Natural Products for Neurodegenerative Diseases

Natural Products for Neurodegenerative Diseases

Editors

Yung Hou Wong, Hong Kong Joseph T.Y. Wong, Hong Kong

48 figures, 2 in color and 2 tables, 2005

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Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

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Vol. 14, No. 1–2, 2005

Contents

5 Editorial

Reviews 6 Natural Products and Derivatives Affecting Neurotransmission Relevant

to Alzheimer‘s and Parkinson‘s Disease Houghton, P.J. (London); Howes, M.-J. (Kew) 23 Regulation of Neuroinflammation by Herbal Medicine and Its

Implications for Neurodegenerative Diseases. A Focus on Traditional Medicines and Flavonoids Suk, K. (Daegu) 34 Search for Natural Products Related to Regeneration of the Neuronal

Network Tohda, C.; Kuboyama, T.; Komatsu, K. (Toyama) 46 Multifunctional Activities of Green Tea Catechins in Neuroprotection.

Modulation of Cell Survival Genes, Iron-Dependent Oxidative Stress and PKC Signaling Pathway Mandel, S.A.; Avramovich-Tirosh, Y.; Reznichenko, L.; Zheng, H.; Weinreb, O.; Amit, T.; Youdim, M.B.H. (Haifa) 61 Unique Properties of Polyphenol Stilbenes in the Brain: More than

Direct Antioxidant Actions; Gene/Protein Regulatory Activity Doré, S. (Baltimore, Md.) 71 Neuroprotective Effects of Huperzine A. A Natural Cholinesterase

Inhibitor for the Treatment of Alzheimer‘s Disease Wang, R.; Tang, X.C. (Shanghai)

83 Author Index 84 Subject Index

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Neurosignals 2005;14:5

Editorial

Natural products have a long history of use for neurological symptoms. For example, Crocus sativus has traditionally been used as an antispasmodic and sedative. Yet the study of the mechanisms of action of neuroactive natural products began only recently. Armed with an arsenal of enabling technologies, researchers are now able to take a closer look at the complex molecular events associated with the therapeutic actions of natural products in neurodegenerative diseases. Extensive information has been generated to inspire the design and development of more efficacious and safe therapies. This issue brings together contemporary perspectives and research findings of leading experts on a vast array of mechanisms underlying the effects of natural products on neuroprotection and neuronal regeneration. The multifunctional properties of constituents of botanicals and their derivatives are discussed here to illustrate the possibilities toward the development of effective multitarget therapeutics for neurodegenerative diseases. The study of neuroactive natural products will indeed advance our understanding of the nervous system, and open up exciting opportunities in drug discovery. Yung Hou Wong, Hong Kong Joseph T.Y. Wong, Hong Kong

© 2005 S. Karger AG, Basel 1424–862X/05/0142–0005$22.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nsg

Review Received: September 27, 2004 Accepted after revision: November 16, 2004

Neurosignals 2005;14:6–22 DOI: 10.1159/000085382

Natural Products and Derivatives Affecting Neurotransmission Relevant to Alzheimer’s and Parkinson’s Disease Peter J. Houghton a Melanie-Jayne Howes b a King’s

College London, London, and b Jodrell Laboratory, Royal Botanic Gardens, Kew, UK

Key Words Alzheimer’s disease W Parkinson’s disease W Cholinesterase inhibitors W Cholinergic receptors W Dopaminergic receptors W Acetylcholine W Dopamine W Galantamine W Huperzine W Physostigmine W L-DOPA W Ergot alkaloids

Abstract The two major neurodegenerative diseases Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterised by low levels in the brain of the neurotransmitters acetylcholine (ACh) and dopamine (DA), respectively. Clinical treatment of these two conditions is palliative and relies, in most cases, on improving stimulation at the relevant receptors by either increasing levels of the endogenous neurotransmitter or by the use of substances which have a similar agonist response. Natural products continue to provide useful drugs in their own right but also provide templates for the development of other compounds. The major advances in the treatment of AD have been the use of acetylcholinesterase inhibitors such as galantamine, huperzine A, physostigmine and its derivatives to increase the levels of ACh rather than the use of cholinergic compounds, although compounds with nicotinic properties have attracted some interest. In contrast, the treatment of PD has relied on the elevation of DA levels by use of L-DOPA, its precursor,

ABC

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and by the administration of dopaminergic agonists, especially the ergot alkaloid derivatives. The use of inhibitors of enzymes that cause breakdown of DA is an avenue which is being explored. As well as the major natural products of clinical interest, the paper discusses the chemistry, activity and usage of the constituents of plants used in traditional medicine for the treatment of diseases presenting symptoms similar to those characteristic for Alzheimer’s or Parkinson’s disease. Copyright © 2005 S. Karger AG, Basel

Introduction

Neurodegenerative disease is a generic term applied to a variety of conditions arising from a chronic breakdown and deterioration of the neurons, particularly those of the central nervous system (CNS). In addition, these neurons may accumulate aggregated proteins which cause dysfunction. Alzheimer’s disease (AD) and Parkinson’s disease or parkinsonism (PD) are the two best-known diseases of this type and will be the main diseases associated with this review. However, some specialists would classify multiple sclerosis and spongiform encephalopathies as neurodegenerative diseases. The latter have received publicity in recent years due to the link between CreutzfeldtJacob disease (CJD) in humans and ‘mad cow disease’. Spongiform encephalopathies have been shown to be

Peter J. Houghton Pharmacognosy Research Laboratories, Department of Pharmacy King’s College London, Franklin-Wilkins Building 150 Stamford Street, London SE1 9NH (UK) Tel. +44 20 7848 4775, Fax +44 20 7848 4800, E-Mail [email protected]

