suited for gene knockdo wn studies using shRN A constructs. Se veral other viral based systems ha ve been described for the transfection of neurons in culture and intact tissues that could ha ve adv antages with respect to toxicity le vels and

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Surface GluR1 (arbitrary units)

β-gal GluR1 HA (α-SNAP) GluR1

8 6 HA (β-SNAP) GluR1

4 2

βg α- al SN A P

0

(a)

(b)

1

2

3

4

(c) infected

control stimulate record

(d)

AMPA EPSC Amplitude (pA)

PICK1

20 pA

control

100 ms (e)

control PICK1

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FIGURE 11.1 (Color figure fol ows page 176.) (a,b) Sindbis virus–mediated overexpression of proteins in cultured neurons. Dissociated hippocampal neurons were infected with Sindbis virus encoding β-gal (left panel), HA-tagged α-SNAP (right panel) or β-SNAP (not shown) and stained for surf ace AMPA receptor using anti-GluR1 antibody (green) and for β-gal or HA-tag (red). White boxes define enla gements shown in lo wer panels. (b) Quantitation of surface AMPAR levels in neurons infected with Sindbis viral constructs for β-gal, α-SNAP. Reproduced with permission from Else vier [25]. (c) Sindbis virus–mediated o verexpression of proteins in CA1 p yramidal neurons in acute cultured hippocampal slices. Lo w-power transmission (C1) and fluorescence (C2) images of an acute hippocampal slice culture overnight with Sindbis virus bicistronically e xpressing EGFP. High-power images (transmission and fluorescence images superimposed) of whole-cell patch clamp recordings fro noninfected control (C3) and neighboring infected (C4) neurons in the CA1 p yramidal cell layer. (d) Schematic sho wing the e xperimental configuration for recording from control an infected neurons. (e) Averaged EPSCs (bottom traces, –70 mV, top traces, +40 mV) from an example pairwise e xperiment for a control noninfected neuron (left) and for a neighboring neuron infected with virus e xpressing PICK1, and summary analysis of all pairwise compar isons ( n = 9) of the ef fects of viral e xpression of PICK1 on EPSC amplitude recorded at a holding potential of –70 mV . Modified with permission from [31]

applicability to experiments that require prolonged and stable neuronal gene delivery [42–47]. For example, both adeno virus and herpes virus-based e xpression systems have been used to study real-time traf ficking of GFP-tagged AMPA receptors in cultured hippocampal neurons [48] and in vivo, respectively [39]. Recently, growing interest has been seen in the use of lenti viruses, which can infect both di viding and post-mitotic cells, for neuronal gene transfer [49]. F or a detailed description of the development and application of the currently used lenti viral vector systems to neuronal gene delivery, see Chapter 13 by Osten, Dittgen and Licznerski.

11.4 CHEMICAL AND PHYSICAL TRANSFECTION APPROACHES 11.4.1 MICROINJECTION

AND

BIOLISTICS

The two main physical approaches for introducing DN A into neurons are biolistics [50–54] and microinjection [55–57]. Both of these approaches in volve the physical introduction of DN A directly into the neuronal nucleus. The biolistic approach involves using a specialized apparatus (the Biorad laboratories Helios gene gun) to accelerate micrometer-diameter gold particles coated in vector DNAs encoding genes of interest at high v elocity into neurons either in culture or slices. F ollowing bombardment, neurons that contain a gold particle in their nucleus are lik ely to express the genes of interest [12,53,54]. Careful optimization is needed to a void damage to the cells or tissue of interest; ho wever, once this has been achie ved, routine transfection of plasmids or knockdown of genes in cultured neurons or intact brain tissues can be obtained. Importantly , gold particles can be coated with more than one plasmid DNA (or other synthetic nucleic acid) allo wing co-transfection of neurons. For e xample, Leitges et al. ha ve ele gantly used biolistic transfection of cultured Purkinje neurons to co-e xpress GFP (to allo w visualization of transfected neurons)

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with plasmids encoding protein kinase C (PKC) isoforms or , alternatively, dsRNAi oligonucleotides targeting PKC [58]. These studies led to the identification of th PKC isoforms critical for cerebellar long-term depression (LTD) [58]. Similarly , using biolistic transfection of cerebellar neuronal cultures from GluR2 sub unit knockout mice with wild-type and mutant AMPA receptor GluR2 sub units, this group demonstrated the importance of AMPA-receptor GluR2-subunit phosphorylation in cerebellar L TD [59]. Biolistic transfection is also particularly good for dispersed transfection of neurons in slices and, similar to viral methods such as Sindbis, allows simultaneous electrophysiological analysis using dual recordings, of transfected and control untransfected neurons in the same slice [60–62]. The ability to co-transfect neurons in brain slices with multiple constructs has been ele gantly used to study the role of rab GTP ase family members in AMPA receptor traffickin [60,62]. A detailed protocol for the biolistic transfection of neurons has been described by McAllister [54]. Whereas biolistics in volves particle-mediated nuclear bombardment, microinjection involves the direct injection of plasmid DN As of interest into the neuronal nucleus (or cRNA or protein into the cytoplasm) using high-resistance glass pipettes. For nuclear microinjection of neurons, DNA can be dissolved in phosphate-buffered saline at a concentration of 0.05 µg/ml using equimolar ratios of e xpression constructs [55–57]. DN A (approximately 1.2 µl) can then be loaded into pre-pulled high-resistance (approximately 30 Mohm) Pyrex glass pipettes and injected into the nucleus of single neurons under visual guidance using a 40 × or 60 × objective in combination with a microinjector (for e xample, the Eppendorf microinjection system). Cells can then be allo wed to e xpress the DN As for 24 to 48 hours or longer before analysis [55–57,63]. One limitation of this approach is that injecting younger, less-developed neurons is more dif ficult, as their nuclei are smalle . With practice, reasonably large numbers of neurons (50 to 100) can be injected in a short period of time (e.g., 15 to 40 min) with the simultaneous introduction of several constructs. However, this technique is only useful for e xperiments requiring relati vely fe w transfected cells. Nuclear microinjection has been ef fectively used in e xperiments requiring precise gene transfer to a single identified neuron [63] or precise contro of neuronal co-transfection of tw o or more plasmid DN As [56,57]. F or e xample, nuclear microinjection of receptor sub unit cDNAs into cultured hippocampal neurons has been used to study the receptor sub unit requirements of neuronal assembly and surface membrane traf ficking of both GA AA receptor and GAB AB receptors [56,57].

11.4.2 CHEMICAL APPROACHES Several chemical approaches ha ve been established for routinely transfecting cultured neurons, although these methods might not be well-suited to transfecting intact tissue (such as brain slices) and, therefore, are less fl xible than viral vectors in this respect. However, major adv antages include relati ve ease of use, the ability to cotransfect more than one construct and no requirement for subcloning into specialized vectors [11,12]. Of the various chemical transfection methodologies, calcium-phosphate and liposome-based approaches ha ve become most popular . In both cases,

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complexes are formed between the DNA and carrier, either DNA/calcium-phosphate co-precipitate or DNA/liposome complexes, which enter neurons by a poorly char acterized process that might be dependent on endoc ytosis [11,64]. Calcium phosphate transfection is a well-established method for introducing constructs into many types of cells including neurons [11].This technique is popular because it is relatively easy to use, multiple constructs can be simultaneously co-transfected and subcloning into specialized v ectors is rarely required [11,12]. Ho wever, a problem with this technique is the v ariable and often lo w transfection ef ficien y (0.1 to 7%) [12,65]. Nonetheless this technique has been v ery ef fectively used by se veral groups, to transfect cultured neurons with man y recombinant DNAs including epitope-tagged receptor subunits, other synaptic proteins and shRN Ai constructs [66–74]. Other chemical transfection methodologies include the use of cationic liposomebased (for e xample, Lipofectamine 2000) and nonliposomal-based lipids (e.g., Effectene) [75,76], which are no w commonly used for gene transfer into cultured neurons [12]. Effectene (Qiagen) and Lipofectamine 2000 (Invitrogen) have become particularly popular for neuronal transfection applications and have been extensively used for studies of neurotransmitter receptor traf ficking and synaptogenesi [12,64,77–80]. Importantly, modifications of the Lipofectamine 2000 neuronal trans fection protocol have increased neuronal transfection efficiencies of cultured cortica neurons from around 3% [64] to around 30% [81]. Detailed protocols for Lipofectamine 2000 mediated neuronal transfection are described in the literature [64,81].

11.5 ELECTRICAL APPROACHES: ELECTROPORATION AND NUCLEOFECTION A major adv antage of virally mediated neuronal transfection strate gies is the ef fi ciency of neuronal infection (up to 95% of cells) giving substantially higher neuronal transfection efficiencies compared to other approaches such as chemical and pysical transfection [12]. However, some disadvantages of the viral approach exist, including that viral particles must be produced for each genetic manipulation to be carried out, which can mak e this a time-consuming and laborious approach. Viral vectors often have a maximal genetic loading capacity [12] and necessary safety requirements for the use of viral vectors must also be taken into consideration. In addition, some commonly used viruses such as Sindbis virus cause the shut-of f of host cell protein synthesis (within 8 hours) and toxicity during prolonged infection times (for example, Sindbis virus infection times of longer than 48 to 72 hours), which could preclude experiments that necessitate longer (several days) transgene expression. As a result, other approaches for high-ef ficien y transfection of neurons, amenable to both cultured cells and intact tissues, ha ve been sought. One emer ging non viral, neuronal transfection approach that holds significant promise for high-e ficien y transfection of DNA, RNA and oligonucleotides into neurons is electric field-medi ated gene transfer, or electroporation. Electroporation, whereby cells are e xposed to a brief application of an electric field, is generally beli ved to result in structural rearrangement of the cell membrane and opening of aqueous pores [82–85], which allows charged molecules to enter the

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cell c ytoplasm. In addition to perforating the membrane, the electric field als provides an electromoti ve force (electrophoresis) that forces char ged molecules through the transient membrane pores. A major advantage of electroporation is the ease with which more than one e xpression construct can be simultaneously introduced into cells. Therefore, this allo ws the introduction of plasmids encoding a protein of interest or shRN Ai constructs and co-transfection with a reporter protein such as GFP , allo wing functional analysis by imaging or electroph ysiological approaches [12]. Electroporation has been successfully used to transfect man y types of neuronal preparation including primary cultured neurons, brain slices and neuronal populations in vivo [12,86,87]. Several groups have reported the use of electroporation to transfect cultured primary hippocampal neurons with DN A, RNA and other macromolecules using either custom or commercially a vailable electroporation de vices [87–89]. For instance, the Biorad Gene Pulser electroporation de vice can be used to transfect newly dissociated hippocampal neurons, with protein expression lasting for up to 4 weeks in culture, although substantial v ariability seems to be reported in efficiencies (2 to 80%) by this method [12,87,88,90]. Biorad-mediated electropo ration of cultured hippocampal neurons is an effective mechanism for overexpression of tagged neurotransmitter receptors ( Figure 11.2a). Electroporation has also been adapted to intact brain tissue, including brain slices and neurons in vivo [86,91–95]. In vivo electroporation is now routinely used to carry out gene transfer e xperiments in chick or mouse embryos and has also been used to transfect neurons in slices of rat hippocampus and mouse corte x [86,94,95]. Recently, a modification of the electroporation technique has been d veloped by AMAXA Biosystems and termed “nucleofection” [96–100]. This electroporationbased technique leads to direct transfer of DN A into the cell nucleus and results in rapid and highly efficient transient transfection of ma y difficult to transfect primar cell types, including neurons [96]. To carry out nuclefoection, neuronal cells are isolated from a particular tissue of interest (e.g., hippocampus or corte x) and dissociated following standard protocols for dispersed neuronal cultures [101,102]. Prior to plating, cells are resuspended in a b uffer supplied by the manuf acturer to which the DNA of interest ha ve been added. This cell/DNA/buffer mix is then transferred to a nucleofection cuv ette and “electroporated” in the AMAXA electroporation device (see AMAXA protocol, www.amaxa.com, for further details). Cells are then plated to the required density as per standard protocols. Over 50 to 60% transfection efficiencies of cultured rat embryonic cortical neurons can be achi ved [102] and high ef ficien y transfection of cultures of other embryonic, neonatal and adult primary neuronal cell types such as hippocampal (Figure 11.2b), striatal, cerebellar , retinal ganglion and dorsal root g anglion neurons has also been reported [102,103]. High enough transfection efficien y can be achieved to allow detection of exogenous protein expression (for example, GFP) by immunofluorescence or Western blotting of cell lysates from transfected neurons (Figure 11.2b and Figure 11.2c) [102]. Such high levels of transfection allow the use of biochemical techniques, such as analysis of receptor trafficking by sur ace biotinylation [102,104]. Nucleofection also appears to be an ef fective technique for carrying out gene knockdo wn in neurons [104]. Using nucleofection, Couv e et al. were able to introduce short hairpin-e xpressing

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RNAi constructs into cultured cortical neurons, allowing target-specific gene knock down in these cells and biochemical analyses [104]. Nucleofection has also been used to introduce siRNA oligos into cultured cortical neurons, achie ving up to 85% reduction in expression of target genes [103]. Nucleofection can therefore be combined with both single cell type analyses such as electrop hysiology (Figure 11.2d) [102] or biochemical approaches such as biotin ylation to assess the consequences on neurotransmitter receptor function of o verexpression or gene knockdo wn of a particular target protein [102,104].

11.6 TRANSDUCING NEURONS WITH BIOLOGICALLY ACTIVE PEPTIDES, PROTEINS AND ANTIBODIES In addition to neuronal gene transfer of nucleic acids (DN A, RNA and oligonucleotides), the direct introduction of other types of biologically acti ve macromolecules (such as proteins or peptides) into neurons is a po werful approach to study the role of a particular neuronal protein of interest in neural function and ph ysiology. Of particular use has been the application of function blocking or protein–protein interaction blocking peptides or proteins to tar get the activity of a particular protein or protein comple x in combination with functional analysis using biochemical, imaging or electroph ysiological techniques.

11.6.1 FUNCTION BLOCKING AND PROTEIN–PROTEIN INTERACTION BLOCKING PEPTIDES, PROTEINS AND ANTIBODIES Macromolecules with function-blocking acti vities are po werful tools for the study of a protein’ s ph ysiological function. Examples include bacterially e xpressed or purified proteins with a catalytic function, such as protein kinase or phosphatas preparations, which can be introduced into cells to increase the enzymatic acti vity within that cell and which ha ve been e xtensively used to study v arious aspects of phospho-dependent regulation of synaptic transmission and plasticity . Of particular use are proteins that can be e xpressed as recombinant fusion proteins in a constitutively acti ve or dominant ne gative form and that can thus be used to increase or block the acti vity of a particular tar get protein or pathw ay. F or e xample, se veral constitutively active kinases, phosphatases or other signalling molecules such as the ras superf amilly small GTP ase proteins, can be readily produced as recombinant fusion proteins in an acti ve or dominant ne gative confirmation and then introduce into cells [2,105]. In addition to the lar ge number of pharmacologically useful peptide toxins, se veral bacterial toxins also ha ve useful biological acti vities [106]. For e xample, toxins that tar get the signalling machinery that re gulates the c ytoskeleton (chlostridial toxins) [107] or that target the membrane-trafficking machiner [108] (e.g., Botulinum and tetanus toxins) ha ve been used to study the role of these processes in v arious aspects of neurotransmitter receptor re gulation and synaptic plasticity [109–112]. A number of antibodies ha ve also pro ved useful for blocking the acti vity of their tar get antigen. Sometimes this is disco vered fortuitously, b ut increasingly antibodies are specifically raised to taget a particular biological activity

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(b)

GFP 1 (a)

2

3

4

(c)

120 100

120

90 100 Number of events

80 70 60 50 40 30

80 60 GFP HAP-1A

40 20

20 10 0

0 GFP

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HAP1a

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(d)

100 150 200 Amplitude (pA) (e)

1.0 Cumulative fraction

Average mIPSC amplitude (pA)

110

0.8 0.6 0.4 GFP HAP-1A

0.2 0.0 0

50

100

150

200

Amplitude (pA) (f)

250

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250

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FIGURE 11.2 (a) Electroporation-mediated o verexpression of proteins in cultured neurons. Cultured hippocampal neuron 21 days old expressing a myc epitope tagged GABAA receptor β3 subunit after transfection by Biorad mediated electroporation. Dissociated hippocampal neurons were electroporated in a Biorad electroporation de vice on the day of dissociation in the presence of 10 µg of myc β3 DN A follo wed by plating onto glass co verslips. (b, c) Overexpression of proteins in cultured neurons by nucleofection. High-efficien y transfection of cultured cortical neurons with GFP cDNA after dissociation using an AMAXA nucleofection device. (b) Field of cultured cortical neurons expressing GFP at 10 days in vitro. (c) GFP in lysates of cultured cortical neurons could be detected by Western blotting. (d–f) Electrophysiological recordings from cultured hippocampal neurons transfected by nucleofection with either GFP or the GAB AA receptor-associated protein HAP1. Ov erexpression of HAP1 increases minitature inhibitory post-synaptic current amplitude in cultured hippocampal neurons, compared to overexpression of GFP alone. (d) Bar graph of the mean mIPSC amplitudes representing GFP (hatched bar) or GFP + HAP1 (open bar) cDN A nucleofected 14-day old hippocampal neurons ( n = 7 for each condition). (e) The distrib ution of mIPSCs recorded from HAP1 nucleofected neurons are shifted to higher amplitudes relati ve to neurons nucleofected with GFP alone. (f) Cumulative probability data for mIPSC amplitudes demonstrating the significant amplitude shift between GFP-nucleofected (filled symbols) and GFP + HAP nucleofected (open symbols) neurons. Modified and reproduced with permission from [102]

(for example, by raising an antibody to a catalytic domain or protein–protein inter action domain of interest). Presumably , these antibodies w ork by steric hindrance or by locking a tar get protein in a particular conformation. The use of se veral function-blocking antibodies ha ve been described to study the protein machinery important for synaptic transmission and plasticity [113–116]. For example, antibodies targeting the activity of the ATPase NSF, the molecular motors kinesin and dynein [114], the e xtracellular domain or tyrosine kinase domain of the insulin receptor [117] or N-cadherin dependent cell adhesion or insulin receptor signalling [116–118] have all been used to study the role of these proteins in synaptic transmission and plasticity. In addition, because protein–protein interactions underlie most biological processes [119] and significant e fort has been directed toward the identification an characterization of the protein–protein interactions important for regulating the activity of neurotransmitter receptors and synapses, the use of peptides that specificall block a particular protein–protein interaction are also being increasingly used. Critical for application of proteins, antibodies and peptides as tools to study the role of a particular signalling pathway or protein complex in synaptic transmission has been the de velopment of methods to introduce (transduce) these reagents into tar get neurons. Here, we first discuss some considerations for the design of protein inte action-blocking peptides and then discuss two main approaches for the introduction of peptides and other macromolecules into neurons for their subsequent analysis in synapse physiology and plasticity.

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11.6.2 IDENTIFICATION OF PROTEIN–PROTEIN BINDING DOMAINS AND DESIGN OF PROTEIN–PROTEIN INTERACTION BLOCKING PEPTIDES Once a protein–protein interaction has been identified and sh wn to be direct, the next step is to identify the molecular determinants (i.e., delineate the amino acid residues that constitute the interaction domain) of at least one if not both of the binding partners (for example, the amino acid residues important for the interaction of a neurotransmitter receptor and receptor associated protein). This information can then allow the design of a short peptide or protein domain that can ef fectively outcompete the protein interaction in a cellular conte xt. The peptide can then be synthesized as a short synthetic peptide or expressed from a vector [41,113,120–122] and the functional consequences of introducing it into neurons tested. Two main protein interaction mapping techniques have been widely used for the identificatio of protein interaction binding sites, the yeast tw o-hybrid (Y2H) system and glutathione S-transferase (GST) fusion protein af finity chromatograp y (Box 11.1). Protein–protein interaction mapping studies allo w the production of a synthetic peptide that can be used to test the functional consequences of blocking the protein–protein interaction of interest (see Section 11.7). In addition, once a minimal binding domain has been identified, the t o-hybrid approach can be used to identify specific amino acid residues essential for the interaction by introducing point muta tions in the binding domains of the interacting proteins and looking for a loss of reporter gene activity. This type of molecular information can be useful as it allo ws the generation and analysis of mutant receptors that can no longer bind a particular associated protein [24]. Similarly to Y2H, GST pull-do wns and co-immunoprecipitation experiments can also be used to identify point mutations that block a particular protein interaction of interest, allowing production and analysis of mutant receptors [24,25]. Once a minimal protein–protein interaction domain has been identified, design ing a short synthetic-interaction blocking peptide that can competiti vely block this particular protein–protein interaction should then be possible (although in some cases, a lar ger protein stretch or intact domain is needed) [1,109,113,121,122,128, 130,132,138,142–151]. GST pull-do wn or co-immunoprecipitation assays are par ticularly useful for confirming the ability of a particular protein–protein interactio blocking peptide to disrupt a protein interaction (a control that cannot be easily carried out with the two-hybrid system). In this case, binding of the full-length GST fusion protein for a particular associated protein is tested in the presence or absence of the function blocking peptide and a control is carried out using a scrambled or mutated version of the same peptide that would be expected to have no effect (Figure 11.3). Similarly, in the case of co-immunoprecipitation experiments, the peptide can be added to the co-immunoprecipitation and tested for its ability to disrupt endogenous protein comple xes.