caused by prions, proteinaceous particles synthesized within the cell which replicate and accumulate in cells and so alter protein structure and therefore function but, as yet, there are no drugs available to treat these conditions. Most commonly, neurodegenerative disease manifests in elderly people and, in advanced industrialised and post-industrialised societies, where life expectancy is long, this group of conditions is a major cause of morbidity and of death, as well as imposing severe strains on the social welfare systems. To illustrate this it is reported that, in the USA, AD now affects at least 5 million people and in the UK in 2001 it affected 750,000 people and was the fourth most common cause of death [1]. However, it is increasingly becoming recognized by the World Health Organisation as a global problem [2]. The common symptoms of neurodegenerative diseases, such as loss of memory and tremor, have been recognised as a feature of increasing age for a long time and are acknowledged in many traditional medical systems. However, it is only comparatively recently that, as distinctive diseases, they have been recognized and received much attention from mainstream medicine. This is most likely due to the fact that, in the past, short life expectancy precluded many surviving to an age where neurodegeneration was likely to affect a significant part of the population. The etiology of neurodegenerative diseases is still largely unknown but, especially for AD and PD, postmortem studies have shown clear links between the disease and a deficiency of neurotransmitters in parts of the brain. Thus, in AD there is a chronic shortage of acetylcholine (ACh) 1 and in PD a deficit of dopamine (DA) 2. Until very recently, the only clinical treatment for both of these conditions was the reversal of these deficiencies by elevation of the levels of the transmitter by agonists, or by inhibition of enzymes involved in their removal from the immediate locality of the synapse. These approaches are discussed more thoroughly below. A variety of natural products have been shown to play roles which would have the desired effects and some of these, or their derivatives, have been brought into clinical use. This paper gives an overview of such compounds, as well as others, with interesting activity which have not been developed as drugs or are still undergoing clinical trials. It should not be forgotten that there are also many traditional medicinal plants with a reputation of alleviating or preventing symptoms of neurodegeneration and that these are used as crude extracts and mixtures [3, 4]. In some cases, such extracts have been shown to relieve neurodegenerative symptoms in animal models or display relevant in vitro activity, but both the modes of action and identity of any compounds responsible have not been fully determined.

Increasingly attention is being paid to the excitatory amino acid neurotransmitters and relevant receptors which are present in the CNS, e.g. the N-methyl-D-aspartate (NMDA) receptor. The use of these in designing new drugs or of explaining the use of traditional medicinal plant extracts is in its infancy and no significant findings have been made of natural products which either bind to the receptors or affect levels of the transmitters.

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Neurosignals 2005;14:6–22

Alzheimer’s Disease

AD is the major disease of a group which is characterised by loss of cognitive function leading to dementia. AD is estimated to account for between 50 and 60% of dementia cases in persons over 65 years of age [5] and is a progressive, neurodegenerative disease that primarily affects the elderly population. It is a major public health concern in developed countries because of the strains imposed on carers and financial resources by the increasing numbers of sufferers. The main symptoms associated with AD involve a decline in cognitive dysfunction, primarily memory loss [6, 7] and in the later stages of the disease language deficits, depression, agitation, mood disturbances and psychosis are often seen [8]. Although AD, as a defined medical condition, has only existed for about 100 years, age-related loss of memory and cognitive decline has been documented for thousands of years in human history. In common with many other conditions, ancient writings which describe the symptoms also suggest remedies, based usually on plant extracts. Thus, Ashwagandha (Withania somnifera) is mentioned in ancient Sanskrit writings from India as a ‘medharasayan’ or promoter of learning and memory retrieval whilst in the sixteenth century Gerard’s Herbal from the UK, sage Salvia officinalis, is described as being ‘good for the memory’. In recent years, the value of a few of these has been demonstrated scientifically and some of the compounds mentioned below have been isolated from traditional medicinal plants. Postmortem studies have shown that AD is characterized by low amounts of the enzyme choline acetyl transferase (ChAT) and enzyme abnormalities which would produce levels of the neurotransmitter ACh 1 (fig. 1) [9, 10] and there also appears to be a depletion of nicotinic function. Attention deficit in AD is reversed with nicotine 3, which is reported to upregulate nicotinic receptors and to increase ACh release, so enhancing cholinergic neurotransmission [11, 12].

7

CH3 CH3

+ N CH3

HO

CH3

NH2

O

CH3

N O

O

H

1 Acetylcholine

CH2 OH

O

HO 2

O

Dopamine

4 Hyoscine (scopolamine)

H N

N CH3

Fig. 2. Scopolamine, a muscarinic receptor antagonist.

3 Nicotine

Fig. 1. Neurotransmitters of the CNS relevant to neurodegenerative

disease and the agonist nicotine.

ACh is certainly associated with cognitive function, since situations where it is blocked from acting on the cholinergic receptors by drugs such as hyoscine (scopolamine) 4 (fig. 2), which is a muscarinic antagonist, result in severe cognitive impairment in the patient. It is still unclear if the low levels of ACh in the CNS are cause or effect as far as AD is concerned, but the repletion of levels has been exploited therapeutically with some success in the last 15 years in the symptomatic relief of AD. The synthetic compound tacrine was the first drug introduced into the clinic and it increased the levels of ACh by inhibition of acetylcholinesterase (AChE), the enzyme responsible for fast breakdown of ACh after its release from the nerve ending. This inhibition results in ACh having a longer half-life and therefore increasing in concentration at the synapse. Tacrine was the first of several AChE inhibitors which have come into clinical use, but it is no longer used since the more recent introductions, generally named second generation inhibitors, are safer and have longer-lasting effects. These recent introductions include at least two based on natural products. It should be stressed that AChE inhibitors only alleviate some of the cognitive symptoms of the disease for a time and ultimately do not arrest the cognitive decline of the patient. Another approach which has been proposed is the employment of cholinergic and, to some extent, nicotinic agonists, but this has not proved to be as useful therapeutically as the inhibition of cholinesterase. More recently prevention of glutamate-mediated neurotoxicity has been a therapeutic target in AD. The Nmethyl-D-aspartate (NMDA) receptor antagonist, memantine, has been introduced in some countries for clinical use in AD patients. It should be noted that other types of activity, besides increase of neurotransmitter levels, are

8

Neurosignals 2005;14:6–22

also being explored as leads for chemotherapeutic treatment of AD. Activities being explored include antioxidant, anti-inflammatory and inhibition of ß-amyloid synthesis, but these are not covered to any extent in this paper.

AD: The Use of Cholinergic Compounds

The rationale underlying the use of cholinergic compounds is that they are agonists of the nicotinic cholinergic receptor and so compensate for the low levels of ACh. The binding of ACh to the receptor is shown diagrammatically in figure 3. It can be seen that the important features required in a molecule are an amine which becomes positively charged at the pH of the immediate environment and so binds to an aspartate in the receptor, a portion of the molecule able to form hydrogen bonds with the asparagine domain of the receptor and small groups able to bind to hydrophobic sites near the aspartate region. In addition, the receptor lies in a pocket which allows only small molecules to enter. Although these compounds have been suggested as valuable agents in treating AD, because they also appear to inhibit fibrillary tangle and amyloid production, success has been limited as far as clinical studies are concerned, although results in animals were initially promising [13]. Two major alkaloidal natural products are known to have this effect, arecoline 5 and pilocarpine 6 (fig. 4). It can be seen that these molecules are small and that they incorporate at least two of the criteria noted above for binding to the receptor. Arecoline is the major alkaloid present in areca or betel nut, the fruit of the palm tree Areca catechu L. (Arecaceae), which is extensively used as a masticatory throughout the Indian subcontinent and other parts of southeast Asia. It is estimated that 500 million people regularly chew betel nut (often referred to as ‘pan’ in India) in a

Houghton/Howes

form which is usually shredded, mixed with lime and wrapped in a leaf of Piper betel L. Excessive salivation occurs, which is a direct result of its cholinergic activity, which also gives the mild CNS stimulation for which the product is principally used. There was some interest in arecoline as a treatment for AD since it showed improvement in memory tests in rats [14]. Arecoline has been shown to bind to the M2 muscarinic receptors but not the nicotinic receptors [15]. A small clinical study showed that, when arecoline was given continually by intravenous infusion in AD patients, it enhanced verbal memory [16]. Derivatives of arecoline have been synthesized in order to improve selectivity for cortical muscarinic ACh receptors and two examples are Lu 25-109 and talsaclidine 8 which are M1 receptor agonists. Although Lu 25-109 showed encouraging results in vitro [17], it failed to improve cognition when tested clinically in patients with mild to moderate AD [18]. Talsaclidine has been shown to increase cholinomimetic central activation in animals and humans without some of the side effects seen with AChE inhibitor therapy, but cognitive function was not significantly improved [19]. Tests on rhesus monkeys did however show some improvement in memory-related tasks but at doses which gave unacceptable side effects [20]. Pilocarpine 6 is one of a series of related alkaloids found in species of the South American plant genus Pilocarpus, known commonly as Jaborandi leaf, which was used in traditional medicine since it induced sweating and urination, features which were perceived as being useful for eliminating toxins from the body. The molecular structure of pilocarpine 6 bears similarities to ACh since the positively charged N atom and the lactone binding to the serine are about the same distance apart. Chewing the leaf causes copious salivation as well as other typical features of cholinergic stimulation such as contraction of the pupils. Pilocarpine has been shown to enhance memory performance in aged rats [21], but no studies on its application in humans for the treatment of AD have been reported and it appears to have been discarded as a potential therapeutic lead. This is possibly due to its poor pharmacokinetic profile as it cannot pass through the bloodbrain barrier, as well as its undesirable side effects.

CH3 Aspartic acid -

CH3

COO

O

O

CH3

N

Hydrophobic sites H O

H

Hydrophobic site

CH3

N

Asparagine

Fig. 3. Binding of acetylcholine to nicotinic receptor.

O CH3 N

OCH3

O N

N

H CH3

CH3

5

H

Arecoline

6

O

Pilocarpine

N N N

N

CH3

O N

N

C

CH

CH3 7

LU 25-109

8

Talsaclidine

Fig. 4. Cholinergic compounds.

To avoid the undesirable effects of excess cholinergic stimulation, ACh is rapidly hydrolysed after release at the synapse by an enzyme named acetylcholinesterase AChE.

A similar enzyme, butylcholinesterase BuChE, also occurs. If the cholinesterase is inhibited, the ACh does not hydrolyse so quickly, and levels of ACh rise. The ‘classic’ cholinesterase inhibitor is the alkaloid physostigmine 9, also called eserine. This was isolated from the Calabar Bean, the seeds of Physostigma venenosum Balf., in the nineteenth century in studies stimulated by the use of the seeds as an ordeal poison in what is now southeastern Nigeria. The toxic effects of calabar bean extract were found to be due to excessive cholinergic stimulation resulting in increased salivation, nausea, bradycardia, muscle cramps and respiratory failure, as well as CNS effects such as agitation. The cholinergic excess was found to be caused by inhibition of the rapid breakdown of acetylcholine by physostigmine.

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AD: The Use of Cholinesterase Inhibitors – Alkaloids

9

H

Aspartate

O

O

CH3

O N O

Histidine

N

CH3

H

N

CH3

CH3

N

O O

CH3

O N

CH3 CH3 N CH3

CH3

CH3

N

N

H3C H3C

OH Serine

O

9

Physostigmine

10 Neostigmine

CH3 H

CH3 N CH3

CH3 CH3

N

Tyrosine

O CH3

O 11 Rivastigmine OH H

OH OH

O

Fig. 5. Interaction between acetylcholine and the acetylcholinester-

CH3O

O

ase active site.

N

O

N CH3 12

In recent years, the structure of AChE has been determined and the mode of binding for AChE inhibitors has also been elucidated. A variety of AChEs exist according to the source species, but they vary only in small details and all contain the active site at the base of a deep cleft in the enzyme. Figure 5 illustrates the particular amino acid residues which are considered to be the most important in the binding process and from this knowledge, structure-activity relationships of AChE inhibitors can be understood at the intermolecular level. The important regions of an inhibitor appear to be a positively charged nitrogen, which binds to an aspartate residue, and a region, separated by a lipophilic area from the positive charge, which can form a hydrogen bond with a tyrosine or serine residue. A positively charged nitrogen is common in many alkaloids at body pH and it is not surprising that many of the most powerful AChE inhibitors are alkaloids. In recent years, however, a number of natural products, mentioned below, which do not contain any nitrogen, have been found to be AChE inhibitors. This raises questions about their binding characteristics to AChE since the key role played by a positively-charged moiety of the molecule bonding with the anionic aspartate residue, can no longer account for the activity. The interactions between the enzyme and such non-alkaloidal compounds have generally not yet been investigated. The cholinesterase inhibitors (fig. 6) cause overstimulation of a number of functions in many animal species and exert a considerable toxic effect even at quite low

10

Neurosignals 2005;14:6–22

Galantamine

13 Hamayne CH3

H CH3

N NH2

O

14 Huperzine A

Fig. 6. Acetylcholinesterase inhibitors.

doses. This has been exploited in the area of insecticides, and the insecticide carbaryl was developed by making synthetic analogues of physostigmine. In therapeutics, cholinesterase inhibition had, until recently, a somewhat limited application in only ophthalmology and the treatment of myasthenia gravis. However, the realisation that early symptoms of AD could be improved by the use of cholinesterase inhibitors awakened renewed interest and, since physostigmine was known to cross the blood-brain barrier, several in vivo studies were conducted which showed that it reduced symptoms of ACh deficiency in the CNS. Physostigmine was reported to protect mice against cognitive impairment caused by oxygen deficit and it improved learning in rats [22]. Clinical studies showed significant cognitive benefits in both normal and AD patients [23], but it had a short half-life, which has prevented its application clinically in AD patients, since this would require multiple daily dosing.