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BOX 11.1 Delineating Protein–Protein Interaction Domains by GST Pull-Down or Y2H Analysis A principal use of the tw o-hybrid system [123] has been to screen for proteins that bind a “bait” protein of interest (such as an ion channel intracellular domain or other neuronal or synaptic protein) [124]. Detailed descriptions of the Y2H system can be found else where [123,124]. Since the tw ohybrid system is an ef fective method for identifying channel and receptor -associated proteins [102,104,113,124–130], that an ob vious extension of the tw o-hybrid approach is to then use the methodology to identify a minimal protein interaction domain is perhaps not surprising. A certain amount of information regarding the binding domain in an associated protein is often obtained from the initial two-hybrid screen if multiple clones of different sizes are identified that encode the same protein. However, further information can be obtained by constructing a set of truncated v ersions of either the “bait” or “fish” and testing them for reporter gene act vity. For example, two-hybrid mapping has been used to determine the re gion in AMPAR and GABAAR intracellular domains important for interaction with se veral of their associated proteins, such as GRIP1, PICK1, SAP97 and NSF for AMPARs and plic1, GAB ARAP and GRIF1 for GAB AARs [113,126,128–134]. Another widely used approach for identifying and characterizing protein–protein interactions is glutathione-S-transferase (GST) fusion protein af finity chromatograp y, also termed GST fusion protein “pull-down.” This approach is based on the construction of a fusion protein between a protein of interest (for instance, the intracellular domain of an ionotropic receptor) and glutathione Stransferase, a 26-kDa enzyme that can bind with high af finity to glutathione in an isopro yl-b-Dthiogalactoside (IPTG)–inducible v ector such as pGEX [135]. The GST fusion protein approach allows both the production of the recombinant fusion protein in bacteria and then its easy purification by affinity chromatograp y on glutathione agarose beads [135]. In addition, the fusion protein can also be complexed with the glutathione ag arose beads to produce a glutathione ag arose bead/GST fusion protein af finity matrix that can be used to study protein–protein interactions. or example, a GST fusion protein can incubate with a mixed population of proteins from a cell lysate (e.g., from transfected cells or neurons) or with a specific purified protein of interest (e.g., another bacteriall expressed protein or protein produced by in vitro translation) and then protein complexes recovered on glutathione coupled beads. The complexes can then be analyzed by SDS-PAGE and detected by Western blotting, autoradiography or protein staining [25,102,127,136–139]. Importantly, GST alone can be used as a ne gative control. GST fusion protein “pull-do wns” can be used for se veral applications, including to “fish” for n vel interactions or confirm protein interactions identified b another method [102,138]. In a similar w ay to the Y2H method, this type of interaction assay can then be extended to map a protein–protein interaction domain or binding site by making GST fusion proteins of truncated versions of the protein or receptor intracellular domain of interest and similarly testing these truncations for the protein–protein interaction. Although the above two approaches for binding-site mapping are some of the most commonly used, several other biochemical approaches ha ve been ef fectively used to identify interacting protein binding sites. Co-immunoprecipitation e xperiments from lysates of heterologous cells e xpressing cDNA expression constructs encoding deletions of one or both of two interacting proteins of interest can be used. Other strate gies include making a prediction of the lik ely amino acid residues based on known sequence information or modelling on other similar protein interactions and then confirming the binding domain by site-directed mutagenesis of the predicted ey residues followed by biochemical confirmation [140,141]

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(a) GST GST-plic

(b)

pepPBα

control

no pep

input

GluR2m

GluR2r

GluR4cr

NSF

(c)

FIGURE 11.3 Examples of diffusion of peptides into neurons via the patch pipette. (a) Composite image (left panel) of hippocampal CA1 p yramidal neuron f lled during whole-cell patch clamp recording in an acute hippocampal slice with 500 µM biotinylated peptide (biotin-NR2aCT) in the recording pipette for 1 hour before fxation. Biotin staining was detected with Alexa568 conjugated streptavidin (scale bar: 10 µm). Close up (top right panel) of the area in the left panel outlined by the white box sho wing proximal dendrites and their spines (scale bar: 2 µm). CA1 neuron (bottom right panel) f lled for 10 min before f xation (scale bar: 10 µm). Reproduced with permission from Elsevier [144]. (b, c) Protein interaction-blocking peptides for the interaction of GluR2 with the AMPA receptor associated protein NSF (b) and the GABAA receptor with the GABAA receptor-associated protein plic1 (c), respectively. (b) Western blot with anti-NSF antibody showing the strong retention of His-NSF by GSTGluR2 C-terminus and the effects of inclusion of inactive (GluR4c) or active (GluR2r or GluR2m) peptides (100 µM) on the retention of His-NSF by GST -GluR2 C terminus. Reproduced with permission from Else vier [113]. (c) Western blot analysis of the strong interaction of myc tagged GABAA receptor a1 subunit to the UBA domain of plic1 expressed as a GST fusion protein (lanes 3 to 5) or GST alone (lane 2), in the presence of 1 µM pepPB (lane 5) or scrambled control (lane 4). Lane 1 represents 10% of the input used. Reproduced with permission from [132]. (d, e) Electroph ysiological ef fects of protein interaction-blocking peptides on e xcitatory synaptic transmission or GABAA receptor currents. (d) Summary graph of the ef fects of dialysis via the patch pipette of an NSF GluR2 interaction-blocking peptide (GluR2m, left panel) or control peptide (GluR2s) on recordings of EPSC amplitude (from 11 cells for each peptide) plotted as a function of time during dialysis of the peptide. Modif ed with permission from Elsevier [113]. (e) Time dependence of the effect of dialysis via the patch pipette of a GABAA receptor plic1 protein interaction-blocking peptide (pepPBα), or scrambled control on 10 µm GABAactivated currents in HEK293 cells e xpressing GABAA receptors (n = 4 to 7 cells for each peptide). Reproduced with permission from [132].

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GluR2m (KRMKVAKNQA)

GluR2s (VRKKNMAKQA) 120 100 80 60 40 20 0

120 100 80 60 40 20 0 (d)

control scrambled pepPBα

GABA-activated currents (normalised)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 (e)

FIGURE 11.3 (Continued.)

11.7 FUNCTIONAL STUDIES USING FUNCTION BLOCKING AND PROTEIN–PROTEIN INTERACTION-BLOCKING PEPTIDES, PROTEINS AND ANTIBODIES Although protein–protein interaction-blocking peptides are useful for confirmin that a binding site has been correctly identified, their primary use is to all w analysis of the physiological function of a particular protein–protein interaction in a process of interest. One method for introducing a function-blocking peptide of interest into neurons depends on e xpressing the peptide or protein domain from a plasmid or viral vector [41,150,151], followed by functional analysis. In addition, several methods that do not depend on neuronal transfection have been used to introduce peptides and proteins into cells, including by microinjection into the cell c ytoplasm, via dialysis from an electrophysiological recording pipette and by protein transduction.

11.7.1 INTRODUCING PEPTIDES AND PROTEINS INTO NEURONS VIA THE ELECTROPHYSIOLOGICAL RECORDING PIPETTE Whole-cell patch clamp electroph ysiology has been e xtensively used to simultaneously dialyze into a neuron a particular biologically acti ve macromolecule while

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monitoring the functional consequences on receptor -mediated currents, basal synaptic transmission or synaptic plasticity ( Figure 11.3a ). Using this approach, the consequences of se veral function-blocking antibodies, including kinesin, dynein, insulin receptor and NSF on e xcitatory synaptic plasticity ha ve been tested (Figure 11.3c) [113,114,116,117]. Similarly, function-blocking peptides that target the activity of a particular signalling pathw ay (e.g., protein kinase A inhibitory peptide) [147,152,153], cellular process (e.g., P4 endoc ytosis blocking peptide) [109,116,117,137] or cytoskeleton/actin dynamics (e.g., cofilin peptide) [154] h ve been functionally studied. The power of coupling protein–protein interaction blocking peptides with whole-cell, patch-clamp recording is highlighted by the significan progress made in functionally characterizing the role played by proteins important for re gulating the acti vity and traf ficking of ey neurotransmitter receptors. F or example, the role of se veral AMPA receptor– and NMD A receptor–associated proteins (including NSF , GRIP1 and PICK1) in e xcitatory synaptic transmission and synaptic plasticity has been studied using protein–protein interaction blocking peptides. Similar approaches ha ve also been used to study the role of inhibitory neurotransmitter receptor -associated proteins such as RA CK1, Plic and MAP1b in controlling inhibitory neurotransmission (Figure 11.3) [132,138,143]. The key roles played by se veral protein-kinase anchoring and scaf folding proteins in controlling neurotransmitter receptor phosphorylation and activity have also been demonstrated by this method [138,140,147,153,155–158]. The ability to precisely identify the molecular determinants of a particular protein interaction (often to a single amino acid) has become a po werful approach to precisely discriminate the functional role of associated proteins that interact with the same binding domain. F or example, the glutamate receptor -interacting proteins PICK1 and GRIP1 associate via the carboxyl terminal PDZ domain of the GluR2 subunit. However, the interaction of GluR2 with GRIP1 is inhibited by phosphorylation of a serine residue (S880) within this domain or when this serine is mutated to alanine. Versions of a GluR2 C-terminal domain peptide that are phosphorylated on S880, designed to mimic S880 phosphorylation (by replacing serine 880 with a charged residue such as aspartate or glutamate) or where S880 is replaced with alanine therefore only block the interaction with PICK1 b ut not GRIP1. This has allowed the production of peptides that can specifically discriminate the interactio of these tw o associated proteins and hence perform e xperiments to specificall determine the role of each in re gulating AMPAR acti vity and synaptic plasticity [121,122,145]. Similarly , the proteins NSF and AP2, both interact via the same membrane proximal binding domain in GluR2. Ho wever, point mutagenesis has identified single amino acids necessary for the select ve binding of these proteins, allowing the generation of peptides that will specifically interfere with only one o these protein–protein interactions. Again, the specificity of this approach has mad possible the discrimination of the role of these tw o associated proteins (NSF and AP2) in synaptic plasticity [68].

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11.7.1.1 Some Considerations Although the concentration of peptide to use in the intracellular recording solution must depend on the af finity of the peptide, ma y groups ha ve reported successful functional results with peptide concentrations in the re gion of 100 to 1000 µm [113,143,145,154,159] or 100 to 200 µg/ml [132,138]. Protease inhibitors (such as pepstatin, antipain, leupeptin or bestatin) at a concentration of 1 to 100 µM or 1 to 10 µg/ml can also be included to limit breakdo wn of the peptides by proteases in the cell c ytoplasm [113,143,159]. Some consideration should be made about the time for a particular peptide or protein in the recording pipette to dialyze/equilibrate into the intracellular milieu of the cell. This time will depend on se veral factors including the size (v olume) of the cell (including its cell body , dendritic arbour and axon), the size of the peptide or protein, the access at the interf ace between the pipette and the cell and the concentration of peptide or protein in the pipette solution. Se veral groups ha ve estimated the time to achieve sufficient di fusion of modulators and protein–protein interactionblocking peptides from the recording pipette into a cell [143,159,160]. The time needed for peptides to dif fuse into the cell across an idealized barrier at the end of the pipette of width w and area A can be calculated as follo ws [159]. The rate of increase of peptide concentration, C, in a cell of v olume V is given by: V

dC DA(C pipette − C ) = dt w

(11.1)

where D is the dif fusion coefficient, and Cpipette is the peptide concentration in the pipette. The pipette series resistance across the barrier is: Rs =

ρw A

(11.2)

where ρ is the resistivity of the pipette solution. From Equation 11.1 and Equation 11.2, V

dC DA(C pipette − C )ρ = dt Rs

Thus, C approaches Cpipette exponentially: C = Cpipette (1–et/τ) with a time constant of τ = (VRs)/(Dρ).

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By interpolating (on a log-log graph of D against molecular weight) literature values for sucrose (MW = 342, D = 5.2 × 1010 m2/sec) and somatostatin (MW = 1638, D = 1.66 × 1010 m2/sec), Marie and Attwell [159] estimated that an 8-mer peptide with a molecular weight near 900 w ould have a dif fusion constant of D = 2.6 × 1010 m2/sec. Assuming the resisti vity of the solution to be ρ = 0.8 m Ω, the series resistance to be Rs = 4 M Ω, and the v olume of the cell (in this case a retinal Müller cell) to be V = 1014 m3, they predicted the equilibration time constant for the peptide to equilibrate between the patch pipette and the cell v olume to be around t = 190 sec [159]. Using a similar approach, diffusion of a 28-mer peptide, with a molecular weight of 3945 and an estimated dif fusion constant D = 1.07 × 1010 m2/sec, into a bipolar cell with an estimated volume of 850 µm (for a 10-µm diameter soma, 100-µm total length of axon, plus dendrites of diameter 2 µm and a synaptic terminal of diameter 3 µm) w as estimated to tak e τ = 248 sec (assuming a series resistance of 25 M Ω and resistivity of the solution to be ρ = 0.8 Ωm) [143]. These estimates suggest that small peptides should v ery rapidly diffuse into the cell and tak e ef fect. In agreement with this result, e xperimental data support the conclusion that biologically active concentrations of peptides can be achieved inside the neuron by this method within a f ew minutes ( Figure 11.3 ) [113,128,132,138, 143,159]. F or e xample, ef fects of small peptides on synaptic currents ha ve been demonstrated within timescales of less than 10 min [57,113,117,132], pro viding good experimental evidence for the rapid ef fects of these reagents.

11.8 TRANSDUCING NEURONS WITH MEMBRANEPERMEANT PEPTIDES AND PROTEINS Although dialysis via the electroph ysiological patch pipette or via microinjection has proven to be an ef fective mechanism for introducing peptides and proteins into cells, this method has se veral limitations, including the v ery low number of cells that can be transduced, limiting many types of biochemical and cell biological assays, and the need to directly access (and potentially perturb) the cells intracellular milieu with a micro-electrode. However, several studies have now demonstrated that modifying peptide and protein sequences to render them permeant to the cell plasma membrane is possible while maintaining their ph ysiological activity [161]. Of par ticular effectiveness have been some polycationic sequences (also called cell-penetrating peptides, protein-transduction domains or membrane-translocating sequences) that are able to transport co valently (or non-covalently) attached biologically acti ve agents and macromolecules [162,163], including peptide sequences [132,142,164,165] and proteins [150,151,164], across the cell membrane in a rapid and concentration-dependent manner [161,163–165]. These types of polycationic sequences ha ve therefore been used as a method for transducing neurons with function-blocking or protein–protein interaction-blocking peptide sequences, allowing cell-based assays of ionotropic receptor function. The first membrane-translo cating sequences were identified in the Antennapedia homeobox protein ( Antennapedia, amino acids 43 through 58 [RQIKIWFQRRMKKWK]) [166] and the HIV -1

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Tat protein (T at amino acids 47 through 57 [YGRKKRRQRRR]) [161,167–169]. More recently, poly-arginine sequences (of 6 to 12 ar ginines) have been sho wn to be just as effective at mediating transport across the cell membrane [144,170]. These membrane-translocating sequences ha ve been ef fectively used to introduce biologically active or protein-interaction blocking peptides into se veral types of neuronal preparation, including cultured neurons, or ganotypic acute brain slices and neurons in vivo, and ha ve been sho wn to be ef fective by this deli very method for blocking protein–protein interactions important for neurotransmitter receptor function (Figure 11.4). For example, peptides designed to mimic an AMPAR internalization domain or to block the interaction of the GAB AAR with associated proteins plic1 and GABARAP have been introduced into cell lines and neurons via an attached Antennapedia sequence and the consequences on receptor trafficking studied [66,132,171] In addition, membrane-permeant peptide sequences are available as commercial kits (for example, Penetratin 1, Qbiogen or the Chariot peptide from ActiveMotif) that can be used to form co valent (for e xample, via thiol groups) or non-co valent complexes with peptides, proteins, antibodies and nucleic acids that can ef fectively introduce these reagents into neurons [161,172–174]. In addition to the use of peptide sequences for cell transduction, se veral groups have shown that other types of modification can also be efectively used for rendering biologically acti ve peptides, proteins and antibodies permeant to cell membranes [161,173–176]. Myristoylation of peptide sequences has proved particularly effective with respect to this goal, and se veral groups ha ve used myristo ylation of peptides to introduce these reagents into various preparations including isolated nerve terminals (synaptosomes), dissociated cultured neurons and brain slices [175,177–179]. For e xample, myristo ylated dynamin blocking p4 peptide has been used to study endocytosis of neurotransmitter receptors, including NMD ARs [179]. Similarly , Sacktor et al. ha ve ele gantly used peptide myristo ylation to introduce a peptide targeting the activity of the atypical protein kinase C (PKM) into acute brain slices to study the role of this kinase in synaptic plasticity [177,178].

11.9 CONCLUSION From the wide v ariety of approaches discussed in this chapter , an e ver-growing number of methods are clearly a vailable for neuronal transfection and transduction. Thus, neuroscientists and synaptic ph ysiologists currently ha ve at their disposal an increasingly sophisticated molecular toolbox for introducing biologically active macromolecules into neurons, greatly facilitating experiments that a few years ago would not have been possible due to technical limitations. Furthermore, this wide array of currently available approaches pro vides researchers significant f xibility to select the method best adapted to a particular e xperiment. The development of a wide range of methods for neuronal gene transfer no w allows routine transfection of most neuronal preparations routinely used by neuroscientists, including dissociated cultured neurons, acute and organotypic brain slices and neurons in vivo. This has facilitated pioneering studies on the molecular machinery important for regulating the construction, maintenance and plasticity of synapses.

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(a)

(b) NR2A

NR2B

NR1 PSD95 NR2A CT PKI IP:

+

+

−

−

+

−

+

−

−

−

+

+

−

+

−

+

NR2A/B Rb IgG Lysate (c)

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FIGURE 11.4 Transduction of neurons with membrane-permeant peptides. (a) Transduction of 10-day old primary cultured neurons with membrane-permeant peptides containing a poly (11)-R sequence. Primary cultured neurons were incubated with PKA inhibitory peptide (1 µM), (FITC-11R-G-FIASGRTGTTNA, left panel) or with an additional nuclear localization sequence (FITC-PKKKRKV-11R-G-FIASGRTGRRNAI, right panel) for 30 min, follo wed by a 30-min incubation in ne w medium. Cells were then fi ed in 4% paraformaldeh yde and analyzed by confocal microscop y (scale bar: 100 µm). Reproduced with permission from [164]. (b) Transduction of EGFP with an 11R protein transduction domain (11R-EGFP) into a hippocampal brain slice. Protein (1 µM) w as incubated with the brain slice for 30 min, followed by a further 30-min incubation in ne w media, three PBS washes and analysis of the EGFP signal by confocal microscop y. Transduction of neurons in CA1 (left panel) and CA4 and dentate gyrus (DG, right panel) could be observed (scale bar: 100 µm). Reproduced with permission from [164]. (c) Example of disruption of the interaction between an ionotropic receptor (the NMD A receptor) and a receptor -associated protein (PSD-95) in acute slices using a membrane-permeant (11R) protein interaction-blocking peptide. Acute hippocampal slices were treated for 5 hours with 10 µM fluorescein-11R-NR2aCT peptide or 10 µ fluorescein-11R-PKI peptide (which ser ed as a negative control), homogenized and extracted with 1% SDS. Immunoprecipiation of NMD A receptors w as performed with a mixture of NR2A and NR2B antibodies (four lanes on left) and also non-immune rabbit IgG as a ne gative control (middle lanes) follo wed by immunoblotting with antibodies to NR2A, NR2B, NR1 and PSD-95, as indicated. Control lysates lanes are also shown (two lanes on right). Treatment of slices with the interaction-blocking peptide clearly blocks co-immunoprecipitation of NMDA receptors with PSD-95 compared to the PKI peptide control. Reproduced with per mission from Else vier [144].

Essentially all neuronal transfection approaches, including viral, chemical, physical and electrical, are suitable for transfection of dissociated cultured neurons with the choice of approach to a certain e xtent determined by the required le vels of transfection efficien y. At present, only viral approaches have so far been consistently shown to guarantee v ery high (95%) transfection ef ficiencies of cultured neurons A future goal will be the continued improvement of neuronal transfection efficiencie using nonviral approaches that, in some circumstances, could ha ve adv antages in terms of toxicity and safety requirements and because these methods do not usually require cloning into specialized v ectors. Recent developments have already greatly improved the ef ficiencies of no viral approaches for dissociated neuronal cultures. Examples include optimization of Lipofectamine 2000 protocols, which no w give transfection efficiencies of dissociated cortical neurons in the r gion of 25 to 30% [81], and the greater than 50% neuronal transfection efficiencies demonstrated usin nucleofection [102], permitting biochemical analyses [102,104]. These significan improvements suggest that further optimization of current methods or the adv ent of new technologies could soon allo w nonviral transfection efficiencies comparable t those that can be achie ved using viral v ectors [172].

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For intact brain tissues, including acute and organotypic brain slices and neurons in vivo , viral, biolistic and electroporation-based transfection approaches ha ve proved most ef fective. An important further goal will be the combination of these methods with v ectors designed to impro ve the spatial or temporal specificity o neuronal transfection. Temporal specificit , particularly in vivo , can be achie ved using either inducible promoters or promoters whose expression is particularly wellsuited for dri ving expression during a particular windo w of neuronal de velopment (for e xample, see Chapter 13 by Osten, Dittgen and Licznerski) [180,181]. In addition, the ability to tar get a particular neuronal (or other) cell population in the brain using cell-type specific promoters is an additional important goal The ability to introduce molecules into neurons (particularly antibodies, recombinant proteins and peptides) by intracellular dialysis via the patch pipette while simultaneously monitoring electroph ysiologically the functional consequences of this interv ention on synaptic acti vity or membrane currents has no w become an established method for studying the role of particular proteins and protein–protein interactions in neurotransmitter receptor regulation, synaptic transmission and plasticity. With the rapidly expanding set of identified protein–protein interactions at th synapse, and the large amount of molecular information deri ved from the biochemical identification of the cognate-binding domains for these protein–protein interac tions, the design and functional testing of protein–protein interaction-blocking peptides of ever greater specificity ( ven where several proteins interact with the same region or binding domain in one of the proteins) [68,121,145] is an important goal. Molecular interv entions via the patch pipette will continue to be one of the k ey methods for functionally characterizing the molecular machinery that underlies the regulation of synaptic activity [121,145]. In the future, the increasing use of wholecell patch clamp electroph ysiology in vivo [182], which can be readily combined with neuronal transduction approaches [180,182], will pro ve a po werful approach for testing in indi vidual cells the ph ysiological functions of particular proteins in a physiological setting [182]. In conjunction with these adv ances, the further refine ment of membrane-permeant protein transduction-domain technologies [161] will further simplify the introduction of peptides or proteins into lar ge numbers of neurons. This development will not only f acilitate biochemical analyses in dissociated neuronal cultures and brain slices [132,144] but should also facilitate molecular interventions using these tools in vivo [142]. These advances will provide the essential framework needed for understanding the functional consequences of protein–protein interactions, from the single amino acid le vel through molecular and cellular function to netw orks of neurons in animals.

11.10 ACKNOWLEDGMENTS We are grateful to D. Attwell for the theoretical estimates of peptide equilibration between an electrophysiological recording pipette and whole-cell clamped cell, and I.L. Arancibia-Carcamo for help with the figures

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Acute In Vivo Expression of Recombinant Proteins in Rat Brain Using Sindbis Virus Hélène Marie and Robert C. Malenka

CONTENTS 12.1 12.2 12.3 12.4

Introduction ...............................................................................................241 Generation of Sindbis Viral Particles .......................................................242 In Vivo Injection of Viral Particles............................................................243 Use of Acute In Vivo Expression of Recombinant Proteins for Cell-Based Assays.................................................................244 12.5 Conclusions ...............................................................................................247 References..............................................................................................................247

12.1 INTRODUCTION The ability to molecularly manipulate protein e xpression in the rodent brain has proven to be a po werful tool in neuroscience. Specificall , the generation of transgenic mouse lines has allo wed scientists to be gin to answer important questions about the functional role of specific proteins. H wever, this technique still suf fers from the lack of strict temporal and spatial re gulation of protein e xpression as well as the possibility of genetic compensation. More recently , the e xpression of heter ologous proteins in or ganotypic brain slices and dissociated neuronal cultures has become another valuable method to study protein function in neurons. In this system, temporal regulation is achieved by acute expression of proteins using various transfection techniques or viruses encoding the protein of choice. A major advantage of this approach is that it allo ws the direct comparison of cellular properties, such as dendritic spine morphology or synaptic function, between neurons e xpressing the protein of interest and neighboring control neurons from the same animal. However, a drawback of this technique is that placing dissociated neurons or brain slices in culture invariably results in a de gree of cell death and abnormal synaptic re wiring that can perturb the results obtained from v arious cellular assays. In this chapter , 241

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we describe the use of Sindbis viral-mediated gene transfer to acutely e xpress proteins in vivo in the rat brain. This relatively new technique allows acute expression of a protein of choice in a temporally and spatially restricted manner . Importantly, the neurons expressing the recombinant proteins are allowed to do so while remaining in their physiological environment in freely behaving animals. Cell-based assays on the infected neurons can then be performed using well-established standard preparations, such as acute brain slices.

12.2 GENERATION OF SINDBIS VIRAL PARTICLES Several viral vectors, including the herpes simple x virus, the lenti virus and Sindbis virus can be used to acutely introduce genes in neurons in vivo. We have chosen the Sindbis virus system because it produces the recombinant protein of interest rapidly and is neurotropic [1]. The Sindbis virus is a member of the alphavirus family. These viruses are small-en veloped viruses with single-stranded RN A genomes [2]. The Sindbis expression system is a transient expression system in which the Sindbis lifecycle is exploited to produce recombinant proteins. Green fluorescent protein (GFP is one obvious marker of choice for monitoring the successful infection of neurons. It can be fused to the recombinant cDNA of interest or encoded as a separate protein as part of a bicistronic cDN A using an internal ribosomal entry site (IRES). We found that either combination allowed enough GFP expression for good visualization of infected neurons within 24 hours of in vivo expression. The generation of infective Sindbis virus particles has been described previously [3,4]. Briefl , the cDNA of interest is cloned into a carefully designed plasmid vector (pSinRep5) under the control of the Sindbis virus subgenomic promoter . This DNA construct is linearized and used to make recombinant RNA in vitro, which is capped, polyadenylated and then introduced into BHK-21 cells by electroporation.This RNA contains both the inserted gene of interest and the Sindbis viral genome components that are essential for the replication of the viral genome. However, it does not contain the genes for the structural proteins that are needed to generate the virus particles. These genes are pro vided by another RNA that is also transcribed in vitro from the linearized helper virus plasmid DH(26S).This helper RNA needs to be co-transfected into the BHK21 cells to pro vide the structural proteins in trans. Because the helper RNA lacks a packaging signal, the particles released by the transfected cells contain only the recombinant RN A and are ready to infect ne w cells but will only under go one round of infection. Such infection is thus termed a “dead-end” infection. After 36 to 48 hours of e xpression, most BHK21 cells should sho w signs of cytotoxicity. At this point, the supernatant of these cells containing the viral particles is collected and concentrated. We found that simple sedimentation of virus particles by centrifug ation yields titers that are suf ficient for in vivo infection. Briefl , the supernatant is first centrifuged at 2000 rpm for 5 min to rem ve cell debris. This supernatant is then ultracentrifuged in a swinging-b ucket rotor (e.g., Beckman SW40) at 30,000 rpm for 90 min (deri ved from [3]) and carefully aspirated from the top, lea ving approximately 200 µl. The remaining, usually in visible, pellet is resuspended, aliquoted in small amounts and stored at 80°C.

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The Sindbis virus has been classified as a Biosafety L vel-2 (BL-2) agent by the NIH Recombinant DN A Advisory Committee due to its lo w level of pathogenicity in humans. Experiments using this virus need to be appro ved by the administrative panel on biosafety of your institution. BL-2 facilities are used to synthesize the viruses and personnel needs to be trained to w ork with BL-2 agents. The viral particles can be inacti vated by or ganic solvents, bleach or autocla ving. The high and rapid onset of e xpression of this virus is accomplished by progressively shutting of f protein e xpression of the infected host cell. This beha vior has raised le gitimate concerns about the c ytotoxicity of Sindbis viruses. Ho wever, we have found that neurons e xpressing these v ectors in vivo for periods up to 30 hours sho wed no electroph ysiological or morphological signs of toxicity . Other investigators ha ve described “non-toxic” in vivo expression up to 48 hours [5,6]. Furthermore, less toxic, modified ersions of the Sindbis v ectors ha ve no w been engineered [7,8]. Alternatively, the lenti viral expression system can be used. Lentiviruses ha ve much lo wer potential for c ytotoxicity b ut this comes at the cost of much lower levels of e xpression of the protein of interest [9]. This system is also less neurotropic and requires, in our hands, up to se ven days of in vivo expression for suitable visualization of the infected neurons. For in vivo injection of viruses, optimizing the conditions of infection and collection of the viral particles is essential. Electroporation of the BHK21 cells should yield more than 80% transfection, as visualized by GFP e xpression. This result can be achieved by obtaining high-quality RNA and optimizing the electroporation conditions. We also found that a fe w rounds of freeze-tha w c ycles of the aliquots did not deteriorate the quality of the virus for in vivo infection.

12.3 IN VIVO INJECTION OF VIRAL PARTICLES We have adapted standard stereotaxic injection techniques to allo w micro-injection of viral particles into the CA1 re gion of hippocampi of young adult rats of postnatal days (PND) 21 through 28. Of course, of the utmost importance is that procedures for humane treatment of animals must be observ ed at all times. Belo w, we describe the procedure that we ha ve de veloped after careful e valuation of the guidelines and options pro vided by our animal f acility veterinary specialists. A PND21–28 rat is anesthetized with a k etamine/xylazine (50/4.4 mg/kg body weight) cocktail by intraperitoneal (IP) injection. As the animal becomes dro wsy 5 to 10 min after the IP injection, we increase analgesia locally by injecting b upivacaine (2 mg/kg) subcutaneously at the site of the incision. Once the animal is in deep anesthesia, as illustrated by the pinch test (i.e., no refl x should be observ ed when vigorously pinching its paw), we shave its head using an electric clipper. This first part of the procedure needs to be performed way from the sur gical area, as shaven hair is a potential hazard for infection. The animal is then mo ved to the clean sur gical area and is immobilized in a stereotaxic apparatus. The scalp is scrubbed using betadine, rinsed with 70% ethanol and, using sterile instruments, a scalp incision is made. The skin can be immobilized away from the skull using hemostatic forceps. A cannula previously filled with vira solution is placed at the site of injection using the antero-posterior and lateral

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coordinates assigned to the CA1 re gion of the hippocampus (see paragraph belo w). Using a hand-held drill, a hole (1 to 2 mm across) is made in the skull and the dura mater is opened using the bent end of a h ypodermic needle. We lower the cannula to the appropriate depth, at which point we inject the viral solution (total v olume of 0.5 µl). The injection is made using a Harv ard Apparatus injection pump at a fl w rate of 0.1 µl/min to minimize tissue damage. When the injection is complete, the cannula is remo ved slowly at a rate of 0.5 mm/min and the skin is sealed with super glue. To minimize post-operation pain, the animal is given additional analgesia in the form of a subcutaneous injection of b uprenorphine (0.03 mg/kg). The animal is then returned to its cage and allo wed to recover. A heating lamp is used to a void post-operative hypothermia and the animal is monitored for heart rate and respiration during recovery. The surgical procedure needs to be performed in a BL-2 appro ved area and the animals need to be housed in a BL-2 f acility overnight. A major task for the successful use of this technique is to find the stereotaxi coordinates that allo w reliable injections into the area of interest. Se veral adult rat brain atlases are currently available [10] but no atlases are found for the de veloping rat brain. Therefore, we obtained the appropriate coordinates for our PND21–28 rats by initially using the adult coordinates for the CA1 p yramidal cell layer of the hippocampus and modifying these coordinates by trial and error . Bregma, the point of intersection of the sagittal suture with the coronal suture, w as used as the point of reference for the lateral and antero-posterior coordinates (see [10] for details on stereotaxic coordinates nomenclature). We use the follo wing coordinates: anteroposterior, 4 mm; lateral, 2.5 mm, vertical: 2.4 mm, for injection in the CA1 pyramidal neuron layer of the hippocampus. If you plan to use an injection pump, we ha ve found the follo wing procedures help optimize the chances of obtaining a successful injection. We start the pump fl wing upon entering the brain and place the injection cannula 0.5 mm abo ve the final site of injection. At that point, the fl w of solution has often been greatly reduced or stopped and therefore we w ait until enough pressure has b uilt in the pump to resume the fl w. We then lo wer the cannula to its final destination. This trick ensures that you do not damage the area to be injected by the sudden gush of viral solution that often occurs when the fl w of solution resumes. We have found that k eeping the weight of the animal constant yields more accurate injections than solely choosing the animal by age. Also, systematically using the coordinates by carefully locating bre gma for each injection is crucial to obtain reliable injections at the desired location. Judging the best place of injection by eye is never reliable.

12.4 USE OF ACUTE IN VIVO EXPRESSION OF RECOMBINANT PROTEINS FOR CELL-BASED ASSAYS Figure 12.1 shows a successful injection of a GFP-e xpressing Sindbis virion in the CA1 layer of a PDN24 rat hippocampus after 24 hours of e xpression. Note that the virus only infects the neurons of the CA1 pyramidal layer. Thus, by expressing GFP

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24 hours in vivo expression

245

CA1 CA3

250 µm

15 µm

FIGURE 12.1 The diagram sho ws a schematic of the e xperimental protocol. Photos sho w low resolution (4 ×, top panels) and high resolution (40 ×, bottom panels) images of a hippocampal slice (left panels sho w DIC images; right panels sho w GFP fluorescence) obtaine from a rat (PND23) injected with GFP-e xpressing Sindbis virion.

along with the protein of interest (either as a fusion protein or via the use of an IRES), identifying the cells that ha ve been infected in li ving tissue using simple epifluorescent light microsco y and performing any number of cell-restricted assays is straightforward. For example, we were able to compare the detailed electroph ysiological properties of infected v ersus uninfected neighbor neurons in acutely dissected hippocampal slices from infected animals. The infected neurons sho wed healthy and stable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor– and N-meth yl-D-aspartate (NMDA) receptor–mediated e xcitatory post-synaptic currents (EPSCs) comparable to those recorded from uninfected neurons in the same slice preparation and also comparable to neurons from uninfected animals. We could maintain recordings for times suf ficient to xamine longterm potentiation ( LTP) and long-term depression ( LTD) ( Figure 12.2a ), and also perform many difficult electrop ysiological assays such as those that require minimal stimulation techniques [11]. Because standard acute hippocampal slices were used, recordings did not suf fer from the dra wbacks that often accompan y the use of organotypic brain slices, such as epileptiform activity and small, unstable responses. Other investigators have found that normal e xtracellular field potential recording could be obtained from areas of the slice containing a high proportion of infected cells, thus confirming the verall good heath of the slices [5]. Furthermore, this

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LTD −200

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Amplitude (pA)

Amplitude (pA)

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2 1

−150 −100 −50

1 0 −5 0

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20 30 Time (min)

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(a)

5 µM

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(b)

FIGURE 12.2 (Color figure fol ows page 176 .) (a) Example of a whole-cell recording obtained from a GFP-infected CA1 pyramidal neuron (PND24 rat) showing long-term potentiation (LTP) of the AMPA receptor-mediated EPSC. Sample traces were a veraged over the baseline period (1) and 45 to 60 min post L TP induction (2). (b) Example of a whole-cell recording obtained from a GFP-infected CA1 pyramidal neuron showing long-term depression (LTD) of the AMPA receptor-mediated EPSC. Sample traces sho wn were a veraged over the baseline period (1) and 35 to 50 min post L TD induction (2). Scale bar: 20 msec, 50 pA.

technique has been successfully applied to other regions of the brain such as the rat, somatosensory “barrel” corte x [5,6]. This approach also f acilitates the study of the ef fects of in vivo molecular manipulations on the morphology of neurons. By adding a fluorescent dye (e.g.

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Alexa 568 fluor ydrazide from Molecular Probes) to the whole-cell pipette recording solution, we were able to fill cells with the dye during electrop ysiological recordings. After fixing and mounting the tissue, this enabled visualization of th detailed morphology of the infected neurons using a Zeiss LSM 510 laser -scanning confocal microscope. For example, by collecting Z-stacks of parts of apical secondary dendrites and reconstructing these in 3-D using Volocity software (Improvision), we could compare the density and morphology of dendritic spines between infected and uninfected neurons ( Figure 12.2b).

12.5 CONCLUSIONS In this chapter , we ha ve briefly described the use of a viral-based gene xpression system to acutely e xpress heterologous proteins in the rat brain in vivo . We believe that this technique of fers several advantages over other approaches that are used to express recombinant proteins in vivo. In particular, it allows evaluation of the effect of acute expression of any protein of interest in a temporally and spatially restricted manner while minimizing the possibility of time-dependent compensations in response to the molecular manipulation. It also permits direct comparison of molecularly-manipulated and neighboring control neurons within the same tissue under close-to-ideal physiological conditions. However, one drawback of the use of Sindbis viruses is that the y often result in high o ver-expression of the recombinant protein. In theory, this could affect the normal functioning of the neuron as well as result in the recombinant protein ha ving effects that the endogenous protein does not. Thus, we encourage the reader to k eep up-to-date with the latest v ersions of the Sindbis viruses that sho w the least c ytotoxicity and to consider the use of other viralexpression systems such as lenti viruses, which permit lo wer-level and longer -term expression of recombinant proteins. Lenti viruses can be particularly adv antageous when using it to express RNAi to knockdown the expression of endogenous proteins.

REFERENCES 1. Washbourne, P. and McAllister, A.K., Techniques for gene transfer into neurons,Curr. Opin. Neurobiol., 12, 566–573, 2002. 2. Strauss, J.H. and Strauss, E.G., The alphaviruses: Gene e xpression, replication, and evolution, Microbiol. Rev., 58, 491–562, 1994. 3. Yuste, R., Lanni, F. and Konnerth, A., Imaging Neurons, A Laboratory Manual, Cold Spring Harbor Laboratory Press: Ne w York, 58.1–58.8, 2000. 4. Bredenbeek, P.J., Frolov, I., Rice, C.M. and Schlesinger, S., Sindbis virus expression vectors: P ackaging of RN A replicons by using defecti ve helper RN As, J. V irol., 6439–6446, 1993. 5. D’Apuzzo, M., Mandolesi, G., Reis, G. and Schuman, E.M., Abundant GFP expression and L TP in hippocampal acute slices by in vivo injection of sindbis virus, J. Neurophys., 86, 1037–1042, 2001. 6. Takahashi, T., Svoboda, K. and Malinow, R., Experience strengthening transmission by driving AMPA receptors into synapses, Science, 299, 1585–1588, 2003.

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7. Jeromin, A., Yuan, L.L., Frick, A., Pfaffinge , P. and Johnston, D., A modified Sindbi vector for prolonged gene expression in neurons, J. Neurophys., 90, 2741–2745, 2003. 8. Kim, J., Dittgen, T., Nimmerjahn, A., Waters, J., P awlak, V., Helmchen, F ., Schlesinger, S., Seeburg, P.H. and Osten, P. Sindbis vector SINrep(nsP2S726): A tool for rapid heterologous expression with attenuated cytotoxicity in neurons, J. Neurosci. Meth., 133, 81–90, 2004. 9. Quinonez, R. and Sutton, R.E., Lenti viral vectors for gene deli very into cells, DNA Cell. Biol ., 21, 937–951, 2002. 10. Paxinos, G. and Watson, C., The Rat Br ain in Ster eotaxic Coor dinates, Academic Press: New York, 1998. 11. Isaac, J.T., Hjelmstad, G.O., Nicoll, R.A. and Malenka, R.C., Long-term potentiation at single fiber inputs to hippocampal CA1 yramidal cells, Proc. Natl. Acad. Sci. USA, 93, 8710–8715, 1996.

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13

Lentivirus-Based Genetic Manipulations in Neurons In Vivo Pavel Osten, Tanjew Dittgen, and Pawel Licznerski

CONTENTS 13.1 Introduction ...............................................................................................249 13.2 Lentivirus-Based Vector Systems..............................................................250 13.3 Lentivirus-Based Heterologous Expressio n..............................................250 13.4 Lentivirus-Based Genetic Manipulation s..................................................252 13.5 Delivery of Lent iviral Particles into the Brai n .........................................253 13.6 Experimental Applications ........................................................................254 13.7 Conclusions ...............................................................................................256 13.8 Acknowledgments .....................................................................................256 References..............................................................................................................256

13.1 INTRODUCTION Various classes of retroviruses, adenoviruses and adeno-associated viruses have been successfully adapted for d evelopment of recombinant vectors with the aim of longterm gene delivery to different cell types in different tissues [1]. Lentiviruses belong to a class of retroviruses that efficiently infect both ividing and non-dividing (postmitotic) cells, making the recombinant lent iviral vectors applicable for stable, longterm gene deli very to neurons [1–4]. The latest, state-of-the-art generations of the lentiviral vectors have a large (about 9 kilobases) transfer capacity, and gene delivery via these v ectors is de void of cellular c ytotoxicity or humoral response. Thus, whereas these vectors are being developed primarily for clinical applications in gene therapy, the y pro vide an e xcellent and easy-to-use tool for gene manipulation in cultured neurons as well as in neurons in vivo, something that has been long missing in basic neuroscience research. Here, we first briefly view the history of the development of the currently used lentiviral vector systems, and then focus on the use of these vectors for gene delivery and gene knockdown in p yramidal neurons in rodent brains. We discuss the e xperimental advantages of stereotactic injections of lentiviral particles, including the high

249

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spatiotemporal control over the introduced genetic manipulation and the fact that only a small population of neurons is a ffected within otherwise intact neuronal networks. In our vi ew, lentiviral vectors provide in ma ny ways an optimal tool for the study of gene functions in small populations or even individual neurons in vivo, which can be combined with p hysiological analysis of the infected neurons either in vivo or in vitro.