Houghton/Howes

As well as inhibiting AChE, physostigmine also inhibits butylcholinesterase (BuChE), another enzyme found in the CNS, so adverse effects associated with BuChE inhibition, such as gastrointestinal disturbance, may also occur with physostigmine. However, BuChE has recently been implicated in the aetiology and progression of AD [24], so inhibition of BuChE may therefore prove to be beneficial in treating AD and physostigmine and other BuChE inhibitors such as rivastigmine may have clinical efficacy superior to AChE-selective inhibitors. To improve its pharmacokinetic profile, there is a considerable history of the synthesis of analogues of physostigmine. These have been applied to the treatment of myasthenia gravis, neostigmine 10 being the most widely used drug for this disease. Neostigmine is a quaternary amine and this feature severely impairs its ability to cross the blood-brain barrier and so be of value in treating AD. Rivastigmine 11 was produced with the express purpose of engineering a better pharmacokinetic profile for usefulness in AD and it inhibits the G1 form of AChE in the cortex and hippocampus, brain areas involved in cognition [25], and it has been shown to improve cognition in AD patients [26, 27]. Clinical studies have borne out the usefulness of rivastigmine (Exelon®) in mild to moderate AD and it has been licensed as a treatment for symptomatic relief of AD since 2000. Galantamine 12 (sometimes referred to as galanthamine) is found in members of the Amaryllidaceae, e.g. the Chinese medicinal herb Lycoris radiata Herb. and the European Galanthus nivalis L. and Narcissus spp. [28]. The ethnopharmacological uses of plants containing this compound are not very clear, but a full report of the history of the development of galantamine from Galanthus nivalis has been recently published [29]. Its cholinesterase inhibitory properties were first exploited in Bulgaria in the mid-twentieth century for the treatment of polio victims, but it only came into prominence as a treatment for AD in the last decade of the twentieth century. Galantamine has been licensed in Europe for AD treatment since 2001. Multi-centre randomised clinical trials showed that it was well tolerated and significantly improved cognitive function when administered to AD patients [30–32]. The cognitive benefits appear to be sustained for at least 3 years, a much longer time than for other drugs of this type [33]. Galantamine is well absorbed when given orally and is also more selective for AChE than BuChE [34]. It also stimulates nicotinic receptors indirectly by its allosteric potentiation of ACh [35], and so may also enhance cholinergic function and memory (see below). This effect suggests that galantamine may

have therapeutic advantages over other AChE inhibitors and its value in vascular dementia as well as AD is recognised in recent studies [36]. Several other alkaloids with AChE inhibitory activity have been recently reported from members of the Amaryllidaceae, but they have not been subjected to vigorous pharmacological and clinical testing. Several other alkaloids isolated from Iberian Narcissus species have been tested for cholinesterase activity [37]. A study of two Crinum species used in Nigerian traditional medicine to help ailing memory resulted in the isolation of four alkaloids of which the most active was hamayne 13, although its IC50 value of 250 ÌM was three orders of magnitude weaker than physostigmine, so it is unlikely that it would be present in sufficient quantity to have a strong therapeutic effect [38]. Whilst physostigmine analogues and galantamine have entered the pharmacopoeia in the Western world, another natural cholinesterase inhibitor, huperzine A 14 has been introduced in China for treating AD. Huperzine A is one of the alkaloids found in the clubmoss Huperzia serrata Thunb. (Lycopodiaceae) which is used in various formulae in traditional Chinese medicine (TCM) to alleviate problems of memory loss, promote circulation and for fever and inflammation [39]. Huperzine A is related to the quinolizidine alkaloids and it reversibly inhibits AChE in vitro and in vivo [40, 41]. Huperzine A has been shown to improve memory in cognitively impaired rats [42] and in gerbils following ischaemia [43]. These observations suggest that huperzine A has clinical potential in cerebrovascular disorders, as well as in AD. Indeed, since it has also been shown to be neuroprotective, AChE inhibition may not be the only explanation for the clinical effects observed. Huperzine A has been shown to be neuroprotective against ß-amyloid peptide fragment 25–53 and free radical-induced cytotoxicity [44] and to attenuate apoptosis by inhibiting the mitochondria-caspase pathway [45]. In a multi-centre, double-blind trial, huperzine A significantly improved memory and behaviour in AD patients, and was reported to be more selective for AChE than BuChE and was less toxic than the synthetic AChE inhibitors donepezil and tacrine [36]. A randomised, placebo-controlled study on over 200 patients, 100 of whom were given 400 Ìg huperzine A daily for 12 weeks and showed significantly higher scores for improvement compared with the placebo group in the various scorings used [46]. Recent progress on many aspects of huperzine A has been reviewed [47]. Other alkaloids have shown AChE activity and most of these have been isolated from plants used in traditional medicine (fig. 7). Coptis chinensis Franch. (Ranunculaceae)

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11

O N+

O

OCH3 OCH3 15 Berberine CH3O

CH3O

N+

N+

CH3O

CH3O

OCH3

O O

OCH3

16 Coptisi ne

17 Palmatine

O

O N

N N

N

N

N

CH3

18 Rutaecarpine

19

Dehydroevodiamine

inhibited AChE in vitro, and reversed scopolamineinduced memory impairment in rats [54]. It also increased cerebral blood flow in vivo in cats, a property which would supplement its usefulness in AD [55]. Solanidine 20 and related compounds and their glycosides are steroidal alkaloids produced in green parts of the genus Solanum, which includes the potato. The toxic properties of these alkaloids have been known for a long time and have been shown to be due to AChE inhibition, with characteristic signs of cholinergic excess such as sweating, palpitations and CNS disturbances including hallucinations [56]. Reports of Solanum species being used to treat AD or related conditions in traditional medicine do not exist and the alkaloids have not been investigated for any usefulness clinically, presumably because of their toxicity. Although it is comparatively easy to explain the fact that some alkaloids inhibit AChE because of their molecular features, it is less easy to correlate chemical structure and activity for some other phytochemical types for which AChE inhibition has recently been reported.