13.2 LENTIVIRUS-BASED VECTOR SYSTEMS The first fficient len iviral expression system was derived from the human immunodeficie cy virus type 1 (HI V-1) and consisted of the foll owing three vectors: a packaging vector expressing the structural Gag and Gag-Pol proteins as well as regulatory and most of the viral accessory proteins; an e nvelope vector expressing heterologous surface glycoproteins — either amphotropic e nvelope of murine leukemia virus or G glycoproteins ofvesicular stomatitis virus (VSV-G); and a transfer vector containing human cytomegalovirus (CMV) promoter for heterologous protein expression [5]. The biosafety of the system was greatly impr oved in the n ext vector generations, first by deletion of all accessory genes from the packagin vector [6] and second, by the d evelopment of the so-called self-inact ivating (SIN) transfer vector, which lacks the transcriptional act ivation sites from the 3 ′ LTR U3 region ( Figure 13.1 ) [7,8]. The inact ivation of the long-terminal repeat ( LTR) transcriptional capacity is particularly important with respect to reducing the risk of v ector mobilization and recombination with latent retro viral sequences in the host cell genome. Next to the HIV -1-based e xpression systems, other primate as well as nonprimate lentivirus-based vectors have recently been de veloped [1,9]. Of these, nonprimate vectors derived from feline immunodeficien y virus (FIV) [10] and equine infectious anemia virus (EIAV) [11] are the furthest developed, and were both shown to efficiently infect neurons in vitro and in vivo. Whereas these vectors offering any advantage in terms of biosafety in comparison to HIV -1-based vectors is not clear , the EIAV vectors appear to be more efficient for gene del very via axonal retrograde transport when pseudotyped with rabies glycoproteins [12]. With respect to transfer capacity and stability of e xpression, the latest FIV and EIA V vectors appear to be comparable to those deri ved from HIV-1 [13,14].

13.3 LENTIVIRUS-BASED HETEROLOGOUS EXPRESSION Recombinant lentiviruses are typically produced (“pseudotyped”) with the VSV-G glycoproteins, mainly because it allo ws easy concentration to high titers by ultracentrifugation [15]. At the same time, because the VSV-G glycoproteins bind ubiquitous phospholipid components of the plasma membrane rather then a specific cel surface receptor, such viruses have an extremely broad host-cell range. The strength as well as cell-specificity of heterologous xpression from the VSV-G-pseudotyped lentiviral v ectors is thus determined by selection of a specific recombinan

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SIN Ientiviral vector ∆U3 R U5

R U5 RRE

promoter CMV pr.

gene

Ψ cPPT

WPRE

SIN Ientiviral vector - proviral form ∆U3 R U5

∆U3 R U5

LTR

promoter

gene

LTR

FIGURE 13.1 Latest generation of the lenti viral self-inacti vating (SIN) v ector. Schematic representation of the SIN lentiviral vector (top) and its integrated proviral form (bottom). The cis-acting elements of the v ector are indicated as white box es. In producer 293T cells, the 5 ′ CMV promoter drives transcription of the vector RNA; RRE (Rev-responsive element) allows efficient nuclear xport of the RNA; and Ψ encapsidation signal mediates RNA targeting into viral particles. Gag, Gag-pol, Rev and Env proteins, necessary for production of viral particles, are expressed from tw o or three helper v ectors provided in trans [7,8,41]. After infection of a host cell, Pol-mediated reverse transcription of the viral RNA results in formation of double stranded (ds) DN A with the U3, R and U5 re gion (long-terminal repeat, L TR) from the 3 ′ now flanking both ′ and 3 ′ ends. Note that due to the deletion TATA box and binding sites for transcription factors Sp1 and NF-B from the U3 region (∆U3), the 5′ region of the proviral form of the v ector lacks an y transcriptional acti vity — hence, the name self-inacti vating [7, 8]. The central polypurine tract (cPPT) forms a “DNA flap” structure that enhances the nuclea import of the dsDNA [42–44]; the LTRs mediate integration of the dsDNA into the host cell’s genome as a pro virus; the w oodchuck hepatitis virus post-transcriptional re gulatory element (WPRE) increases nuclear e xport of the gene of interest mRN A dri ven from the internal promoter of choice [18,45,46].

polymerase II promoter that dr ives the expression of the gene of interest. We have recently tested several promoters for expression in rat cortical pyramidal neurons in vivo [16]. This work showed that Synapsin I promoter was the most efficient durin the second postnatal week, whereas calcium/calmodulin-dependent protein kinase II (-CaMKII) promoter was the strongest from the third postnatal week on. Within 1 week from infection, the level of expression of enhanced green fluorescent protei (EGFP) was sufficient for in vivo two-photon imaging at the resolution of dendritic spines and axonal branches [16]. Both promoters drove EGFP expression selectively in neurons versus glia, and the α-CaMKII promoter was select ive for pyramidal neurons versus interneurons (some mis expression was obser ved, and is to be expected, due to int egration of the viral vector backbone to gene-rich transcriptionally active regions) [17]. The expression in cortical neurons was stable for at least 2 months (the longest period tested), and it may beexpected to stay stable throughout the life of the animal [18]. Finall y, the intrinsic properties of the infected neurons, as analyzed by electrophysiology in vitro in slices and in vivo in anesthetized animals, were not di fferent from uninfected control cells [16]. Thus, the vectors are

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well-suited for high-resolution imaging of structural synaptogenesis of cortical neurons in young as well as adult animals. Furthermore, combination of EGFP expression with expression of a second gene or d ownregulation of endogenous genes via RNA interference (see bel ow) makes possible the use of such vectors for studying gene functions in synaptogenesis in vivo .

13.4 LENTIVIRUS-BASED GENETIC MANIPULATIONS In research applications, to be able to co- express two proteins — a protein function of which one wishes to study and a fluorescent protein (typically EGFP) to labe the infected cells — is often important. An easy approach is to use a bicistronic vector, where the second protein isexpressed downstream of a viral internal ribosome entry site (IRES) ( Figure 13.2a) [19]. H owever, the IRES-based initiation of translation tends to be considerably less e fficient, resulting in an unequal expression of the two proteins. Combination of two promoters, either in tandem [20] or in a reverse orientation separated with a “stuffer” sequence [21], was described as an alternative approach for expression of t wo proteins from lent iviral vectors. H owever, we observed strong transcriptional interference between our Synapsin I and α-CaMKII recombinant promoters when adapted, in either orientation, into the self-inactivating lentiviral vector [22]. Recentl y, an el egant solution to this problem was published by the laboratory of Luigi Naldini, which sh owed that a synthetic bidirectional promoter composed of a minimal core of the CMV promoter (in one direction) and a cellular promoter (ubiquitin or phosphoglycerate kinase promoter in the opposite direction) dr ive expression of t wo proteins at a strength and cell-specificity det rmined by the cellular promoter [23]. We are presently testing this system for the αCaMKII promoter (Figure 13.2b). Gene d ownregulation (knockd own) via R NA interference (R NAi) has become an extremely popular approach to achi eve a loss-of-function manipulation in ma ny different cell types [24,25]. Brief y, introduction of double-stranded short-interfering RNAs (siRNAs, typically 21 base pairs) into a cell causes an act ivation of a multiprotein complex termed RISC (RNA-induced silencing complex), and a subsequent degradation of cellular mR NAs containing a homologous r egion to the siR NA sequence. Recently, expression of siR NAs in mammalian cells was achieved from plasmids containing a polymerase III promote r, e.g., the small nuclear R NA U6 promoter [26]. In this approach, siR NA sequence is expressed as a fold-back short hairpin R NA (shR NA) that is post-transcriptionally processed into typical siR NA via cellular RNase Dice r. We as well as others [27,28] h ave established lent iviral vectors containing one or t wo polymerase III promoters for shR NA expression together with a polymerase II promoter forexpression of a fluorescent protein (Figur 13.2c) [16]. Thus, next to the option of heterologous expression of one or two genes (above), achieving a knockdo wn of endogenous genes in neurons is no w possible — ag ain in a combination with e xpression of a fluorescent protein to label th infected cells.

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Neurospeciﬁc (bicistronic) vector

α-CaMKII promoter IRES (a) Neurospeciﬁc bidirectional vector

CTE

CMV α-CaMKII pr. promoter (b)

Vector expressing shRNAs for gene knock-down GFP U6 shRNA

Ubiquitin promoter (c)

FIGURE 13.2 Lentiviral vectors for genetic modification in neurons. (a Vector with 1.3-kb promoter fragment of α-CaMKII drives strong expression in pyramidal neurons in cortex and hippocampus. Insertion of IRES site ma kes for bicistronic vector for co- expression of t wo genes (however, note that the expression of the second gene after IRES will be considerably lower). The slashed lines indicate cloning sites for insertion of recombinant genes. The cisacting elements (white bo xes) are as in Figure 13.1. (b) Bidirectional vector for expression of two genes at equal strength from a synthetic minimal CMV -α-CaMKII promoter, created from a recently described CMV-ubiquitin construct [23]. Note that the second post-transcriptional enhancer, constitutive transport element (CTE), is inserted for the re verse-orientation gene [23]. (c) Vector for siRN A-based gene knockdo wn. One or tw o polymerase III U6 promoters expressing shRNAs are inserted in front of the ubiquitin promoter expressing GFP. Note that since the U6 promoter is ubiquitously acti ve in all infected cells, we used the ubiquitin promoter to e xpress GFP in a similar w ay.

13.5 DELIVERY OF LENTIVIRAL PARTICLES INTO THE BRAIN Lentiviral vectors were shown to efficiently infect neurons in vivo in rodent as well as primate brains in the nigrostriatal system and hippocampus [5,6,29–34]. An important aspect to consider with respect to lent ivirus delivery to the brain parenchyma is the extent of diffusion of the viral particles in the brain extracellular space (ECS). The ECS between cells in the vertebrate central nervous system is estimated to be between 20 to 40 nm [35,36]. In contrast, HI V-1 particles are quite la rge — approximately 80 to 100 nm in diameter (database of the International Committee

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on Taxonomy of Viruses, www.ncbi.nlm.nih.gov/ICTVdb) — and the particle size thus should strongly limit their free di ffusion in the brain (note that the VSV-G pseudotyped and wild-type HIV-1 particles seem to be of the same size) [37]. Fro m our experience, injections of di fferent volumes (ranging from about 25 to 200 nl ) of lentiviruses into the rat cort ex or hippocampus (animals greater than P8) resul t in infection within a spherical r egion of a similar size, approximately 400 to 60 0 µm diameter (Figure 13.3a). This result supports the notion that the viruses encounter substantial hindrance in the ECS and can achi eve only a limited spread. ( We have not tried la rger injections, as these would li kely cause some damage in the cell dense cortical or hippocampal r egions.) Because the VSV-G coat binds directly to membrane phospholipids [15], th e particles can be endoc ytosed not only into cell somata but also into dendrites an d axons within the injection area. From our experience with injections in the roden t barrel cort ex, high-rate infection in the superficial cortical layers (layer 2/3) s typically accompanied by infection of few (10 to 20) deep layer-5 pyramidal neurons that send their apical dendrites through layer 2/3 all the way to the layer 1 (Figur e 13.3c). This behavior suggests that dendritic uptak e of the virus and its subsequent transport to the soma is possible but rather inefficient. At the same time, finding ny infected neurons farther away from the injection site is extremely rare, indicatin g that axonal upta ke or retrograde transport almost ne ver occu r. Interestingl y, e ven when pseudotyped with rabies glycoproteins, HI V-1 vector-based particles are very inefficient in retrograde infection [37,38]. In contrast, rabies-pseudotyped EI V vectors appear to undergo efficient retrograde transport [12]. Present y, no explanation exists for this inconsistenc y.

13.6 EXPERIMENTAL APPLICATIONS Due to the limited spread of lent iviruses in the vertebrate ECS, injection-based delivery of the lent iviral v ectors is not practical for gene manipulations in la rge populations of neurons — for example, in an extensive cortical region or large portion of the hippocampus. On the other hand, stereotaxic injections of recombinant lentiviral particles provide a high spatiotemporal control over the genetic manipulations, making the tar geting of small neuronal populations with distinct cellular functions easy, such as a subpopulation of p yramidal neurons in the CA1 hippocampal region or in the corte x (Figure 13.3b and Figure 13.3c), at a defined time in the postnata development. In addition, the f act that only a small number of neurons is infected means that gene functions can be altered in a w ay that w ould result in a lethal phenotype or in an acti vation of compensatory mechanisms if the entire brain or whole brain subregions were altered. This might allow the study of in vivo functions of genes that otherwise w ould be possible to study only using neuronal cultures. The types of experiments that can be easily carried out with the lentiviral vectors include, for example, studies of protein trafficking when wild-type and mutant form of GFP-tagged proteins are e xpressed in vivo and analyzed for subcellular distrib ution “post-hoc” from fi ed-brain sections, or studies of cellular ph ysiology when gene functions are altered via viral infections in vivo and analyzed in vitr o for changes in synaptic transmission or synaptic plasticity in acutely prepared brain

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A1

A2

255

C

300

B CA1

600

CA3 900

FIGURE 13.3 Infection of neurons in vivo . (a) Examples of sparse and dense infections in the barrel corte x. Injection of a small v olume (about 25 nl, first panel) or a la ge v olume (about 200 nl, second panel) of the same viral stock results in a correspondingly sparse or dense infection of neurons, ho wever, within approximately same area. The dashed circle is 600 µm in diameter; scale bar: 100 µm. Viral injections were done in rats at P21 (at a depth 500 µm below the brain surf ace), and analysis of e xpression at P32 by confocal microscop y from fi ed 100-µm brain sections cut tangentially to the barrel corte x. Both images are maximal projections of 20 confocal sections separated by 2-µm z-steps. Expressed protein is RFP-tagged K+ channel. (b) Example of an infection of hippocampal CA1 pyramidal neurons. The injection w as done in mouse at P21, and analysis of e xpression at P45 by confocal microscopy from fi ed 300-µm brain sections cut horizontally . The image is a maximal projection of fi e confocal sections separated by 2-µm z-steps; scale bar: 100 µm. Expressed protein is GFP-tagged Homer1a. (c) Example of a dense infection in the barrel corte x, layer 2/3. The injection w as done in rat at P21, and analysis of e xpression at P28 by confocal microscopy from fi ed 100-µm brain sections cut coronally.The image is a maximal projection of fi e confocal sections separated by 2-µm z-steps; scale bar: 100 µm. The depth from brain surface is indicated on the left site (µm). Note that the densely infected layer 2/3 cells send their cortico-cortical axons down through deeper layers, with some branching in layer 5 (belo w 900 µm); also note fe w labeled layer 5 neurons, possibly infected via their apical dendrites. Expressed protein is GFP.

slices. We have recently sh own that the Synapsin I and α-CaMKII promoter-based vectors can also be used for in vivo two-photon time-lapse imaging of morphological dynamics of dendritic spines and axonal projections of infected EGFP- expressing

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cortical neurons [16]. Similarl y, imaging of EGFP-tagged proteins could be used for time-lapse imaging of trafficking of synaptic proteins in vivo, an application that is yet to be explored. Finally, perhaps the most exciting use of the lent iviral vectors for studying gene and cellular functions in neurons could be in combination with in vivo two-photon ta rgeted patching (TPTP) [16,39]. We h ave recently sh own a “proof of principle” for TPTP from lent ivirus infected cells by recording sensoryevoked responses and recept ive field maps from EGFP expressing layer 2/3 pyramidal neurons in the somatosensory barrel cort ex in anesthetized animals [16,40]. The method of in vivo targeted whole-cell recording from genetically altered cortical neurons can thus be applied to study gene and cellular functions in ind ividual neurons in the intact cort ex, either during early postnatal d evelopment or in the adult in a cortical region of choice.

13.7 CONCLUSIONS In summary, the approach of lentivirus-based gene manipulations in neurons in vivo, as described here, o ffers a number of experimental ad vantages for cellular neuroscience, including the ease of viral production [29], the la rge transfer capacity of the vector and the lack of any cellular or humoral response associated with injection of the virus into the brain. Stereotaxic delivery of lentiviruses to specific brain egions provides an alternative to traditional mouse genetics with high spatiotemporal control over the genetic manipulation and a short time from experimental design to data collection and analysis.

13.8 ACKNOWLEDGMENTS We thank Peter H. See burg for his generous support. D r. Dittgen is supported by GIF grant (I-733-60.13/2002) to Pavel Osten. We thank Damian J. Haydon-Wallace for comments on the manuscript.

REFERENCES 1. Kootstra, N.A. and Verma, I.M., Gene thera py with viral vectors, Ann. Rev. Pharm. Toxicol., 43: 413–439, 2003. 2. Ailles, L.E. and Naldini, L., HI V-1-derived lent iviral vectors, Curr. Top. Microbio. Immun., 261: 31–52, 2002. 3. Deglon, N. and Aebischer, P., Lent iviruses as vectors for CNS diseases, Curr. Top. Microbio. Immun ., 261: 191–209, 2002. 4. Wiznerowicz, M. and Trono, D., Harnessing HIV for thera py, basic research and biotechnology, Trends Biotech., 23(1): 42–47, 2005. 5. Naldini, L., Blome r, U., Galla y, P., Ory, D., Mulli gan, R., Gage, F.H., Verma, I.M. and Trono, D., In vivo gene delivery and stable transduction of nond ividing cells by a lentiviral vector, Science, 272(5259): 263–267, 1996. 6. Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L. and Trono, D., Multiply attenuated lentiviral v ector achie ves ef ficient gene del very in vivo , Nat. Biotec h., 15(9): 871–875, 1997.

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AMPA Receptor Phosphorylation in Synaptic Plasticity: Insights from Knockin Mice Hey-Kyoung Lee

CONTENTS 14.1 14.2

Introduction ...............................................................................................261 AMPA Receptors and Changes in Phosphorylation during LTP and LTD.............................................................................................262 14.3 Changes in GluR1 with L TP and LTD .....................................................263 14.4 GluR1 Phosphorylation: Early Expression Versus Late Maintenance of LTP? ................................................................................264 14.5 Different Mechanisms of LTP in Young Versus Old ................................266 14.6 Role of PKA in L TP .................................................................................267 14.7 GluR1 Phosphorylation Sites in L TD and Receptor Traffickin .............268 14.8 Potential Interaction between GluR1 and GluR2 Phosphorylation Sites during LTD ...........................................................268 14.9 Conclusion.................................................................................................270 14.10 Acknowledgments .....................................................................................271 References..............................................................................................................271

14.1 INTRODUCTION Synaptic plasticity in the brain has been implicated to play a role in major brain functions, including learning and memory , developmental plasticity, recovery after injury and drug addiction. The current understanding of the mechanisms of synaptic plasticity deri ves from molecular and cellular analysis of long-term potentiation (LTP) and long-term depression (LTD). LTP and LTD are readily elicited from many brain re gions with dif ferent induction and e xpression mechanisms. At least tw o

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different induction mechanisms for LTP and LTD exist, one that depends on acti vation of N-meth yl-D-aspartate (NMD A) receptors and another that does not. The expression of NMD A receptor-dependent and receptor -independent LTP and L TD seem to ha ve o verlapping b ut dif ferent signalling mechanisms [1]. Most of the molecular details on NMD A receptor -dependent LTP and L TD ha ve come from studies in the CA1 re gion of the hippocampus. At least in this re gion of the brain, regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors seems to underlie post-synaptic changes associated with NMDA receptordependent LTP and LTD. Especially, evidence exists that changes in AMPA receptor phosphorylation is one of the mechanisms critical for the e xpression of NMD A receptor-dependent bidirectional synaptic plasticity . This re view will summarize the recent findings from our ork using gene “knockin” mice lacking specific phosphorylation sites on the GluR1 su unit of AMPA receptors, and discuss the implications of our results that elucidate the basic mechanisms of NMDA receptor-dependent synaptic plasticity.

14.2 AMPA RECEPTORS AND CHANGES IN PHOSPHORYLATION DURING LTP AND LTD Fast e xcitatory synaptic transmission in the central nerv ous system (CNS) uses glutamate, which acts on various post-synaptic ionotropic glutamate receptors. Ionotropic glutamate receptors are divided into AMPA receptors, NMDA receptors and kainate receptors depending on their agonist preferences. In most CNS synapses, AMPA receptors mediate the majority of basal synaptic transmission, whereas NMDA receptors are acti vated under conditions that produce significant post-syn aptic depolarization. Kainate receptors can participate in synaptic transmission at certain synapses and are also known to play a modulatory role [2]. AMPA receptors are tetramers comprised of combinatorial assembly of four different subunits GluR1 to GluR4 (or GluR-A to GluR-D) [3–5]. Different subunits show distinct spatial and temporal distribution in the brain and confer distinct properties to theAMPA receptor complexes. For example, the GluR2 sub unit prevents Ca 2+ permeability of the ion channel and contrib utes to the linear I-V relationship of the current flux [6–12] All four sub units of AMPA receptors ha ve se veral identified phosphorylatio sites on their intracellular carboxy terminal [13]. Functions of some of the GluR1 and GluR2 phosphorylation sites ha ve been link ed to LTP and LTD. GluR1 phosphorylation at serine-831 (S831) and serine-845 (S845) w as shown to change with both LTP and L TD [14–17]. Recently , using mice that specifically lack these t o phosphorylation sites, we demonstrated that these tw o sites are essential for L TP and LTD [18]. We will discuss this result in more detail to suggest a model of ho w these tw o phosphorylation sites can contrib ute to bidirectional synaptic plasticity . As for the GluR2 subunit, phosphorylation at S880 has been implicated in mediating LTD in both cerebellum and in hippocampal CA1 re gions [19–21]. Because most AMPA receptors in the hippocampal CA1 are heteromeric complexes of GluR1 and GluR2 [5], interactions between the C-tail phosphorylation sites are lik ely. We will review data suggesting how these two subunits can interact to regulate LTP and LTD.

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14.3 CHANGES IN GLUR1 WITH LTP AND LTD The first to promote a specific ypothesis on the modifications of AMPA receptors in synaptic plasticity was Lynch and Baudry (1984). They proposed that an increase in Ca 2+ by NMDA receptor acti vation leads to more functional AMPA receptors at synapses. The predicted increase in the AMPA receptor component of synaptic transmission following LTP has been observ ed by either using specific antagonist [22–24] or by measuring post-synaptic responsi veness to e xogenously applied AMPA receptor agonists [24–26]. An increase in AMPA receptor function could be due to up-regulation of functional synaptic receptors or by modification (i.e., phos phorylation) of e xisting synaptic receptors and e vidence is found to support both mechanisms. Recent studies indicate that the tw o mechanisms might be interrelated [18,27]. The first co vincing demonstration that an increase in the number of functional synaptic receptors occurs comes from Malino w’s group. They found that L TP is associated with an insertion of AMPA receptors to synapses by visualizing GFPtagged GluR1 sub units [28] and by measuring synaptic responses from “electrophysiologically-tagged” GluR1 homomeric receptors [27,29,30]. Homomeric GluR1 AMPA receptors were functionally deli vered to synapses after L TP induction, whereas homomeric GluR2 or GluR3 AMPA receptors were inserted constituti vely [29]. Subunit-specific tra ficking rules are determined by the intracellular carboxy tail of each sub unit [29], suggesting a role of intracellular interacting proteins that bind the carboxy-tails. Recent e vidence supports a model in which the insertion of GluR1-containing receptors occur indirectly via insertion at e xtrasynaptic sites followed by a lateral mo vement into the synaptic sites [31,32]. Interesting to note is that some forms of L TP are absent in GluR1 knock out mice [33–35] (b ut see [36] for further support of a role for GluR1 in L TP). LTP and L TD are also kno wn to depend on protein kinase and protein phosphatase acti vity, respecti vely [1]. Therefore, that changes in phosphorylation of synaptic proteins would mediate the expression of LTP and LTD was suggested early [37,38]. One of the post-synaptic proteins that change phosphorylation state with LTP and L TD is the AMPA receptor. LTP induction increases phosphorylation of GluR1 on serine 831 [14,17], although serine 845 can also increase if LTP is induced following LTD induction [17]. A link between GluR1 phosphorylation and insertion of AMPA receptors into synapses also seems to e xist [27,39]. Ample evidence is found that CaMKII is in volved in LTP [40,41]; hence, one likely candidate phosphoprotein that could mediate L TP was thought to be GluR1, specifically by phosphorylation on the S831 site [14,17,42]. Hwever, mutating S831 to an alanine does not af fect the acti vity-dependent insertion of GluR1 [30]. Alternately, mutation of GluR1 S845 to an alanine is reported to pre vent activity-dependent insertion of GluR1 to synapses [27]. The latter result implies an in volvement of protein kinase A (PKA) signalling for GluR1 insertion by L TP-inducing stimulation. As will be discussed later , PKA has been implicated to play a role in L TP; however, the exact role of PKA has been debated. At this moment, the role of S831 in LTP is unclear, except that it could perhaps underlie the increase in conductance

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of AMPA receptors follo wing LTP [43]. Ho wever, whether an increase in AMPA receptor conductance following LTP exists has recently been challenged [44]. As for LTD, evidence exists that it is associated with dephosphorylation of the GluR1 subunit of AMPA receptors [15–17]. The dephosphorylation of AMPA receptors associated with LTD was specific to S845 [15–17]. H wever, S831 dephosphorylation could also be observed when LTD was followed by LTP induction [17]. As will be discussed later , the dephosphorylation of S845 seems to be one of the mechanisms that lead to removal of synaptic AMPA receptors following LTD induction [18,39], along with GluR2-dependent mechanisms [19,45–53].

14.4 GLUR1 PHOSPHORYLATION: EARLY EXPRESSION VERSUS LATE MAINTENANCE OF LTP? Recently, we provided further evidence that GluR1 S831 and S845 phosphorylation sites are indeed in volved in L TP using mutant mice specifically lacking the t o phosphorylation sites [18]. These mice were generated in Hug anir’s laboratory by replacing the genomic GluR1 sequence with a tar geting v ector construct that has both S831 and S845 mutated to alanines. We found that L TP is still present in the GluR1 phosphomutants; however, the magnitude w as less than that from wild-type littermates. This result contrasts with the GluR1 knock out mice data, where L TP induced with tetanus is absent [33,35]. Collecti vely, this data indicates that at least two mechanisms for LTP expression exist, one that depends on GluR1 and the other that requires phosphorylation of this sub unit. This data supports a model in which GluR1 insertion occurs upon LTP induction, after which the sub units are phosphorylated, allowing for stable incorporation at synapses ( Figure 14.1). Stabilization of newly inserted GluR1 is thought to depend on its attachment to post-synaptic density (PSD) proteins. A specific ypothesis put forth by Lisman proposed that activation of NMDA receptors upon LTP leads to autophosphorylation of CaMKII, which then binds the intracellular C-tail of NMDA receptors at the PSD [54]. These CaMKII molecules then act to recruit “slot” proteins for AMPA receptor insertion. An attractive candidate that can serv e as a “slot” protein is a member of a membrane-associated guan ylate kinase (MA GUK) f amily protein, SAP97 [54]. SAP97 was shown to indeed interact with the e xtreme GluR1 C-tail, which has a type I PDZ lig and motif (TGL) [55–57]. SAP97 is kno wn to interact with the intracellular population of AMPA receptors [58]; ho wever, it is also co-localized at PSDs together with GluR1 [59]. CaMKII phosphorylation of SAP97 seems to allow it to move into spines [60]. Therefore, SAP97 is a likely candidate that can mediate CaMKII-dependent insertion of AMPA receptors to synapses. In support of this, mutation of the GluR1 PDZ lig and (TGL to AGL) that disrupts its interaction with SAP97 prevents activity-dependent insertion of homomeric GluR1 AMPA receptors [30]. Furthermore, o ver-expression of SAP97 increases AMPA receptor -mediated miniature post-synaptic currents (mEPSCs) [61]. Therefore, insertion of GluR1containing AMPA receptors upon L TP seems to depend on the GluR1 interaction with PDZ proteins, a lik ely candidate being SAP97.

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% FP slope

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FIGURE 14.1 A proposed mechanism of L TP. (a) Extrapolation of L TP data from adult GluR1 double phosphomutants. Exponential decay curves were fit on verage LTP data from wild-types and GluR1 double phosphomutant homozygotes. Note that the decay time constant (t) of L TP from homozygotes is less than that from wild-types. This suggests that a defici exists in L TP stabilization in the GluR1 double phosphomutants. (b) A model proposed to explain the LTP deficits seen in adult GluR1 double phosphomutants. TP induction leads to insertion of GluR1 from rec ycling endosomes, which might not depend on phosphorylation of S831 or S845. The residual LTP seen in the GluR1 double phosphomutants are still lik ely dependent on GluR1 because no L TP in the GluR1 knock outs is found. Phosphorylation of GluR1 on S831 or S845 is necessary to stabilize the ne wly inserted AMPA receptors at the synapse. Phosphorylation of S845 can occur by association of GluR1 with PKA via SAP97 and AKAP interaction as discussed in the te xt.

Interestingly, SAP97 interacts directly with AKAP79/150, which brings PKA in close proximity to the GluR1-containing AMPA receptor comple xes [56]. In addition, the formation of this GluR1-SAP97-AKAP comple x significantly increase phosphorylation of GluR1 at S845 [56,62]. This result provides an attractive molecular mechanism in which phosphorylation may stabilize synaptic GluR1.We propose the following model for L TP induction, which is based on a model suggested by Lisman’s group [40,54] and on our interpretation of the data from the GluR1 phosphomutants. We surmise that L TP induction produces “slots” for GluR1-containing AMPA receptor insertion to synapses. A recent study suggests that the insertion happens from AMPA receptors on rec ycling endosomes [63].

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CaMKII-dependent phosphorylation of SAP97 can traf fic these intracellular pool of AMPA receptors to the PSD. The ne wly inserted AMPA receptors are then phosphorylated at S845 by the recruitment of AKAP79/150 by SAP97. The S845 phosphorylation in turn can stabilize the GluR1-containing AMPA receptors at the PSD. Our model predicts that synaptic GluR1 are highly phosphorylated at S845 under basal conditions. Consistent with this idea, PKA acti vation does not enhance basal synaptic AMPA receptor function [15,64,65]. In contrast, application of a PKA inhibitory peptide (PKI) depresses synaptic AMPA receptor responses [15]. Moreover, GluR1 subunits isolated from the PSD cannot undergo further phosphorylation by PKA [66], despite the f act that when looking at the total pool of GluR1, PKA activation can greatly increase S845 phosphorylation [16,67–69]. Collecti vely, this data suggests that the synaptic AMPA receptors could be already fully phosphor ylated at the PKA site, and the maintenance of their phosphorylation requires an ongoing PKA activity. Our hypothesis is that the ongoing PKA acti vity is provided by the recruitment of PKA by GluR1-SAP97-AKAP complex formation at synapses. In support of this, post-synaptic injection of inhibitory peptide that pre vents PKA binding to AKAP causes a “run down” of synaptic AMPA receptor-mediated currents [62,64]. The stability of phosphorylated AMPA receptors at synaptic locations can be interpolated from reports that dephosphorylation of GluR1 S845 is associated with an increase in receptor internalization [18,39]. We will discuss this topic in more detail later.

14.5 DIFFERENT MECHANISMS OF LTP IN YOUNG VERSUS OLD One of the findings from the GluR1 phosphomutants is that the deficit in TP was only present in adult animals (2 to 3 months of age) and not in young animals (3 to 4 weeks old) [18]. This result is in line with the observation that LTP defici in GluR1 knock outs are also only seen in adults [33–35], and suggests that a developmental switch e xists in the mechanisms of L TP such that it is initially independent of GluR1 and becomes GluR1-dependent. This interpretation is seemingly at odds with a pre vious study by Malino w’s group sho wing L TPdependent GluR1 insertion in “young” or ganotypic hippocampal slice cultures [28–30]. However, the dif ference could be due to the f act that Malino w’s group was transfecting GFP-tagged GluR1, which forms homomeric receptors. Thus, the discrepancy might shed light on additional re gulatory mechanisms in nati ve heteromeric AMPA receptor comple xes. Developmental changes in L TP mechanisms are well-documented [70–74]. A wealth of e vidence is found that L TP in mature animals require CaMKII acti vity [74–76]. However, in neonates (P7-8), LTP is not dependent on CaMKII but on PKA activity [73]. Interestingly, around the second week of postnatal age, L TP is shown to depend on concurrent activation of CaMKII and PKA or CaMKII and PKC [72]. These results indicate that the protein kinase activity required for LTP changes during postnatal development.

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At the level of AMPA receptors, the expression of different subunits show distinct temporal patterns during de velopment. F or instance, GluR1, GluR2 and GluR3 subunits sho w a gradual increase in protein le vel during postnatal de velopment, whereas the GluR4 sub unit is e xpressed at a high le vel during the early postnatal week and gradually diminish o ver time [35,71]. In addition, a minor isoform of GluR2, which has a long C-tail (GluR2 long), shows a peak expression level between the first and second postnatal week [70]. Because di ferent sub units and splice variants confer distinct properties on AMPA receptor function and traf ficking, th mechanisms of regulation will likely change depending on the sub unit composition of synaptic AMPA receptors. In accordance with this f act, recent data suggests that in neonates, spontaneous acti vity can deli ver homomeric GluR4 receptors to synapses, which is dependent on the phosphorylation of a PKA site (S842) [27]. However, the synaptic deli very of GluR4 homomeric AMPA receptors is absent when the animals are older than P10 [70,71]. Coincidentally , the insertion of GluR2long into synapses is sho wn to account for at least 50% of the potentiation following LTP induction between the first and second postnatal week [70].Therefore, GluR4- and GluR2 long-dependent mechanisms lik ely play a dominant role in L TP expression in young animals. As the e xpression le vel of GluR1 and α-CaMKII increases as the animals mature, L TP then becomes more dependent on these tw o molecules.

14.6 ROLE OF PKA IN LTP The role of PKA in adult L TP has been mainly restricted to the late phase L TP (L-LTP). Initial studies using bath application of PKA inhibitors sho wed that only the late maintenance phase of L TP (3 hours or more after induction) is block ed [77,78]. These results led to the conclusion that the early phase of T LP is independent of PKA activity. However, studies e xist demonstrating that e ven the early phase of LTP (less than 1 hour after induction) could be inhibited by PKA inhibitors [65,79,80]. In any case, the effect of PKA inhibitors on LTP, whether the inhibition occurs earlier or later , seems to be by af fecting the stability of potentiation: L TP is initially induced to a normal magnitude b ut fails to stabilize. The interpretation of the role of PKA in L TP has been that it acts to “g ate” plasticity by counteracting protein phosphatase activity [65,80,81]. Another line of evidence supporting the involvement of PKA in LTP comes from studies using drugs to increase intracellular cAMP le vels. Increasing intracellular cAMP with either activators of adenylyl cyclase or cAMP analogs produces synaptic potentiation, which is long-lasting [82–84]. Recently , a chemical method to induce LTP was developed using a cocktail of drugs aimed at increasing intracellular cAMP levels [85]. Evidence is also found that a transient increase in PKA acti vity exists following LTP induction [86]. Because the increase in PKA acti vity was transient, PKA likely triggers a cascade of e vents leading to stabilization of L TP. According to our model, one of the molecules that could mediate the stability of LTP is AMPA receptor via its phosphorylation of GluR1 on the S845 site.

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14.7 GLUR1 PHOSPHORYLATION SITES IN LTD AND RECEPTOR TRAFFICKING LTD is associated with a dephosphorylation of GluR1 S845 [15–17], and this w as subsequently shown to be necessary for L TD [18]. The role of GluR1 phosphorylation sites in L TD is probably by mediating AMPA receptor internalization upon dephosphorylation [18,39]. LTD has long been sho wn to depend on the acti vity of v arious protein phosphatases [87,88]. In addition, LTD is known to dephosphorylate a post-synaptic PKA substrate [15], one of which is GluR1 on S845 [15–18]. The dephosphorylation of GluR1 S845 is lik ely mediated by a protein phosphatase cascade in volving protein phosphatase-1 (PP1) and calcineurin (PP2B) [16,17]. Both PP1 and PP2B are localized to post-synaptic sites via interacting molecules [89,90], which brings them in close vicinity to possible synaptic substrates, including AMPA receptors. Synaptic localization of protein phosphatases is critical for LTD because disrupting the interaction between protein phosphatases and their synaptic anchors pre vent LTD [91]. Dephosphorylation of GluR1 S845 follo wing L TD induction can be studied biochemically using a chemical method for inducing L TD (ChemL TD) [15,16]. Using this chemL TD method, GluR1 S845 dephosphorylation w as sho wn to be linked to the internalization of synaptic AMPA receptors [18]. chemL TD-induced internalization of AMPA receptors was absent in mice lacking both S831 and S845 phosphorylation sites, indicating that the phosphorylation sites are critical for the activity-dependent internalization [18]. Because chemL TD is associated with a dephosphorylation of the S845 site [15,16,18,92], the interpretation is that this phosphorylation site is critical for AMPA receptor internalization follo wing LTD. In addition, the f ate of the internalized AMPA receptors seems to depend on the phosphorylation state of GluR1 S845. F or e xample, dephosphorylation of S845 traffics the internalized receptors to the lysosome for d gradation, whereas phosphorylation at this site allo ws reinsertion into the plasma membrane [39]. Accordingly, persistent dephosphorylation of GluR1 S845, as observ ed follo wing L TD induction [16,17], could lead to degradation and down-regulation of synaptic AMPA receptors. Indeed, LTD-induced degradation of AMPA receptors has been observ ed [93].

14.8 POTENTIAL INTERACTION BETWEEN GLUR1 AND GLUR2 PHOSPHORYLATION SITES DURING LTD Despite the evidence linking GluR1 phosphorylation sites and AMPA receptor internalization following LTD [18,39], the majority of studies point to the GluR2 subunit as playing a critical role for activity-dependent AMPA receptor internalization [94]. In addition, a recent study suggests that GluR2 plays a dominant role in determining the fate of AMPA receptors internalized by activity [53]. However, these observations do not preclude a role for GluR1 in either initiating or stabilizing internalizedAMPA receptors. Because the majority of synaptic AMPA receptors are heteromers of

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GluR1 and GluR2 [5], that the two subunits could coordinate the activity-dependent internalization of AMPA receptors is possible. Activity, either in the form of glutamate receptor agonists or synaptic activation, can internalize AMPA receptors [94]. Activity-dependent internalization of AMPA receptors occur via clathrin-coated pits and require the action of dynamin [49,95,96]. AMPA receptors destined to internalize are tar geted to clathrin-coated pits via interacting with a clathrin adaptor protein AP2 [48,49,95]. Although the outcome of different forms of activity leading to AMPA receptor endocytosis is the same, the molecular mechanisms seem to vary [96]. For instance, AMPA receptors internalized by AMPA treatment end up in the lysosomes, whereas NMD A-induced internalization targets internalized AMPA receptors to the rec ycling endosomes [39,53]. Evidence suggests that acti vity-dependent internalization of AMPA receptors relies on the interaction of carboxy-terminal tail of GluR2 sub unit to se veral of its binding partners [19,21,46,49,53,96,97]. The GluR2-dependent internalization of AMPA receptors seems to rely on phosphorylation of S880 on the e xtreme carboxy-terminal, which conforms to a type II PDZ ligand [52,98,99]. Phosphorylation of GluR2 on S880 shifts the balance of GluR2 interaction with GRIP/ABP to Pick-1 [98,99]. Pick-1 w as originally identified as protein interacting with PKC [100,101]. Pick-1 is thought to brin PKC into the vicinity of synaptic AMPA receptors [102], probably by its ability to form homodimers [101,103]. Because GluR2 S880 can be phosphorylated by PKC [98,99], Pick-1 is in a position that can mediate increases in GluR2 S880 phosphorylation. Subsequent studies demonstrated that GluR2 S880 phosphorylation is in volved in LTD both in cerebellum [20,104,105] and in hippocampal CA1 [19,21,51]. The role of PKC in cerebellar L TD has been well-characterized [105–109]. Hence, a likely scenario for cerebellar L TD is that its induction leads to PKC acti vation and recruitment of PKC to synapses via Pick-1, which then phosphorylates GluR2 S880 to mediate endoc ytosis [20,104,105]. In contrast, hippocampal L TD is associated with a decrease in PKC activity [110,111]. Therefore, that GluR2 S880 is phosphorylated by PKC during L TD is unlik ely. The identity of the in vivo protein kinase responsible for phosphorylating GluR2 S880 in the hippocampus is currently unknown. Although overwhelming evidence exists indicating a critical role of GluR2 for AMPA receptor endocytosis associated with LTD, it is likely not the only mechanism. In support of this idea, knock out mice lacking GluR2 or double knock out of GluR2 and GluR3 still exhibit LTD [112,113]. These results indicate that even in the absence of the GluR2 sub unit, LTD can still be e xpressed, presumably, by the remaining GluR1 subunit. Thus, that tw o independent mechanisms for L TD expression exist seems plausible: GluR1-dependent and GluR2-dependent. At present, how these two mechanisms would interact with each other is not clear. One possibility is that GluR1 dephosphorylation allo ws GluR2-dependent receptor internalization to tak e place (Figure 14.2a ). Alternatively, GluR1 dephosphorylation could stabilize or all ow degradation of AMPA receptors internalized by GluR2-dependent mechanisms (Figure 14.2b). In either case, both scenarios can e xplain the observ ation that GluR1 phosphomutants lack L TD [18] and that blocking GluR2-dependent mechanisms

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P

P

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OH P

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P

O

O H

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HO

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LTD P

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O OH

OH

P

O H

P

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(b)

FIGURE 14.2 Two alternative mechanisms for LTD. (a) LTD induction leads to dephosphorylation of GluR1 sub units at S845 sites. This dephosphorylation signals the acti vation of internalization machinery, which could be mediated by GluR2-dependent mechanisms. Recent data suggest that AMPA receptor endocytosis can occur at extrasynaptic endocytic “hot spots” [114,115]. Dephosphorylation of S845 can allo w lateral traf ficking of the AMPA receptors from synaptic sites to these endoc ytic “hot spots. ” Once at these sites, GluR2-dependent mechanisms could aid in clathrin-coat mediated endoc ytosis of the AMPA receptors. (b) Alternatively, LTD induction might first lead to internalization ofAMPA receptors via GluR2dependent mechanisms. In this case, phosphorylation of GluR2-S880 can allo w mobilization of synaptic AMPA receptors to endocytic sites and internalization via clathrin-coat dependent mechanisms. The internalized receptors need to be dephosphorylated at S845 on the GluR1 subunit to stay in the intracellular pool. AMPA receptors that do not under go dephosphorylation of the GluR1 are lik ely recycled back to synapses [39]. Dephosphorylated receptors could be tar geted to the lysosome for de gradation [39].

prevent LTD expression [19,21,94]. Although conceptually GluR1 and GluR2 likely interact to mediate L TD, whether this interaction e xists in vivo is unclear at this point and a waits further investigation.