CH3 H CH3 H

CH3

N

H

H H

CH3

H

HO 20 Solanidine

Fig. 7. Minor alkaloidal cholinesterase inhibitors.

has been used in TCM for several conditions including agerelated cognitive and memory decline. Some alkaloids found in this species, such as berberine 15, coptisine 16 and palmatine 17, are reported to also be anti-ChE [48, 49]. Coptis chinensis extract improved a scopolamine-induced learning and memory deficit in rats [50] and this is likely to be due to the alkaloids present raising ACh levels. Berberine 15 has been shown to be selectively active against AChE compared with BuChE [51] and it has been shown to improve scopolamine-induced amnesia in rats [52]. Rutaecarpine 18 is the major alkaloid found in Evodia rutaecarpa (Juss.) Benth. (Rutaceae), the unripe fruit of which is used in TCM for cardiotonic, restorative and analgesic effects. Pharmacological activities relevant to AD have been identified with the extract and with rutaecarpine. Rutaecarpine inhibited COX-2 activity in vitro, and was anti-inflammatory in vivo [53]. Dehydroevodiamine 19, another alkaloid found in the same plant,

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AD: The Use of Cholinesterase Inhibitors – Terpenoids and Other Types of Compound

Neurosignals 2005;14:6–22

Terpenoids comprise a very large group of natural products and comprise two or more branched 5 carbon units, formed from a common precursor named mevalonic acid. Skeletons consisting of multiplets of 2, 3, 4 or 6 of these linked together in many different ways are found in a variety of mostly cyclic compounds named monoterpenes (10 carbons in the skeleton), sesquiterpenes (15 carbons), diterpenes (20 carbons) and triterpenoids (30 carbons), respectively (fig. 8). These compounds tend to be lipophilic, so they are able to cross the blood-brain barrier, and the monoterpenes, and some of the sesquiterpenes, are volatile, and so effects could occur through inhalation. These compounds are responsible for the strong odours and flavours of many herbs, spices and traditional medicines. Many such molecules are increasingly recognized as having a variety of roles in living organisms, including signalling between members of the same species, thus comprising part of the group of compounds known as pheromones, and protective or attractant roles in flowering plants against herbivores and pollinators respectively. An effect on CNS activity by the volatile substances in perfumes and other odoriferous materials has attracted interest in recent years and one of the first findings that monoterpenes had AChE inhibitory effects was made only in the mid 1990s in studies investigating

Houghton/Howes

historical records that monoterpene-containing plants were ‘good for the memory’ [57]. One such group of plants was the various European species of Salvia, commonly known as sage. An ethanolic extract and oil of S. officinalis L. (Labiatae) and S. lavandulaefolia Vahl. (Labiatae) were investigated for anti-ChE activity and it was found that all gave inhibition of AChE at quite low concentrations [57]. The cholinesterase inhibition shown by the S. lavandulaefolia oil was shown to be partly due to the cyclic monoterpenes 1,8-cineole 21 and ·-pinene 22, which were shown to inhibit AChE in vitro, with some contribution from other constituents, perhaps by acting synergistically [58, 59]. However, the monoterpenes were considerably less active, by a factor of at least 103, than the alkaloidal AChE inhibitors such as physostigmine 9 [58]. Since the effects of the oil were better than those of individual monoterpenes, further in vivo and clinical studies, described below, were carried out on the essential oils, which consist of a mixture of monoterpenes, rather than isolated compounds. Oral administration of S. lavandulaefolia essential oil to rats decreased striatal AChE activity in both the striatum and the hippocampus compared to the control rats. Thus, it appeared that constituents of the S. lavandulaefolia oil, or their metabolites, reach the brain and inhibit AChE in select brain areas, consistent with evidence of inhibition of the brain enzyme in vitro [60]. Clinical studies on human volunteers and even patients with AD have been reported in recent years. The effect of sage in twenty participants using a placebo-controlled, double-blind, balanced, cross-over design showed significant effects on cognition associated with the lowest dose of Salvia, including improvements in both immediate and delayed word recall scores [61]. A small trial with 11 patients showing mild to moderate symptoms of AD showed that oral administration of the essential oil of S. lavandulaefolia significantly improved cognitive function in one of the three different methods of assessment used [62]. Another, more thorough, trial in Iran on 48 patients with similar symptoms of AD showed that those treated with an extract of S. officinalis gave significantly better values in measurements of cognitive function [63]. Melissa officinalis L. (Labiatae) leaf is another species that contains monoterpenes in its essential oil. It has been used as a medicinal plant for more than 2000 years and has a reputation of for promoting long life and for restoring memory [64]. Recent studies have focused on the reputed cognitive effects of M. officinalis. A recent randomised, placebo-controlled, double-blind, balanced

crossover study showed an improvement in cognitive performance and mood in 20 healthy young participants, following treatment with dried leaf of M. officinalis shown by previous in vitro tests to be cholinergically active [65]. In another study, an extract of M. officinalis was administered to patients with mild to moderate AD for 4 months and gave a significantly better outcome on cognitive function than placebo [66]. Although the constituents present have not been investigated, numerous monoterpenes have been identified in the essential oil of M. officinalis, including citral 23 (a mixture of the isomers geranial and neral) and it is known that these are weak inhibitors of AChE [67]. The root of another species of Salvia, S. miltiorrhiza Bung. (Labiatae) is extensively used in TCM to stabilise the heart and calm nerves [48]. S. miltiorrhiza has been the subject of thorough investigation, and consequently numerous pharmacological activities that may be relevant in CNS disorders, including AD, have been identified. S. miltiorrhiza has been employed for the treatment of cerebral vascular disease, and there are several studies to

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CH3

CH3 CH3

H3C

CHO

H3C

O

CH3

CH3

H3C 22 α-Pinene

21 1,8-Cineole

O

CH3

23 Citral

O

CH3

O

CH3

O O

O

H3C

CH3 24 Dihydrotanshinone

CH3

25 Cryptotanshinone

CH3 H3C

CH3

CH3 H

HO CH3

H

COOH

CH3

H CH3 26 Ursolic acid

Fig. 8. Terpenoidal cholinesterase inhibitors.

13

CH3 H3C H CH3 O

CH2OH

O O O

OH OH

O

OH

CH3 H O

OH

27 Sitoindoside IX CH3 CH2OH H3C H CH3 H O

O

O

CH3 H O

OH

28 Withaferin A

Fig. 9. Steroidal derivatives with anticholinesterase inhibitory activ-

ity.

investigate possible mechanisms for the protective effect of S. miltiorrhiza against cerebral ischaemia. There is evidence that S. miltiorrhiza root extract may protect neurons from ischaemia and attenuate dysfunction of neuropeptides of importance in neurodegenerative disease [68]. An inhibitory effect on AChE has been recently demonstrated and has been shown to be due to the diterpenes present known as tanshinones [69]. Dihydrotanshinone 24 was shown to be the most active (IC50 = 1.0 ÌM) with cryptotanshinone 25 (IC50 = 7.0 ÌM ) also showing activity. A feature which appears to be necessary for activity is the saturated bond in the furan ring of the molecules. Screening of 139 different Indian medicinal plants and spices for AChE inhibitory activity led to Origanum majorana L. (Labiatae) showing the highest activity. The active component was identified as the triterpene ursolic acid 26, a fairly common compound, which exhibited an IC50 value of 7.5 nM, less than an order of magnitude weaker than that of the positive control tacrine [70]. This result is of interest in view of the widespread occurrence of ursolic acid and may account for the traditional use of several plant species for memory improvement and ADrelated conditions.