14.9 CONCLUSION The use of gene knockin mice lacking specific phosphorylation sites on the GluR subunit has pro vided insights into the molecular mechanisms of L TP and LTD. In addition, it allows testing of the in vivo role of the specific GluR1 phosphorylatio sites. One important advantage of using GluR1 phosphomutants is that it allo ws for a straightforward interpretation of data by directly studying a do wnstream target of

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the signalling cascades. In contrast, studies on intermediate signalling molecules (i.e., protein kinases and phosphatases) are limiting because of the pleiotropic nature of their actions. The future use of mice lacking specific phosphorylation sites o residues on AMPA receptors will allo w for a more detailed understanding of the molecular mechanisms of bidirectional synaptic plasticity . Especially, further w ork is needed in understanding ho w dif ferent sub units interact with each other in a heteromeric complex to re gulate AMPA receptor function.

14.10 ACKNOWLEDGMENTS The author w ould like to thank A. Kirkwood for helpful discussions.

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62. Tavalin, S.J., Colledge, M., Hell, J.W ., Langeber g, L.K., Hug anir, R.L. and Scott, J.D., Regulation of GluR1 by theA-kinase anchoring protein 79 (AKAP79) signalling complex shares properties with long-term depression, J. Neurosci., 22(18): 3044–3051, 2002. 63. Park, M., Penick, E.C., Edw ards, J.G., Kauer , J.A. and Ehlers, M.D., Rec ycling endosomes supply AMPA receptors for LTP, Science, 305(5692): 1972–1975, 2004. 64. Rosenmund, C., Carr, D.W., Bergeson, S.E., Nilaver, G., Scott, J.D. and Westbrook, G.L., Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons, Nature, 368(6474): 853–856, 1994. 65. Blitzer, R.D., Wong, T., Nouranifar, R., Iyeng ar, R. and Landau, E.M., Postsynaptic cAMP pathw ay g ates early L TP in hippocampal CA1 re gion, Neuron, 15(6): 1403–1414, 1995. 66. McGlade-McCulloh, E., Yamamoto, H., Tan, S.E., Brickey, D.A. and Soderling, T.R., Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II, Nature, 362(6421): 640–642, 1993. 67. Roche, K.W., Tingley, W.G. and Huganir, R.L., Glutamate receptor phosphorylation and synaptic plasticity. Curr. Opin. Neurobiol., 4(3): 383–388, 1994. 68. Mammen, A.L., Kameyama, K., Roche, K.W. and Huganir, R.L., Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II, J. Biol. Chem ., 272(51): 32528–32533, 1997. 69. Banke, T.G., Bowie, D., Lee, H., Hug anir, R.L., Schousboe, A. and Traynelis, S.F., Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase, J. Neurosci., 20(1): 89–102, 2000. 70. Kolleker, A., Zhu, J.J., Schupp, B.J., Qin, Y., Mack, V., Borchardt, T., K ohr, G., Malinow, R., Seeburg, P.H. and Osten, P., Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors, Neuron, 40(6): 1199–1212, 2003. 71. Zhu, J.J., Esteban, J.A., Hayashi, Y. and Malinow, R., Postnatal synaptic potentiation: Delivery of GluR4-containing AMPA receptors by spontaneous activity, Nat. Neurosci., 3(11): 1098–1106, 2000. 72. Wikstrom, M.A., Matthews, P., Roberts, D., Collingridge, G.L. and Bortolotto, Z.A., Parallel kinase cascades are in volved in the induction of L TP at hippocampal CA1 synapses, Neuropharmacology, 45(6): 828–836, 2003. 73. Yasuda, H., Barth, A.L., Stellwagen, D. and Malenka, R.C., A developmental switch in the signalling cascades for L TP induction, Nat. Neurosci., 6(1): 15–16, 2003. 74. Kirkwood, A., Silva, A. and Bear, M.F., Age-dependent decrease of synaptic plasticity in the neocorte x of alphaCaMKII mutant mice, Proc. Natl. Acad. Sci. USA , 94(7): 3380–3383, 1997. 75. Silva, A.J., Stevens, C.F., Tonegawa, S. and Wang, Y., Deficient hippocampal long term potentiation in alpha-calcium-calmodulin kinase II mutant mice, Science, 257(5067): 201–206, 1992. 76. Giese, K.P., Fedorov, N.B., Filipk owski, R.K. and Silv a, A.J., Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in L TP and learning, Science, 279(5352): 870–873, 1998. 77. Frey, U., Huang, Y.Y. and Kandel, E.R., Ef fects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons, Science, 260(5114): 1661–1664, 1993. 78. Matthies, H. and Reymann, K.G., Protein kinase A inhibitors prevent the maintenance of hippocampal long-term potentiation, Neuroreport, 4(6): 712–714, 1993. 79. Musgrave, M.A., Ballyk, B.A. and Goh, J.W ., Coacti vation of metabotropic and NMDA receptors is required for L TP induction, Neuroreport, 4(2): 171–174, 1993.

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97. Osten, P., Khatri, L., Perez, J.L., Kohr, G., Giese, G., Daly, C., Schulz, T.W., Wensky, A., Lee, L.M. and Zif f, E.B., Mutagenesis re veals a role for ABP/GRIP binding to GluR2 in synaptic surf ace accumulation of the AMPA receptor , Neuron, 27(2): 313–325, 2000. 98. Matsuda, S., Mika wa, S. and Hirai, H., Phosphorylation of serine-880 in GluR2 by protein kinase C pre vents its C terminus from binding with glutamate receptor interacting protein, J. Neurochem., 73(4): 1765–1768, 1999. 99. Chung, H.J., Xia, J., Scannevin, R.H., Zhang, X. and Huganir, R.L., Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins, J. Neurosci., 20(19): 7258–67, 2000. 100. Staudinger, J., Zhou, J., Bur gess, R., Elledge, S.J. and Olson, E.N., PICK1: A perinuclear binding protein and substrate for protein kinase C isolated by the yeast tw ohybrid system, J. Cell Biol., 128(3): 263–271, 1995. 101. Staudinger, J., Lu, J. and Olson, E.N., Specific interaction of the PDZ domain protei PICK1 with the COOH terminus of protein kinase C-alpha, J. Biol. Chem., 272(51): 32019–32024, 1997. 102. Perez, J.L., Khatri, L., Chang, C., Sri vastava, S., Osten, P . and Zif f, E.B., PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surf ace le vels of the AMPA-type glutamate receptor subunit 2, J. Neurosci., 21(15): 5417–5428, 2001. 103. Xia, J., Zhang, X., Staudinger , J. and Hug anir, R.L., Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1, Neuron, 22(1): 179–187, 1999. 104. Matsuda, S., Laune y, T., Mika wa, S. and Hirai, H., Disruption of AMPA receptor GluR2 clusters following long-term depression induction in cerebellar Purkinje neurons, EMBO J., 19(12): 2765–2774, 2000. 105. Chung, H.J., Steinberg, J.P., Huganir, R.L. and Linden, D.J., Requirement of AMPA receptor GluR2 phosphorylation for cerebellar long-term depression, Science, 300(5626): 1751–1755, 2003. 106. Linden, D.J. and Connor, J.A., Participation of postsynaptic PKC in cerebellar longterm depression in culture, Science, 254(5038): 1656–1659, 1991. 107. De Zeeuw, C.I., Hansel, C., Bian, F ., Koekkoek, S.K., v an Alphen, A.M., Linden, D.J. and Oberdick, J., Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the v estibulo-ocular refl x, Neuron, 20(3): 495–508, 1998. 108. Xia, J., Chung, H.J., Wihler, C., Hug anir, R.L. and Linden, D.J., Cerebellar longterm depression requires PKC-re gulated interactions between GluR2/3 and PDZ domain-containing proteins, Neuron, 28(2): 499–510, 2000. 109. Goossens, J., Daniel, H., Rancillac, A., van der Steen, J., Oberdick, J., Crepel, F., De Zeeuw, C.I. and Frens, M.A., Expression of protein kinase C inhibitor blocks cerebellar long-term depression without af fecting Purkinje cell excitability in alert mice, J. Neurosci., 21(15): 5813–5823, 2001. 110. Hrabetova, S. and Sacktor, T.C., Bidirectional regulation of protein kinase M zeta in the maintenance of long-term potentiation and long-term depression, J. Neurosci., 16(17): 5324–5333, 1996. 111. Thiels, E., Kantere wicz, B.I., Knapp, L.T ., Barrionuevo, G. and Klann, E., Protein phosphatase-mediated regulation of protein kinase C during long-term depression in the adult hippocampus in vivo, J. Neurosci., 20(19): 7199–7207, 2000. 112. Meng, Y., Zhang, Y. and Jia, Z., Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3, Neuron, 39(1): 163–176, 2003.

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Genomic and PostGenomic Tools for Studying Synapse Biology Josef T. Kittler and Peter L. Oliver

CONTENTS 15.1 15.2

Introduction ...............................................................................................279 ORFeome Projects: The Mammalian Gene Collection and Other Providers of Full-Length cDN A Collections .................................280 15.3 Inferring New Gene Function from Protein-Protein Interaction Datasets .................................................................................................... 283 15.4 Whole Genome shRNAi Libraries............................................................286 15.5 The Mouse as a Cornerstone for Inte grating Genomic and Postgenomic Resources ...........................................................................288 15.5.1 A Mouse Knock out for Ev ery Gene ............................................289 15.5.2 Phenotype-Driven Mouse Mutagenesis .......................................290 15.5.2.1 Spontaneous Mouse Mutants .......................................290 15.5.2.2 Stargazer, Lurcher and Hotfoot Mutants: Insights into Glutamate Receptor Gating, Assembly and Trafficking ...........................................291 15.5.2.3 Large-Scale Neurological Random Mutagenesis Screens ....................................................293 15.5.2.4 New Strategies for the Generation and Identification of Mouse Mutant ..................................294 15.6 Conclusion.................................................................................................295 References..............................................................................................................295

15.1 INTRODUCTION Over the last 10 years, lar ge-scale genomic sequencing has resulted in the completion of the mouse and human genomes and those of se veral invertebrate model organisms, including the nematode worm and fruit fl . Of the approximately 30,000 279

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proteins that are lik ely to e xist in a particular mammalian genome, as man y as 30% of these are estimated to be represented in the brain. Ho wever, reductionist approaches (such as one gene or one protein at a time) ha ve to date pro vided functional information for only 10 to 15% of predicted proteins. Consequently , a large gap exists between the number of kno wn genes and the identification of th corresponding proteins’ function. Similarly , the synaptic function is not kno wn for the vast majority of all proteins expressed in the brain, or even the 500 to 1000 components of the post-synaptic density (PSD) re vealed by proteomic analyses [1–6]. Therefore, one long-term goal of neuroscience research is to understand the role each and every gene plays in regulating aspects of nervous system function and synaptic ph ysiology. The availability of genomic data sets is greatly f acilitating the implementation of additional “genome-wide” projects, providing new research tools and reagents to the wider scientific communit . Although varied in approach, these programs ha ve all benefited from the vailability of genomic sequence information and ha ve the common goal of determining functions for the entire set of genes of a particular organism. These resources therefore have direct applicability to gene function determination in the nerv ous system. Se veral of the man y projects include: the identification and cataloguing of the entire collection of xpressed proteins (transcriptome) for a particular or ganism, including human, mouse, rat, fly and orm, and the determination of their e xpression pattern and subcellular localization; the identification of the protein constituents (proteome) of particular tissues, cell types o subcellular compartments (for instance, the PSD); the systematic identification o the entire complement of protein-protein interactions for a particular or ganism or biological process within that or ganism; the systematic knockdo wn/disruption of every gene in a particular or ganism by RNA interference (RNAi) or gene knock out approaches; and the production of lar ge sets of ph ysiologically rele vant mutant phenotypes in several model organisms, including the mouse.These ongoing projects are beginning to pro vide new data sets and resources that can be directly accessed by the neuroscience community and which, o ver the ne xt few years, will pro vide an expanding source of information and reagents that can be directly implemented into studies of neuronal ph ysiology. The a vailability and standardization of such datasets will benefit biologists in the long term, whereby less time producing simila reagents on a small scale will be required. In addition, these resources will also allow a “system’s biology” approach to understanding brain function and the re gulation of the synapse [7]. Here, we review some of the newly available genomic and post-genomic resources, which could ha ve a direct rele vance for current and future studies of synapse biology .

15.2 ORFEOME PROJECTS: THE MAMMALIAN GENE COLLECTION AND OTHER PROVIDERS OF FULLLENGTH cDNA COLLECTIONS A number of commercial and academic groups, including Origene, the Mammalian Gene Collection (MGC), the Cancer Genome Anatomy Atlas, the Brain Molecular

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Anatomy Project and se veral ef forts by RIKEN, are currently establishing fulllength, open reading frame (ORF) cDN A collections for a number of or ganisms including rat, mouse and human [8–10].The mammalian gene collection, established at the end of the last decade, is a trans-NIH initiati ve to generate full-length ORFs for every gene in several species, most notably in rat, mouse and human. This effort has in volved the systematic analysis of e xpressed sequence tag (EST) sequences from a large set of cDNA libraries (over 110 human and 80 mouse) generated from various tissues, cell lines and de velopmental stages [9–12]. Because the random selection approach for obtaining full-length ORFs has be gun to reach saturation point, MGC has also more recently implemented the tar geted reco very of cDN A clones to allow isolation of genes that, because they are underrepresented in presently available cDNA libraries, are absent from the MGC collection [11]. Currently , the MGC collection consists of almost 13,000 human genes and o ver 11,000 mouse genes and is aiming to reach full co verage of all ORFs in the human, mouse and rat genomes. At present, the MGC ORF collection has been obtained from libraries constructed with a v ariety of dif ferent backbone v ectors, several of which are v ery limited with respect to possible do wnstream functional applications. Ho wever, an increasing number of MGC clones are a vailable in vectors that are compatible with the expression of in vitro cell-free systems (using T7 or SP6 promoters) or expression in mammalian cells with c ytomegalovirus (CMV)-based promoters and that can therefore be used for downstream applications such as protein-protein interaction or expression studies. In addition, several vectors are compatible with recombinatorial cloning approaches allowing rapid transfer into other vectors; for more information, see the MGC website ( Table 15.1). Importantly, MGC clones are available without restriction to the academic scientific community through the IM GE consortium distribution netw ork. In the U.S., distrib utors include the American Type Culture Collection, Invitrogen, Inc. and Open Biosystems. In Europe, MGC clones can be obtained through MRC Geneservice and the RZPD German Resource Center for Genome Research. In addition, se veral websites, including those at Open Biosystems, MRC Geneservice, the MGC website and the National Center for Biotechnology Information (NCBI), provide easy-to-use web-based search tools to identify and obtain clones of interest. Other groups that are establishing collections of full-length cDNA clones include the RIKEN collections of ESTs and full-length mouse ORFs. The RIKEN mouse cDNA clone collection is being de veloped by the Genome Exploration Research group at RIKEN’ s Genomic Sciences Center using proprietary , full-length cDN A technology [13–17]. They have established the RIKEN FANTOM (functional annotation of mouse cDNA) clone set that comprises over 60,000 functionally annotated, full-length open reading frames [14]. RIKEN F ANTOM clone sets and indi vidual FANTOM clones, in addition to RIKEN ESTs (2,000,000 clones) are distrib uted to the research community by DN AFORM (Table 15.1). In addition to academic/governmental initiatives, several commercial entities are also constructing full-length clone collections pro viding both whole cDN A collections and clones on an indi vidual basis. Among these are Origene, which has one of the lar gest collections (T rueclone) containing 24,000 human clones constructed using proprietary technology that is well-suited for the capture of lar ge RN A

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TABLE 15.1 Full-Length Open Reading Frame Clone Resources Mammalian Gene Collection RIKEN Mammalian Gene Collection clones

RIKEN clones Origene Trueclone clones Mammalian Gene Collection Invitrogen GATEWAY vectors Hartley Lab GATEWAY vectors

Cloning Projects http://mgc.nci.nih.go http://genome.gsc.riken.go.jp Clone Providers http://mgc.nci.nih.gov/info/Buy http://www.atcc.org.catalogue/molecular/index.cfm http://clones.invitrogen.com/cloneranger.php http://www.geneservice.co.uk/products/ http://www.rzpd.de http://www.dnaform.co.jp http://www.origene.com GATEWAY Compatible Vectors http://mgc.nci.nih.gov/Vectors http://www.invitrogen.com http://www.geneservice.co.uk/products/plasmidvectors

transcripts and Invitrogen (Table 15.1). Trueclone clones are available in a uniform vector expression system that can be used for s everal d ownstream applications, including expression in cell-free and mammalian expression systems. However, a number of limitations are currently found with ma ny of the clones available from these full-length cD NA projects described ab ove. Most notabl y, a significant majority of the clones in these collections contain ′ and 3 ′ untranslated regions (UTRs). The presence of these UTRs precludes the subcloning and expression of the corresponding proteins as fusion proteins with N- or C-terminal tags [18,19]. In addition, only some of the backbone vectors used in full-length cloning projects are compatible with an expression setting (for instance, the expression of in vit ro cell-free systems or in mammalian cells) or with recombinatorial cloning approaches. As a result of these limitations, several groups are now using polymerase chain reaction (PCR) with ORF-specific primers (designed on the basis of compu tational predictions) to produce n ew ORF collections containing only a transcriptsopen reading frame in standardized vectors systems that also allow for rapid recombinatorial cloning [19–21]. For example, the laboratory of Dr. Marc Vidal has already produced ORFeome collections containing almost 20,000 genes from C. el egans and 8000 from humans [19,22,23]. Both these vectors are compatible with the GATEWAY recombinatorial system and can be subcloned into any expression vector using traditional restriction enzyme and li gase cloning methods. The full clone collections or ind ividual clones from ORFeome projects such as C. el egans ORF clones release 1.1 or Human ORF v1.1 described ab ove are readily available to the academic community from suppliers such Open Biosystems.

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In the near future, comprehensi ve collections of cDN As covering all expressed genes in rat, mouse, human and other or ganisms will provide a way for researchers interested in a single cDNA clone or family of proteins to rapidly and cheaply obtain a full-length, open reading frames for their genes of interest (approximately $40/clone within a few days from some providers). In addition, because ORF clones will increasingly be a vailable in v ectors compatible with recombinatorial cloning approaches, this will also allo w researchers to rapidly subclone a gene of interest into an ever-growing set of acceptor vectors allowing easy transfer into prokaryotic, mammalian, viral or insect e xpression systems. F or example, a set of 50 dif ferent acceptor v ectors compatible with the In vitrogen Gate way recombination system (and, therefore, with man y ORF clones) has been constructed by the laboratory of Dr. James Hartle y (SAIC-Frederick, International Corporation). These vectors that comprise different combinations of fusion protein and tags for expression in various settings are available to the research community ( Table 15.1 ). These FL-ORF resources can therefore be used for se veral types of applications, such as o verexpression studies in cell lines or neurons, to produce proteins for structural studies or antibody production or for protein-protein interaction studies. In addition, the availability of FL-ORF clones that can be rapidly transferred to other acceptor vectors in a high-throughput (HT) approach using recombinatorial cloning technology will greatly f acilitate the use of these clone sets in do wnstream applications such as HT yeast tw o-hybrid (Y2H) or other protein interaction assays such as protein-protein interaction arrays [22,24]. For instance, this will allow screens to be carried out with the intracellular domain of a receptor or other synaptic protein of interest for protein interactions ag ainst clone sets representing the entire transcriptome, or functionally rele vant transcriptome subset such as all the members of a particular protein family (e.g., kinases) or subcellular structure (e.g., the PSD). Other HT screens using these resources could include localization studies with tagged FLORFs to screen for all proteins localized to a particular neuronal subcellular compartment such as axons, dendrites, synapses or spines [25].