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The withanolides are a group of compounds related to the steroids which are found in some genera of the Solanaceae, notably Withania somnifera (L.) Dun. The root of this plant is one of the most highly regarded herbs in Ayurvedic medicine where it is known as ‘ashwagandha’ and has a history of use for almost 4,000 years. It is classed among the rejuvenative tonics known as ‘Rasayanas’ and several groups have described the cognitive enhancing potential of extracts of the roots in experimental animals. Some individual compounds have also been investigated and the sitoindosides IX 27 and X (fig. 9) have been shown to augment learning acquisition and memory in both young and old rats [71]. The mechanisms to explain this effect are unclear, but may involve modulation of cholinergic neurotransmission. An extract containing the sitoindosides VII–X and withaferin A 28 was administered to mice and effects on the neurotransmitter systems in the brain were observed. The results from this study showed that the extract enhanced AChE activity in the lateral septum and globus pallidus areas of the brain and also enhanced muscarinic M1 receptor binding in cortical regions, but it did not affect Á-aminobutyric acid (GABA)A, benzodiazepine receptor binding, nor NMDA or amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor subtypes [72]. The extract containing the sitoindosides VII–X and withaferin A also reversed the reduction in cholinergic markers (e.g. ACh, choline acetyltransferase; ChAT) in rats [73]. These activities could explain the reputed cognition enhancing effects of W. somnifera root because of preferential action on cholinergic neurotransmission in the cortical and basal forebrain, brain areas involved in cognitive function. Based on this information, it could be speculated that the sitoindosides and withaferin A could have potential in AD therapy, but more studies need to be carried out before they can be used with any degree of confidence in being able to achieve this or whether the crude extract might produce a better effect. In addition to their cholinergic activity, the glycowithanolides showed anxiolytic and anti-depressant activities in rats [74], which may be applicable in the symptomatic treatment of AD. W. somnifera root and some constituents are also reported to have anti-oxidant properties which may also be relevant in AD therapy [75]. Anti-inflammatory effects of the extract of roots have been demonstrated in rats [76] and the extract has also been shown to reduce levels of the pro-inflammatory interleukins IL-1 and TNF-·, which are considered to be involved in senile plaque formation and neurodegeneration [77].

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There are many types of phenolic compounds, but the only ones which have been shown to possess AChE inhibitory activity are neolignans from Magnolia officinalis. The root and stem bark of Magnolia officinalis Rehd. Et Wils. (Magnoliaceae) have been used in TCM to treat anxiety and nervous disturbances. M. officinalis contains the biphenolic lignans, honokiol 29 and magnolol 30 (fig. 10). Both lignans increased ChAT activity and inhibited AChE activity in vitro, and increased hippocampal ACh release in vivo [78]. Honokiol and magnolol show anxiolytic effects [79] and these appear to be due to their ability to potentiate GABAergic neurotransmission [80]. These two compounds also appear to have antioxidant antiinflammatory and neuroprotective properties and such polyvalency in activity is of interest in their potential use in treating AD [81, 82].

OH R OH

CH2

H2C

29 R = H Honokiol 30 R = OH Magnolol

Fig. 10. Neolignans with anticholinesterase

inhibitory activity.

O

OH N CH3 31 Lobeline

AD: Use of Nicotinic Compounds

A link between smokers and a lower incidence of AD has been noted and this is thought to be associated with increased nicotine intake, although some recent reports present a contrary view in linking smoking with an increased incidence of AD [83, 84]. Nicotine 3 is reported to have cognition-enhancing effects and these may be due to nicotinic receptor stimulation but also protection against AD by other mechanisms such as inhibition of ßamyloid formation [85], inhibition of the neurotoxic effects of excitatory amino acids (e.g. glutamate) and enhancement of the effects of nerve growth factor (NGF) [86]. There are several other alkaloids which are nicotinic agonists at the cholinergic receptor [87] (fig. 11). Lobeline 31 from Lobelia inflata interacts with the nicotinic receptor [88] and could also be exploited to influence cholinergic function in AD. Other alkaloids such as sophoramine 32 and cytisine 33, found in members of the Leguminosae, have nicotinic actions. Cytisine is used as a pharmacological tool because of its strong binding affinity to nicotinic receptors, but it does not appear to have been developed for any pharmaceutical purposes, probably because of its toxicity.

O

O

N

N H H

N

H

N H

32 Sophoramine

33 Cytisine

Fig. 11. Nicotinergic alkaloids.

Parkinson’s disease is named after the English surgeon who first described a syndrome which he called the ‘shaking palsy’. It affects a large number of people (about 20,000 in the UK) and is most commonly seen in older

patients, although an estimated 5% of sufferers are under 40 years old [89]. Its characteristic feature is an increasing tremor in resting limbs and a rigidity, known as dyskinesia, particularly exhibited as a shuffling gait, but it is also associated with the degeneration of cognitive function and memory. It is thought that oxidative stress in the substantia nigra plays a significant role in the loss of neurons which produce DA [90]. This results in the characteristic deficiency of DA in the substantia nigra seen in the brain of PD patients post mortem, the other characteristic histopathological observation being the presence of deposits called Lewy bodies in the surviving neurons. The major therapeutic approach to PD has been to elevate the levels of DA by either inhibition of monoamine oxidase (MAO), which metabolises DA to less active compounds, or by increasing the concentration of the precursor of DA by administering L-hydroxyphenylalanine (LDOPA) 34 (fig. 12). Another approach involves the use of compounds which are agonists on the DA receptors.

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Parkinson’s Disease

15

COOH

Ph alanine 617

COOH CH3

NH2

NHNH2

Aspartate 311

Serine 505

+ NH3

HO HO

Ph alanine 307

Ph alanine 616

HO OH

OH

HO Serine 508

34 L-DOPA

Tryptamine 613

35 Carbidopa

Ph alanine 509

NHR HO

Fig. 13. Interaction between dopamine and amino acid residues in

the dopamine receptor. HO OH

36 37

R = CH 3 Adrenaline R=H Noradrenaline

CH3

Tryptamine 617

Tryptamine 307

CH3

HO

O NHCH3

NH2

Serine 505

Aspartate 311

OH + NH2

HO

CH3

Ph alanine 616

HO 38 Ephedrine

39 Cathinone

Serine 508

Tryptamine 613 Ph alanine 509

Fig. 12. Dopamine and related compounds.