15.3 INFERRING NEW GENE FUNCTION FROM PROTEIN-PROTEIN INTERACTION DATASETS Protein-protein interactions underlie most biological processes [26], including much nervous system biology. For example, the NMDA receptor at excitatory synapses is estimated to be in a comple x with as man y as 180 other proteins [3–5]. From the direct interaction of proteins with neurotransmitter receptors to the comple x networks of protein-protein interactions that organize the PSD, protein-protein interactions play a crucial role in re gulating the acti vity and strength of synapses [3–5]. Identifying and characterizing the entire complement of protein-protein interactions in a neuron, synapse or receptor comple x can therefore help to understand man y aspects of nerv ous system function and can also pro vide ne w insights into the function of a particular gene of interest. Since its first description by Fields and Song [27], the Y2H system has provided an invaluable approach for the identification of a la ge number of protein-protein

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interactions important for the normal and pathophysiological function of the nervous system. For example, Y2H screens with the intracellular domains of neurotransmitter receptors such as AMPA-type glutamate receptors and GA BA receptors h ave identified everal proteins important for the tra fficking and localization of these neu rotransmitter receptors such as GRIP1 and GA BARAP, respectively [28,29]. More recently, proteomics approaches (greatly facilitated by genome and transcriptome datasets) h ave helped to catalogue the entire synaptic components list of the preand post-synaptic domains [3–6,21,30,31]. These approaches h ave identified m ny of the components that r egulate membrane trafficking and synaptic t rgeting of the main neurotransmitter gated ion channels, in addition to isolating constituents of the synapse and the molecular machinery underlying synaptic plasticity and learning and memory. The goal is to identify all the direct (binary) protein-protein interactions for a particular brain (o rganism), neuron, synapse or receptor compl ex. The life-science community will contri bute considerably to this e ffort by carrying out la rge-scale protein-protein interaction mapping e fforts for the whole transcriptome of a particular organism. These studies will pr ovide new information for researchers about a particular protein of interest (e.g., th ey could r eveal pr eviously unkn own protein interactions and hence protein functions) but will also all ow a “system ’s biology” approach to be formulated to globally study all the protein-protein interactions important for a particular process, for example, synaptic plasticity [7]. Impressively, such “genome-scale” protein interaction mapping efforts (interactomes) have already been described for s everal i nvertebrate model o rganisms and with the arr ival of similar interactome maps for mammalian genomes in the near future, interactomes will emerge as a p owerful tool for better understanding ma ny biological processes [32,33]. The first genome-wide eukaryotic protein-protein interaction map, or interac tome, available to the scientific community was for the yeast Saccharomyces, defining tens of thousands of ovel protein interactions [34–39]. More recentl y, in t wo landmark studies, the interactome maps of t wo multicellular o rganisms, Caenorhabditis el egans and Drosophila melan ogaster, h ave been reported [24,40–42] (Table 15.2). Despite di fferences in nervous system complexity, comparative genomics of the eukaryotic genomes suggests that striking conser vation

TABLE 15.2 shRNAi Resources Hannon Lab Bernards Lab

Arrayed shRNAi Projects http://katahdin.cshl.org:9331/RNAi_web/scripts/main2.pl http://biomedicalgenetics.nl/Members/Bernards/Berns.TableS1.xls

MRC GeneService Open Biosystems

shRNAi Clone Providers http://www.geneservice.co.uk/products/ http://www.openbiosystems.com

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exists between man y components of indi vidual biological processes, including much of the molecular machinery essential for neuronal function. Because important protein-protein interactions (including those for ion channels and synapseassociated proteins) are lik ely to be conserv ed between species, the information generated from in vertebrate interactome maps could pro vide a v aluable tool for identifying no vel conserv ed protein-protein interactions that are important for mammalian nervous system function [43]. To be able to use the information from in vertebrate protein-protein interaction maps (Table 15.2) to identify potential mammalian protein-protein interactions for a particular protein, identifying the orthologous genes between the tw o genomes of interest is first necessar . This can be achie ved by hand simply by carrying out a BLAST search with a mammalian protein, taking the most homologous (orthologous) match in yeast, w orm or fly and using this i vertebrate orthologue to directly search one of the web-based search engines pro vided for the dissemination of these invertebrate interactome datasets (Table 15.2). Using this type of approach, we have identified s veral novel mammalian protein interactions with the GAB AAR associated protein GABARAP. More recently, several groups have used nearest-neighbor analysis and the InP aranoid algorithm on a genome scale to search for sets of orthologous protein-protein interactions [32,43]. Using this approach, Lehner and Fraser et al. have generated a “putative” first draft human protein-protein interactio map based on the data available from the lower eukaryotic protein-protein interaction maps [32,44,45]. The work by Lehner and Fraser describes a network of over 70,000 predicted physical interactions between approximately 6,200 human proteins, predicting interactions for as man y as a third of human genes, including almost 500 human disease genes and 1500 genes of unkno wn function [32,44]. These original invertebrate protein-protein interaction datasets and the predicted draft human protein-protein interaction map can be directly searched online for novel protein-protein interactions to a protein of interest (T able 15.2). The information generated in this study and that of the original in vertebrate protein-protein interaction maps can provide a rich source of information about no vel protein-protein interactions for neuroscience research and could be particularly useful for generating ne w information about unknown synaptic genes identified from proteomics of the PSD or recepto complexes (Table 15.2). In addition to the gro wing number of medium- and lar ge-scale efforts used to identify binary (direct) protein-protein interactions using HT techniques including Y2H and other in vitro interaction approaches such as protein arrays, a large amount of low throughput molecular interaction data is also continually being reported. One recent estimate of such small-scale results reported in 110 journals suggests that almost 2000 interactions are published monthly , almost as man y as the estimated 2600 molecular interactions reported per month from HT approaches [46]. As a result, a v ery rapidly gro wing v olume of publicly a vailable e xperimental data on physical protein-protein interactions is to be found. In an attempt to curate and report this information in a usable manner , bioinformatics ha ve e volved in maintaining web-based protein-protein interaction databases and accompan ying softw are, with “open database” policies making their information freely accessible to the research community. Several of these databases are pro viding their information in

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machine-readable archi ves, pro viding platforms to easily search these databases directly while also allo wing the information to be accessed/interf aced with other third-party bioinformatics tools and softw are [39]. An increasing number of databases exist (for a list of databases, see Table 15.2 ) including the Biomolecular Interaction Network (BIND), which now contains over 100,000 records of molecular interactions [46] (including protein-protein but also protein-DNA and protein-RNA) from both HT data submissions and hand-curated information g athered from the scientific literature [47–49]. In addition, BIND is part of a net ork of other collaborating interaction databases with similar goals — the International Molecular Interaction Exchange (IMEx) consortium, which includes the database of interacting proteins (DIP), Molecular INT eraction database (MINT), IntAct and MIPS. Other databases include the mammalian Protein-Protein Interaction database (PPI), the General Repository for Interaction Datasets (GRID), and the Human Protein Inter action Database (HPID) [21,46–50]. The above examples and others have the common goal to catalog all e xperimentally determined interactions between proteins, combining information from a v ariety of sources to create a single, consistent set of protein-protein interaction datasets a vailable to the research community .

15.4 WHOLE GENOME shRNAi LIBRARIES An effective method for studying the role of a particular gene or set of genes in a biological process of interest is to study the functional consequences of disrupting or silencing the gene. The observ ation that small double-stranded RN A (dsRNA) molecules (also termed small interfering dsRNAs, or siRNAs), designed to be identical to a tar get gene will silence that gene through the specific destruction of it mRNA, has lead to RNA interference (RNAi) rapidly becoming the method of choice for gene silencing in a v ariety of mammalian cells including neurons [44,51–56] (see also Chapter 10 by Jacob for a detailed description of the methodology and application in neurobiology). Two main approaches e xist for RN Ai in mammalian cells: either dsRNAs are synthesized chemically or , alternatively, a vector directing the transcription of a short hairpin RN A (shRNA) is used. The transcribed shRN A is processed by enzymes in the cell to generate an siRNA that will silence the target gene. Because shRNAs are encoded by DN A vectors, they have the advantage that they can be introduced into mammalian cells (including neurons) or used to generate transgenic mice, using standard transfection techniques or viruses, including adenovirus, lentivirus and retro virus [53–55]. shRN A vectors also allo w both transient or stable transfection under either constitutive or inducible promoter systems [53–55] and also allow for the long term e xpression of the siRN As, which facilitates monitoring the consequences of long-term silencing of a particular gene. Whereas RNAi in model organisms such as the nematode w orm or fruit fly ha been used on a “genome scale” to study the consequences of silencing almost all genes in those or ganisms, RN Ai in mammalian systems has to date mainly been applied to the analysis of single genes or small-scale attempts to tar get genes in a particular gene f amily or signal transduction pathw ay [57,58]. Recently , however, in tw o landmark studies, the tools to allo w genome-wide RN Ai screens of gene function in mammalian cells ha ve been reported. Using similar retro viral shRNA

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TABLE 15.3 Protein-Protein Interaction Maps and Databases Yeast: PathCalling Yeast interaction database C. elegans: Worm Interactome Version 5 (WI5 i View) Fruit fly: Drosophila interaction database Human: Sanger Center interaction map

Biomolecular Interaction Net work Database (BIND) Database of Interacting Proteins (DIP) Molecular Interactions Database (MINT) General Repository for Interaction Datasets (GRID) Mammalian Protein-Protein Interaction Database (PPI) Human Protein Interaction Database (HPID) IntAct Protein-Protein Interaction Database Munich Information Center for Protein Sequences (MIPS)

Searchable Maps http://portal.curagen.com/cgi-/bin/com.curagen.portal. servlet.PortalYeastList http://vidal.dfci.harvard.edu http://portal.curagen.com/cgi-bin/interaction/flyHome.p http://www.sanger.ac.uk/PostGenomics/signaltransduction/ interactionmap Databases http://www.bind.ca http://dip.doe-mpi.ucla.edu http://160.80.34.4/mint/index.php http://biodata.mshri.on.ca/grid/servlet/Index http://fantom21.gsc.riken.go.jp/PPI http://www.hpid.org http://www.ebi.ac.uk/intact/index.jsp http://defiant.inf.ed.ac.uk:800 http://mips.gdf.de

vector-based approaches, the groups of Rene Bernards (Netherlands Cancer Institute) and Gr eg Hannon (Cold Spring Harbor Laboratories) reported the construction of arrayed shR NA libraries from chemically synthesized oligonucleotides, ta rgeting approximately a third of all known human genes [56,59]. The Bernards group human shRNAi library is based on the pSuper R NAi system d eveloped by Brummelkamp et al. [60] and ta rgets approximately 8000 human genes [59]. The pSuper shR NA vector system uses the polymerase III Histone H1-R NA promoter to produce 21 nucleotide siR NAs and can be used as a standard shR NA vector or to produce shRNA-expressing retr ovirus, all owing either transient or stable transfection of mammalian cells ( Table 15.3). Another human shRNAi vector library has been constructed by Gr eg Hannons’ laboratory (Table 15.3). Importantl y, Hannons’ group has also constructed an shRNAi library to ta rget the genes in the mouse genome that has direct applicability to many in the neuroscience community. At present, the Hannon human shRNAi library comprises over 23,500 sequence-verified sh NA expression cassettes covering over 16,300 human genes, whereas the mouse shR NAi library comprises over 13,800 sequence-verified sh NA expression cassettes ta rgeting over 9,800 mouse genes

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[56]. The mouse and human libraries are being continually e xpanded with the goal of producing shRN A constructs for e very gene in the mouse and human genome. The backbone plasmid designed by the Hannon laboratory (pShag Magic Version 2.0) has several useful features. It can be used as a standard v ector or packaged into a retrovirus and has puromycin selecti vity, facilitating the generation of stable cell lines. In addition, the vector has been adapted for recombinatorial cloning using the recently developed MAGIC system [56,61]. The inclusion of MAGIC system compatibility allows the rapid transfer of the pShag Magic shRN A expression cassette into other destination v ector delivery systems such as lenti viruses or adeno viruses. Importantly, these shRNA libraries are a vailable to the scientific communit , either as the entire v ector collection, as gene f amily sets or on an indi vidual clone basis through cDNA collection providers including MRC Geneservice and Open Biosystems. The human shRNA sets can be used for RN Ai experiments in human-derived neuronal cell lines and could also potentially be used for gene silencing in human neural stem cells. Of perhaps greater use will be the mouse library , which can be used for silencing e xperiments in mouse cultured neurons and mice in vivo . The availability of these libraries to the research community provides a valuable resource for easily obtaining shRNAi constructs to any human or mouse gene of interest and will also provide for the possibility to carry out lar ge-scale loss-of-function genetic screens in mammalian cells. F or example, the use of these libraries in combination with HT imaging approaches [25] will allow functional screens to identify molecules important for processes such as neurite outgro wth, neuronal polarity formation and spine and synapse formation.

15.5 THE MOUSE AS A CORNERSTONE FOR INTEGRATING GENOMIC AND POSTGENOMIC RESOURCES The sequencing of the human genome has revealed the presence of 25,000 to 35,000 genes, although functional information is available for only a fraction (around 15%) of them. An important goal for neuroscience research will be to annotate the human genome with functional information re garding the role of these genes in brain function and neurological disease and, for a number of reasons, the mouse will play a vital part in this research. First, in contrast to other currently a vailable genetically tractable model organisms, many similarities are found between mouse and human development, anatomy, ph ysiology, beha vior and disease. In addition, the mouse genome has a high le vel of synten y to the human sequence, with almost all mouse genes ha ving homologs in humans. The completion and refining of the mous genome and rapid e xpansion of se veral FL-ORF cloning and sequencing projects are pro viding a lar ge amount of high-quality sequence data for both the mouse genome and transcriptome, allowing a highly accurate picture of the entire complement of expressed mouse genes to be established. In conjunction with this, an e vergrowing number of tools exists for the targeted manipulation of the mouse genome, the implementation of which is being greatly facilitated by completion of the Mouse Genome Project. Of particular use has been the tar geted mutagenesis of a specifi

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gene of interest by homologous recombination in embryonic stem (ES) cells, allowing the addition (knock-in) or remo val (knock out) of an y gene of choice [62,63]. To complement this targeted approach, recently established large-scale mutagenesis approaches are generating an ever-expanding collection of mutant ES cells and mice. In combination with other lar ge-scale mouse genomics ef forts [1,64–69], such as neuronal e xpression and protein-protein interaction data and arrayed libraries of FL-ORFs and shRN Ai vectors for e very mouse gene, this pro vides a v ast resource directly available to the neuroscience community .

15.5.1 A MOUSE KNOCKOUT

FOR

EVERY GENE

The use of knock out mice, particularly the ability to both spatially and temporally control the elimination of a particular gene, has already led to man y new insights and discoveries regarding the role of particular synaptic proteins or neurotransmitter receptors within the context of synaptic transmission, neuronal plasticity and behavior [62]. More recently , knock-in technology , whereby a particular gene can be replaced with a modified ersion of the same gene, for instance, an ion channel lacking a particular phsophorylation site, protein-protein interaction domain (such as PDZ lig and) or allosteric modifier site, has been used (see also Chapter 14 by Lee). This type of approach has been ele gantly used to study the role of α-amino3-hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptor phosphorylation and associated proteins in various aspects of plasticity, learning and memory [70,71]. Similar strategies have allowed a detailed analysis of the role of GAB AAR subunit heterogeneity in aspects of mouse beha vior and neural netw ork function [72–74]. From such examples, mouse transgenic approaches have clearly played a crucial role in studies of neuroph ysiology; ho wever, despite adv ances in gene tar geting technology, over 90% of all human genes still have no corresponding mouse mutant line. To address this so-called “phenotype gap,” a knockout mouse or mouse mutant representing every gene would be a valuable resource for academic research and the pharmaceutical industry [75–78]. Based on the efficien y of standard gene-targeting methodologies, this resource w ould not be a viable proposal; ho wever, recent advances in ES cell manipulation has made this goal a reality . This “gene-trap” approach is based on the random disruption of genes by inte gration of a selectable marker, with the adv antage that a small sequence tag can be generated for each individual insertion, allo wing instant identification of the ta geted locus. The gene-trap vector, typically containing a splice donor and acceptor site to f acilitate integration, is introduced into ES cells by electroporation follo wed by selection for the reporter gene. A number of independent groups have begun to generate thousands of indi vidual ES cell lines with their corresponding sequence tag [75,76,78,79]. Some limitations to the gene-trap approach e xist, for instance, gene-trapping has been shown to be biased towards larger transcription units and the true nature of the null-allele can also be of concern, with many trapped genes showing “leaky” expression that can only be determined e xperimentally once the mouse line has been rederived. Notwithstanding these limitations, lar ge sets of gene-trap mutagenized ES cells, representing mutations in tens of thousands of genes, are currently vaailable to the neuroscience communit y, in ma ny cases without cha rge ( Table 15.4 ).

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TABLE 15.4 Gene Trap and ENU Resources Gene Trap Searchable Databases German Gene Trap Consortium http://www.genetrap.de Mammalian Functional Genomics Center http://www.escells.ca BayGenomics http://baygenomics.ucsf.edu Lexicon Genetics http://www.lexgen.com ENU Mutagenesis Mouse Genome Project http://www.mouse-genome.bcm.tmc.edu MRC-Harwell ENU Mutagenesis Program http://www.mgu.har.mrc.ac.uk/mutabase Jackson Laboratory http://www.jax.org.nmf Tennessee Mouse Genome Consortium http://www.tnmouse.org Northwestern University http://genome.northwestern.edu GSF (Germany) http://www0.gsf.de/ieg/groups/genome/enu.html

Researchers can identify cell lines containing gene-trap insertions rel evant to their genes of interest by searching online databases using homology searches orkeywords for a specific gene These mutant ES cell lines can then be used to produce the corresponding mutant mouse o r, with the increasing availability of protocols to differentiate mouse ES cell into neurons in vitro [80,81], gene-trap ES cell resources can also be used to produce neural stem cell lines with a mutation of interest.

15.5.2 PHENOTYPE-DRIVEN MOUSE MUTAGENESIS With the time and cost constraints of co nventional gene ta rgeting, additional HT approaches for the generation of mouse mutant models are clearly required. Other disadvantages of traditional targeted mutagenesis include embryonic lethality and functional redundancy due to genetic compensation. Considering these limitations and the fact that spontaneous mouse mutants have provided a great deal of valuable insights into neuronal function, a number of la rge-scale mouse mutagenesis projects h ave been established that are pr oviding a rich resource of n ew mutant lines rel evant to neurop hysiological research. A major ad vantage of the use of spontaneous mutant or randomly mutagenized mice is that noa priori assumptions are made about the role of a particular gene or expected phenotypic outcome. Instead, a potentially interesting or clinically rel evant phenotype dr ives the research process, facilitating the discovery of novel gene function. The significan insights revealed regarding ionotropic glutamate receptor r egulation gained from studies of naturally occurring spontaneous mouse mutants suggests that the generation of la rge numbers of mouse mutant lines will pr ovide n ew information about synapse r egulation and plasticit y. 15.5.2.1 Spontaneous Mouse Mutants Historically, the identification of naturally occurring mutants relies on experienced research staff and technicians to identify unusual beh avior among the thousands of

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inbred mice handled e very week. Once a mouse has been isolated and inheritance of the trait confirmed, pathological characterization and cloning of the mutated gen can commence by mapping in a genetic cross. Such positional cloning strate gies were a considerable undertaking until the late 1990s; without access to genomic sequence, a highly detailed genetic map w as required in combination with o verlapping large-insert genomic clones (yeast or bacterial artificial chromosomes) for gen identification and ventual sequencing [82]. Man y dif ferent types of spontaneous mutations in mice ha ve been identified, including single nucleotide substitutions large deletions resulting in the loss of multiple genes as well as smaller intragenic deletions, DNA insertions and chromosomal rearrangements such as in versions and translocations [63,83]. Although genetic mapping is still the cornerstone of mouse cloning, the time tak en to identify mutations has reduced considerably in recent years thanks to the advent of automated sequencing in addition to web-based genome annotation interfaces, allowing candidate genes in a defined inter al to be identifie in seconds as opposed to months or e ven years. 15.5.2.2 Stargazer, Lurcher and Hotfoot Mutants: Insights into Glutamate Receptor Gating, Assembly and Trafficking A striking example of spontaneous mouse mutants that ha ve revealed key information about the functional re gulation of neurotransmitter receptor function and synaptic transmission are the stargazer, lurcher and hotfoot mutants. The stargazer (stg) mouse w as initially described in 1990 [84] and tw o allelic v ariants, waggler and stargazer 3J , ha ve since been identified. Stargazer mutants e xhibit seizures and cerebellar ataxia [85–88] and, due to intronic disruptions, the y ha ve dramatically reduced levels of mRNA for the stargazin protein, a 36-kDa four-pass transmembrane protein with structural similarity to the skeletal muscle voltage gated calcium channel gamma sub units [89]. Although initially demonstrated to be a calcium channel accessory subunit [89], calcium currents appear to be normal in stg/stg cerebellar granule cells. However, the observation that stargazer mice lack functional AMPARs on cerebellar granule cells suggested the star gazin protein could in addition play an essential role in the synaptic targeting of these glutamate receptor subtypes essential for fast synaptic transmission [90]. Further studies ha ve revealed that in cerebellar granule cells in stg/stg mice, AMPARs are mainly retained intracellularly as a result of which mossy fiber to granule cell synapses instg/stg mice have very few AMPARs and no synaptic currents [91]. Additional experiments confirmed the essential rol of stargazin, as AMPAR responses in stg/stg granule cells can be rescued by expression of e xogenous stargazin [91]. Star gazin directly associates with AMPARs and, in addition, interacts with the synaptic scaf fold protein PSD-95 via a carboxyl terminal PDZ ligand at the C-terminus of stargazin [91]. This interaction is essential for stargazin’s ability to recruit AMPARs to synapses. In addition, star gazin plays a critical role in the surf ace targeting and transport of AMPARs [92–94], which in part could be due to star gazin’s interaction with the unfolded protein response in the endoplasmic reticulum (ER) and the protein nPIST in the Golgi [95]. Star gazin defines a amily of transmembrane AMPAR re gulatory proteins (T ARPS) that comprise stargazin, γ-3, γ-4 and γ-8 b ut not related proteins, and mediate surf ace