PD: Use of Dopaminergic Agonists

The structure-activity relationships of compounds which are agonists at the DA receptor are generally accepted to be the two ortho OH groups on the aromatic ring which bind with serine residues 505 and 508, the aromatic ring enabling hydrophobic interactions with a phenylalanine at 617 and, in the terminal position of the ethyl amino group attached to the aromatic ring, a nitrogen with a positive charge which interacts electrostatically with an aspartate residue (fig. 13). It is noteworthy that the adrenergic receptor is very similar in some respects (fig. 14) and so it is possible that compounds with a structure favouring binding to one of these receptors, also have some effect on the other. DA itself is quite unstable and cannot cross the bloodbrain barrier. However, it can be formed within the brain by conversion of its precursor L-DOPA. This compound

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Fig. 14. Interaction between adrenaline (epinephrine) and amino acid residues in the adrenergic receptor.

is now administered routinely to PD patients. It is found in commercially viable amounts in various species of bean, notably Mucuna spp., and this has been used as a commercial source, although the drug is now mainly obtained by synthesis. L-DOPA is often given together with another analogue of DA, carbidopa 35, which is not dopaminergic but which inhibits dopa-decarboxylase and so, by maintaining levels of L-DOPA in the blood, prolongs its activity. When L-DOPA is given over long periods of time, it is common for a sudden decline in sensitivity to occur from time to time and this is called the ‘on-off’ effect. This is probably due to a number of factors including depletion in the ability of the substantia nigra cells to store DA and desensitization of the receptors. Other dopaminergic agents, some of which are mentioned below, are often used as adjuvants to reverse the ‘off’ effect. It is interesting that the powdered seeds of Mucuna pruriens L. have been used in Ayurvedic medicine for dis-

Houghton/Howes

eases of the nervous system [91] and this product has shown to reduce adverse effects, such as the ‘on-off’ effect, in patients [92]. A commercial extract of M. pruriens HP200 was shown to be twice as effective as the equivalent dose of L-DOPA in rats [93]. This may be due to the presence of other, more active, compounds. However, a later study showed that when the same preparation was given to rats over a 52-week period, it elevated DA levels in the cortex but not in the striatum nigrum, this calling into question whether the observed improvements in parkinsonian symptoms were due to the hypothesis originally proposed, i.e. that the L-DOPA in the Mucuna extract was converted to DA and reached the parts of the brain where a deficiency is associated with PD [93]. The transmitter dopamine DA is one of a group of compounds known as phenylpropylamines, which includes some important neurotransmitters such as adrenaline (epinephrine) 36 and noradrenaline (norepinephrine) 37. The phenylpropylamines also include several naturally-occurring compounds known as protoalkaloids, since their N atom is not part of a heterocyclic ring. Most protoalkaloids appear to have a greater adrenergic than dopaminergic effect, although it appears that they can stimulate both types of receptor. The major naturally-occurring compound of this type used therapeutically is ephedrine 38, obtained from some species of Ephedra (Ephedraceae), and these plants which have been used for many centuries in TCM. Ephedrine is used principally for its adrenergic sympathomimetic properties in drying up secretions, but it is well-known for its CNS-stimulant side effects, which may be due also to dopaminergic properties. In contrast, the synthetic phenylpropylamines known as amphetamines are primarily employed as CNS stimulants and there appears to be some association between their use and alleviation of the dyskinesia often associated with PD. A natural compound very similar in chemistry and pharmacology to the amphetamines is cathinone 39, a major active constituent of Catha edulis Forsk. (Celastraceae). The fresh young leaves of this plant are known as ‘khat’ and are used as a stimulant masticatory in Ethiopia, Yemen and surrounding countries and by the diaspora of those communities in Europe [94]. Cathinone has been shown to be present mainly in the young leaves only and, since there have been anecdotal reports of reduction in PD-like tremors induced by neuroleptic drugs in some regular chewers of khat, it might be that this is due to a similar effect of cathinone as that observed for amphetamines [95]. Probably the second most important group of compounds used for the treatment of PD are derivatives of the

Natural Products and Neurotransmission

HO

N H

CH3 H N

HO O

H3C O

H3C H

N

N H

O CH3

N N

O O

N

O

CH3

O

N

CH3

H

H

N

N

CH3

Br H 40 Ergotamine

H 41 Bromocriptine

NHC 2H5 SCH3 N

C 3H7 H

O O

O

CH3

N(C 2H5)2

N

N

CH3

N CH2

N

H

N

CH3 H

H

N

N H

N

H

H

42 Pergolide

43 Cabergoline

44 Lisuride

Fig. 15. Ergot alkaloid derivatives used to treat parkinsonism.

ergot indole alkaloids (fig. 15). Ergot, Claviceps purpurea Tulasne (Clavipitaceae), is a fungus which infects the ears of cereal crops, notably rye Secale cereale L. Ergot has a long history of poisoning animals and humans who eat the cereal flour contaminated with ergot, but also has been exploited in traditional medicine in some parts of Europe to aid childbirth, since an extract causes contraction of the uterus towards the end of pregnancy and also constricts blood vessels, thus reducing bleeding which often occurs at birth. The effects of ergot on the CNS has also been well-documented and outbreaks of ‘madness’ involving hallucinations have been shown to correlate with times of heavy contamination of rye flour in the communities affected. The activity of ergot has been shown to be due to the alkaloids present such as ergotamine 40. All of the alkaloids contain the indole ring structure known as lysergic acid and similarities with the three neurotransmitters noradrenaline (norepinephrine), DA and serotonin can be seen in this (fig. 16). This probably explains the wide spectrum of activity of these alkaloids and so it has been found necessary to alter the chemical structure to produce compounds which more specifically bind to only one of the receptors. A large amount of derivatives of ergot alkaloids have been synthesized for differing therapeutic effects

Neurosignals 2005;14:6–22

17

NHR NH2

NCH3

O

NHR

NH2

NH2

HO

H

O

NCH3 H

HO OH

N H Ergot alkaloid

OH Noradrenaline

OH

N H

OH

N H

Dopamine

Serotonin (5-HT)

Ergot alkaloid

Fig. 16. Ergot alkaloid skeleton showing parts similar to neurotransmitters.

HO HO HO

H N

CH3

N

HO

H

CH3

45 Apomorphine

46 Salsolinol

H

CH3O N

N

CH3

H

H 47

Ibogaine

CH3 N

O

48 Benzatropine

N N

CH3O

CH3

N N

CH3O

H 49 Harmine

CH3

H 50 Harmaline

Fig. 17. Other alkaloids with dopaminergic and monoamine oxidase

inhibitory activity.