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expression of AMPARs [96]. Additional studies ha ve no w re vealed that star gazin can form an acti vity-dependent but tight comple x with a lar ge proportion of nati ve AMPARs and can be viewed as a AMPAR auxilliary subunit [97–99]. In agreement with this, the TARP/stargazin protein f amily is important for re gulating se veral aspects of AMPAR trafficking [100–102]. More recent studies h ve also re vealed that TARPs can directly re gulate the function and acti vity of AMPARs including their deactivation and desensitization kinetics [103,104]. Insights into the mechanisms of e xcitatory synaptic transmission, glutamate receptor trafficking and ating have also been gained from studies of the lurcher and hotfoot mouse mutants. First described in 1960, the heterozygous lurcher (Lc/+) phenotype is characterized by a w obbling, ataxic g ait observ able in the second postnatal week caused by a loss of cerebellar Purkinje cells [105]. Lc/+ animals live a normal lifespan; however, homozygous ( Lc/Lc) mice only survive for a few hours after birth due in part to significant neurodgeneration in the hindbrain and brainstem [106]. The mutation responsible for the Lc phenotype was identified by positiona cloning in a highly conserv ed domain in transmembrane domain III (TM3) of the mouse glutamate receptor gene GluR2 (Grid2). Although a member of the lig andgated ionotropic iGluR f amily, both Grid2 and related Grid1 f ail to display ion channel function alone or with other iGluRs nor do the y bind glutamate, leading to their classification as orphan receptors [107].Grid2 knockouts (Grid2–/–) also suffer from ataxia and impaired synaptic plasticity, although no Purkinje cell degeneration occurs; however, heterozygous mice ( Grid2+/–) are essentially normal, suggesting a gain-of-function mechanism for the Grid2Lc allele [108]. Electrophysiological studies on P10–11 cerebellar slices re vealed that Lc/+ Purkinje cells ha ve a significantly higher depolarized resting potential and mem brane conductance than wild-type, indicati ve of lar ge constitutive inward current [109]. To confirm whether this phenomenon as a direct consequence of the mutant allele, reconstitution of homomeric Grid2Lc channels in Xenopus laevis oocytes was carried out, producing a dramatic depolarization in the resting potential [109]. Substitution of the large organic cation N-methyl-D-glucamine (NMDG) for most of the e xternal Na + significantly decreased this depolarization, sh wing a constitutive Na + current was responsible. Moreo ver, this study demonstrated that wildtype Grid2 receptors are also lik ely to form functional homomeric channels in vivo. The lurcher mutation is situated at the e xtreme extracellular end of TM3 in Grid2, within a highly conserv ed motif found in all iGluRs; introduction of the lurcher mutation into an AMPA receptor (GluR1Lc) and kainate receptor (GluR6Lc) does not affect the formation of functional channels and renders them constitutively active in the absence of ligand binding [110]. Despite this, glutamate was still able to acti vate currents through GluR1Lc whereas channel desensitization and deactivation kinetics were significantly sl wed. The gating-related region of iGluRs has since been proposed to be mediated by four TM3 helices that move apart to f acilitate pore opening. In the presence of the lurcher mutation (Alanine to Threonine), the larger side chain of the substituted amino acid prevents complete closure of the g ate, resulting in a constituti ve current and slo w inactivation even in the absence of a lig and [110–114].

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At least 16 spontaneous examples of the hotfoot GluR2 mutants have been found to date [115]. The earliest of those were characterized by mild ataxia; neuroanatomical analysis of the cerebellum re vealed a small proportion of Purkinje cells with ectopic spines de void of pre-synaptic innerv ations [116]. Although direct comparisons between the hotfoot mutants in vivo has been some what compounded by differing genetic backgrounds [115], the y have provided a useful tool for the study of receptor assembly at the synapse. Unlik e the dominant lurcher mutations, the hotfoot alleles are typically recessi vely inherited loss-of-function mutations that do not result in Purkinje cell death. F or example, the ho4J ataxia mutation results in the loss of almost half of the extracellular amino-terminal leucine/isoleucine/valinebinding protein (LIVBP)-lik e domain, causing retention of Grid2 molecules in the ER and consequently disrupting its normal traf ficking and oligomerisation [117] Indeed, this re gion of Grid2 is defined as a hot-spot for mutations, with smalle deletions in other alleles (ho7J, ho11J, ho12J and ho9J) causing similar ER processing and transport defects [118]. Furthermore, such mutations also reduced the inter action between indi vidual Grid2 molecules, pre venting the formation of functional channels. From structural modeling, the deletions in the LIVBP-lik e domain were predicted to be present in the re gions required for dimerisation, suggesting that weakly or non-associated Grid2 proteins w ould be mis-folded, causing retention in the ER [118]. An N-terminal traf ficking signal has been reported for GluR1 an GluR2 [119], thus the hotfoot mutations might additionally cause loss or improper presentation of a similar motif. Further supporting e vidence for these h ypotheses came from the introduction of the ho4J mutation into GluR1, which also disrupted both receptor trafficking and oligomerisation [111–113] That such a lar ge number of spontaneous mutant lines (lurcher and hotfoot) in a single gene are a vailable is certainly unusual. Whether this simply reflects th overt yet nonlethal nature of the mutant phenotypes or the relati vely large size of the gene is unclear. Yet this battery of Grid2 mutants has revealed important insights regarding ionotropic glutamate receptor g ating and assembly at the synapse in an in vivo model [110–113]. Similarly , the star gazer mouse mutant has re vealed a critical role for the star gazin protein in AMPAR surface and synaptic tar geting as well as channel kinetics. Undoubtedly , the star gazin, lurcher and hotfoot naturally occurring mutants will continue to pro vide further insights into the modulation of synaptic function by glutamate. 15.5.2.3 Large-Scale Neurological Random Mutagenesis Screens As the e xamples above show, valuable functional information can be pro vided by mouse mutant models or comparati ve studies of an allelic series [88,120,121]. However, access to such naturally occurring resources is v ery rare and studies in vivo, particularly those based on beha vioral parameters, are often confounded by background affects. To address this, a number of genome-wide random mutagenesis screens have been established [67,122–129] in s everal countries (Table 15.4). The chemical mutagen N-eth yl-N-nitrosourea is commonly used for this purpose: male mice from one inbred strain are treated, causing point mutations to occur in the

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spermatozoa. These mice are then crossed to wild-type females of a different inbred strain. In the search for dominantly inherited traits, all the of fspring of this cross (F1 population) are then screened for phenotypes of interest [130]. The main advantage of this approach o ver targeted knockouts is that more than one mutant representing a single gene can be made (an allelic series) with no bias as to the e xpected outcome, f acilitating the assignment of no vel functions to kno wn genes [122,131,132]. In addition, a wide range of mutant outcomes are possible, including loss-of-function and g ain-of-function alleles that might re veal more about the neurodevelopmental or beha vioral role of a particular locus than the corresponding knockout [122,129,133,134]. The mutagenesis programme at the Mammalian Genetics Unit, Harwell, U.K., is typical among other screens in that a battery of simple yet wide-ranging beha vioral tests are carried out on all F1 animals at 6 weeks of age [125,127]. Co vering parameters such as locomotor acti vity, wire maneuverability and grip strength, this protocol is designed to pick up deficits in sensory and autonomic function. Additional, more directed screens can also be applied at this stage, such as those for circadian rhythm defects [135] or anxiety-related behaviors [136]. Histopathological [129] or gene-expression profiling [137] can also be carried out to further refine t phenotypic characterization. The same mutagenesis method can be applied to identify recessively inherited phenotypes, although a further generation of breeding is required [138,139]. Once inheritance of the trait of interest is confirmed, generatin a genetic map to identify the chromosomal position of the mutation is then necessary followed by candidate gene sequencing [138,139]. F or example, the po wer of this methodology can be illustrated by an allelic series of mutations in the v oltage-gated sodium channel, Scn8a. The knockout suffers from paralysis and juv enile lethality, but a less se vere phenotypic outcome is observ ed in one of three ne w recessive Nethyl-N-nitrosourea (ENU)-derived lines with mutations in the pore loop domain, facilitating functional studies of this re gion of the channel on an identical genetic background [140]. 15.5.2.4 New Strategies for the Generation and Identification of Mouse Mutants One drawback of the phenotype-dri ven approach abo ve is that identification of th mutated gene still requires genetic mapping and candidate sequencing that, despite the a vailability of comprehensi ve genome sequence annotation, is the undoubted bottleneck of this methodology . A number of strate gies ha ve been de veloped to overcome this problem, each with its o wn advantages and limitations. For example, ENU-mutagenized males can be mated to females carrying either a defined chro mosomal deletion or in version (balancer chromosome) that includes a coat color marker for the detection of the v arious progen y classes [130,141]. Ne w recessive traits are identified in those mice carrying one mutated allele where the second i within the deleted or in verted re gion. Consequently , in both cases, the mutation resides within known genomic boundaries, significantly reducing the time require to identify the causati ve gene. With recent adv ances in mutation detection from multiple samples, carrying out gene-driven screens is now feasible. A bank of DNA

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from thousands of ENU mutagenized mice [142,143] or ES cells [144] is screened for mutations from a whole gene (for instance, a neurotransmitter receptor or synaptic protein) or genomic region of interest and the corresponding archi ved frozen sperm or clone is then used to rederi ve the selected mouse line. Although no guarantee exists of an y measurable phenotype in the resulting mutant, this method allo ws a functional domain to be screened in a matter of weeks. Indeed, the resource of around 5000 mutant lines currently a vailable is sufficient to isolate one mutation i any given gene [142].

15.6 CONCLUSION The a vailability of a gro wing number of lar ge-scale resources has a number of advantages for biologists, and the infrastructure is no w in place to mak e these resources freely accessible to ensure that they are used efficiently by neuroscientist and the scientific community as a whole. Firstl , these resources can pro vide a researcher with a gro wing number of tools to specifically address the function of protein of interest, be it a kno wn channel associated protein, unkno wn hit from a Y2H or other protein-protein interaction screen or an unkno wn protein from the PSD proteome. These tools include shRN Ai constructs that can be transfected into neurons, the ability to screen a protein for unknown interactions using protein-protein interaction maps and databases and the a vailability of full-length clones that can be rapidly transferred into a wide array of v ectors for do wnstream studies such as protein interaction or localization studies. In addition, the combination of both random and targeted approaches is generating an increasingly large number of mouse mutants and mutant cell lines. With the adv ent of more sophisticated conditional knockouts and substantial allelic series of mutants, the functional information gained from such resources is expanding exponentially, making obtaining a mutant or series of mutants for a protein of interest substantially easier . In combination with lar ge datasets of protein-protein interaction data, these resources will also allow a systems biology approach to neuroph ysiologically rele vant questions [7]. The recent approach of combining systematic interactome mapping with genetic perturbation analysis in C. elegans as applied to the TGF-β signaling network provides a powerful approach for developing a systems biology approach to signaling molecules [145]. In the near future, lar ge-scale mouse resources will similarly allo w an inte grated approach to comple x biological questions [7].

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4b

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PKC PICK1 NSF SNAP ABP/GRIP

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Stg

NMDAR synaptic activity

FIGURE 1.1 Schematic representation of AMPAR trafficking. athway (a) represents GluR1 traf ficking and path ay (b) represents GluR2 traf ficking. AMPAR comple xes e xit from the ER/Golgi associated with star gazin (1). GluR1containing AMPAR traffic through the secretory path ay (2a) and are inserted at extrasynaptic sites through an activitydependent mechanism (3a). Stargazin binds PSD95, which localizes the comple x at the synapse where the receptor can bind other scaffolding proteins such as RIL and 4.1N (4a). Star gazin can then be released. GluR2-containing AMPAR traffic through the secretory path ay (2b) and are inserted at synaptic sites through a constituti ve mechanism (3b). PICK1 facilitates the transport of the receptor possibly both to and from the plasma membrane b ut is remo ved from the AMPAR by the NSF/SNAP when the receptor reaches the plasma membrane. NSF/SN AP/ PICK1 forms a transient complex with GluR2-containing AMPAR (not sho wn on this schematic). AMPAR are stabilized at the membrane by scaffolding proteins. Phosphorylation by PKC pre vents interaction between ABP/GRIP and GluR2 (4b) and f avors the PICK1/GluR2 interaction (5b). The AMPAR are then internalized following a coupling with PICK1, andAMPAR/PICK1 complex are destabilized by NSF/SN AP when the receptor reaches its destination (6b). GluR2-containing AMPAR can then interact with an intracellular pool of ABP/GRIP, possibly in the endosome (7b), and get rec ycled to the membrane through interaction with PICK1 (8b) or tar geted to lysosomes (9b) and de graded (10b).

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Excitation

EGFP EYFP

ECFP

(a)

550

650

Wavelength (nm)

(b)

FIGURE 7.2 (a) Topology of the GFP folding pattern [51]. α-helices and connecting loops distrib ute 11 anti-parallel β-sheet strands (arro ws) and form the sides of the barrel that surrounds the GFP chromophore. (b) Excitation and emission spectra of some fluorescent proteins: Aequorea victoria GFP variants, dsRed and hcRed.

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X

Fluorescence Intensity

X

Y

Y

X

100 50

Y

Time (a) X

Fluorescence Intensity

X

Y

Y

100 50

Time (b)

FIGURE 7.3 (a) An example of FRAP of EGFP-GluR1 in dendritic spines of cultured hippocampal neurons. The area of dendrite between points X (proximal) and Y (distal) w as photobleached and subsequent fluorescence rec very was measured at 5-sec interv als. The chart represents typical result for quantification of the fluorescent intensity measur ments. (b) An example of FLIP of EGFP-GluR1 in dendritic spines of cultured neurons. Fluorescence at point Y was measured after repeated photobleaching at point X. The chart shows typical changes in the fluorescence intensity at th point of measurement (Y). These measurements allow estimation of the speed of traf ficking of the bleached molecule along the dendritic shaft [66].

Extracellular

Ι

I

ΙΙ

ΙΙΙ

0.1 NMDA

∆F/F0

0 II

0.1 Punctate Diﬀuse

0.2 −50 II

Intracellular

(a)

250 550 Time(s)

(b)

FIGURE 7.5 (a) Ecliptic pHluorin protein is fused to the N-terminal domain of an AMPAR subunit and is positioned inside the acidic lumen of transport v esicles in the secretory pathw ay. Fluorescence is quenched until e xocytosis and exposure of the pHluorin to the neutral e xtracellular environment, where a rapid increase in fluorescence occurs. This fluorescence is lost when pHluorin-tagged receptors are internalized into acid endo ytotic vesicles. (b) NMDA-induced endocytosis of AMPARs in cultured hippocampal neurons [67]. I: Fluorescence under basal conditions from synaptic (red) and e xtrasynaptic (blue) pHluorin-GluR2; II: Rapid endoc ytosis of e xtrasynaptic pHluorin-GluR2 after NMD A receptors activation; III: Subsequent slo w removal of synaptic pHluorin-GluR2, presumably by lateral dif fusion out of the synaptic area. See te xt for details.

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A1

A2

A3

A4

b2 b1

b3

B

D

C

0.5 0.4

*

0.04 MSD (µm2)

MSD (µm2)

**

0.05

0.3 0.2 0.1

0.03

***

0.02 0.01

0.2 0.4 0.6 0.8

1

0.2 0.4 0.6 0.8

Time (s)

1

Time (s)

E

F 100 Mobile receptors (%)

Mobile receptors (%)

100 75 50 25

GlyR GABAAR AMPAR NMDAR N-Cam

75 50 25

FIGURE 8.2 Characteristics of neurotransmitter receptor lateral diffusion. (a) Example of GlyR motion over the neuritic surface of spinal cultured neurons. Images were e xtracted from a sequence of 850 frames (acquisition time, 75 msec). a1 to a4 correspond to frames 6, 118, 150 and 629, respecti vely. QD fluorescence spots (green) and FM4-64-labele synaptic boutons (red). One QD (arro w), first located at bouton b1, di fuses in the e xtrasynaptic membrane (a1 to a3) and associates with bouton b2 (a7). Another QD (arro whead) remains associated with synaptic boutons b3 and blinks at image a4. Scale bar , 2 µm. (b) MSD v ersus time, calculated for a continuous sequence of images between frames 54 and 161, which sho ws the e xtrasynaptic motion. (c) MSD v ersus time, calculated for a continuous sequence of images between frames 503 and 597, when the QD is located at the periphery of bouton b2. Error bars sho w mean ± S.D. (d) QD-GlyR dif fusion during long recording. Projection of time-lapse recording (1 Hz, 20 min) of QD-GlyR trajectories (green) o verlaid with FM4-64 staining (red) and bright-field image. Extrasynaptic QD-GlyR (*) xplored large surfaces of the membrane, and synaptic QD-GlyRs were stable (**) or mobile (***) in a confined domain aroun the synapse. Scale bar , 5 µm. Fractions of mobile (D > 7 × 103 µm2/sec) molecules at e xtrasynaptic (e) and synaptic (f) sites. Only slowly mobile synaptic N-Cam molecules were considered. Similar fraction of mobile molecules is found in the e xtrasynaptic membrane for all neurotransmitter receptor types. Note that neurotransmitter receptors are slo wer in the extrasynaptic membrane than the transmembrane adhesion molecule, N-Cam. Note the smaller fraction of mobile inhibitory receptors compared to e xcitatory receptors at synapses. Agonist-evoked block

Synaptic block

EPSC amplitude (pA)

EPSC amplitude (pA)

NMDA / MK-801

500 400 300 200 100 0

0

20 10 Time(min)

(a)

30

MK-801

400 300 200 100 0

-5

0

5 10 15 20 Time(min)

(b)

FIGURE 9.3 Lateral mobility of NMD A receptors. (a) Agonist-evoked block of synaptic receptors. Whole-cell application (top panel and arro ws in lo wer panel, for 1 sec) of NMD A in the presence of MK801 resulted in complete and irreversible block of the NMDA receptor-mediated EPSC. (b) Selective block of evoked synaptic NMDA receptors (top panel) by MK801 application (solid bar , lower panel) also completely block ed the EPSC. Ho wever, following removal of MK801, the EPSC sho wed a 30–40% reco very over the course of se veral minutes. Filled circles indicate MK801 application. In both top panels, acti vated receptors are red, inacti ve are blue and the shaded area represents that o ver which the agonist is dispersed. Adapted and redra wn from [52].

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infected

control Surface GluR1 (arbitrary units)

β-gal GluR1 HA (α-SNAP) GluR1

stimulate

8 6 4 2

record

HA (β-SNAP) GluR1

βg α- al SN A P

0

(a)

(d)

(b) 2

3

4

20 pA

1

PICK1 AMPA EPSC Amplitude (pA)

control

control PICK1

100 ms (e)

(c)

FIGURE 11.1 (a,b) Sindbis virus–mediated o verexpression of proteins in cultured neurons. Dissociated hippocampal neurons were infected with Sindbis virus encoding β-gal (left panel), HA-tagged α-SNAP (right panel) or β-SNAP (not shown) and stained for surface AMPA receptor using anti-GluR1 antibody (green) and for β-gal or HA-tag (red). White boxes define enla gements shown in lower panels. (b) Quantitation of surf ace AMPAR levels in neurons infected with Sindbis viral constructs for β-gal, α-SNAP. Reproduced with permission from Elsevier [25]. (c) Sindbis virus–mediated overexpression of proteins in CA1 p yramidal neurons in acute cultured hippocampal slices. Lo w-power transmission (C1) and fluorescence (C2) images of an acute hippocampal slice cultured vernight with Sindbis virus bicistronically expressing EGFP. High-power images (transmission and fluorescence images superimposed) of whole-cell patch clam recordings from noninfected control (C3) and neighboring infected (C4) neurons in the CA1 p yramidal cell layer. (d) Schematic showing the experimental configuration for recording from control and infected neurons. (e)Averaged EPSCs (bottom traces, –70 mV , top traces, +40 mV) from an e xample pairwise e xperiment for a control noninfected neuron (left) and for a neighboring neuron infected with virus e xpressing PICK1, and summary analysis of all pairwise comparisons ( n = 9) of the ef fects of viral e xpression of PICK1 on EPSC amplitude recorded at a holding potential of –70 mV. Modified with permission from [31] LTD −200

1 2

Amplitude (pA)

Amplitude (pA)

LTP −300 −200 −100 1 0

−5 0

2 10

20

30

40

50

60

Time (min)

2 1

−150 −100 −50

1 0 −5 0

2 10

20 30 Time (min)

40

50

(a)

5µM

5µM

(b)

FIGURE 12.2 (a) Example of a whole-cell recording obtained from a GFP-infected CA1 p yramidal neuron (PND24 rat) showing long-term potentiation (L TP) of the AMPA receptor-mediated EPSC. Sample traces were a veraged over the baseline period (1) and 45 to 60 min post L TP induction (2). (b) Example of a whole-cell recording obtained from a GFP-infected CA1 p yramidal neuron sho wing long-term depression (L TD) of the AMPA receptor-mediated EPSC. Sample traces sho wn were a veraged over the baseline period (1) and 35 to 50 min post L TD induction (2). Scale bar: 20 msec, 50 pA.