18

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and one of these has been dopaminergic receptor stimulation for use in treating PD. Bromocriptine 41, pergolide 42, cabergoline 43 and lisuride 44 are examples of compounds which have been developed in this way and are now used clinically. The pharmacological differences between the compounds are not very great. All are D2 dopamine receptor agonists, although pergolide also has agonist effects at D1 and D3 receptors. All four drugs are wellestablished in treatment and numerous reviews exist on their clinical efficacy and application [96, 97]. Bromocriptine and lisuride were the first of these drugs to be introduced and have comparatively short half-lives. Pergolide has a half-life of 8 h [98] and cabergolide of 68 h, making the latter especially useful for administration only once a day [99]. The ergot-derived drugs were originally used in advanced cases of PD, but there is increasing interest in their use in the first instance since, unlike L-DOPA, they do not need to be metabolized to the active compound and so are not affected by any degeneration in the dopaminergic terminals [100]. Another alkaloid with agonist activity is apomorphine 45, derived from the opium alkaloids (fig. 17). It acts on the D1 and D2 receptors, but, because it is a powerful emetic when given orally, its use is restricted to parenteral administration. It is most commonly given when patients are not exhibiting adverse effects to L-DOPA and as a diagnostic agent for dopaminergic responsiveness. The isoquinolinoid salsolinol 46 has been investigated extensively over the last 10 years. This compound is found in the cocoa bean, the seeds of Theobroma cacao L. (Sterculiaceae), and also in chocolate derived from this plant. The addictive properties of chocolate have been ascribed to the dopaminergic activity of this compound [101], but it also occurs as an endogenous catechol in the brain. Salsolinol has been shown to be dopaminergic at the D2 receptors and also to have a protective effect on neurodegeneration [102]. The N-methyl derivative, which can be synthesized in the

Houghton/Howes

brain, has a role in the pathogenesis of PD [103], and this complicates the usefulness of giving salsolinol as a protective substance to prevent or alleviate PD. Mention should be made of another indole alkaloid, ibogaine 47, obtained from the African plant Tabernanthe iboga Baill. (Apocynaceae), the roots of which were used as a stimulant and, in larger doses, as a hallucinogen in central Africa. This compound has enjoyed much attention recently because of its claimed usefulness in combating addiction, particularly to cocaine, although the basis of its activity is not clear. Ibogaine was shown to release DA in isolated striatal tissue in mice [104], but it has also been shown to inhibit the release of catecholamines by blocking the nicotinic receptor [105] and to block NMDA receptors. These activities preclude its usefulness in treating neurodegenerative disease, but much remains to be done in the study of its antiaddictive potential. The tropane alkaloids, especially hyoscine (scopolamine) 4, have been used to treat PD since they antagonise cholinergic activity at the muscarinic receptors in the striatum and so increase DA activity. The naturallyoccurring alkaloids, found in various genera of the Solanaceae such as Atropa, Hyoscyamus, Datura and Duboisia, are not much used for this purpose, but synthetic derivatives and analogues such as benzatropine 48 are used. These also inhibit DA reuptake, so increasing the levels of DA and compensating for the DA deficiency associated with PD.

R OH O

HO

OH OH

O

51 R = H Kaempferol 52 R = OH Quercetin

OCH3 OCH3 O

CH3O CH3O

OCH3 O 53

Tangeretin

OH OH HO

O

OH O

OH

OH

O

OH OH

54 Epigallocatechin -3-gallate

Fig. 18. Flavonoids with monoamine oxi-

dase inhibitory activity.

PD: Monoamine Oxidase Inhibitors

Monoamine oxidase (MAO) is a major enzyme responsible for the fast breakdown of DA and related compounds at the synapse. Inhibitors of this enzyme, known as MAOIs, cause a net increase in DA levels and, although their major therapeutic use has been as antidepressants, they have potential use in PD. This has not generally been realised because of the side effects associated with elevation of peripheral DA levels. The ß-carboline alkaloids harmane 49 and harmaline 50 are found in several traditional medicine plant species, including Banisteriopsis caapi (Spruce ex Griseb.) Morton (Malpighiaceae), a liana used in Brazil as an ingredient in the hallucinogenic drink ‘ayahuasca’. It is thought that the MAOI properties of the B. caapi constituents prevent metabolism of amines from other plants used to make ayahuasca. Following reports of the successful use of B. caapi root extracts for treating PD patients in Ecuador, it was shown that, in addition to the MAOI properties, the alkaloids harmane and harmaline

stimulated the release of DA from striatal cells [106]. These compounds would therefore have a double effect in helping improve DA levels, and this may underlie the reputed improvements in PD patients when the extract was taken. The role of flavonoids in the diet as important antioxidant contributors has received much attention and their neuroprotective properties because of this effect has been demonstrated by several workers. However, they have also been demonstrated to have MAOI activity and this has been proposed as part of the explanation of the use of the common herb St John’s Wort, as an antidepressant [107]. This dual role has now been proposed for a variety of flavonoids such as kaempferol 51 from the leaves of Ginkgo biloba, a widely used herbal product which has been suggested as a preventative against neurodegeneration [108] (fig. 18). Quercetin 52 has also shown to inhibit

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19

MAO-B [109] and reverse the effects of induced catalepsy, which mimics the bradykinesia often seen in PD [110]. Tangeretin 53 has been shown to inhibit MAO-B and to cross the blood-brain barrier in a rat model and consequently reduce DA depletion, so it may have therapeutic potential [111]. A compound related to the flavonoids, the polyphenol (–)-epigallocatechin-3-gallate 54, found in green tea, has a similar polyvalent activity which may be sufficient to have a protective effect in PD and other conditions [112]. Since most of these flavonoids occur in reasonably large amounts in common fruits and vegetables, the question is raised as to whether the apparent increase in incidence of neurodegenerative disease is related to some extent with the decline in consumption in the diet of such foods in some sectors of the industrialized world.

Conclusions

Several natural products useful compounds themselves, or have provided lead compounds, for present and potential treatment of Alzheimer’s and Parkinson’s diseases. Some compounds have reached widespread clinical use, whilst the interest others at present lies more in their providing explanation for traditional uses of plants. Rational drug design according to the characteristics of the receptors involved is now a possibility, and natural mole-

cules can be subjected to ‘fine-tuning’ by chemical derivatisation and synthesis of analogues to bind more closely and more exclusively to one type of receptor. However, investigation of traditional plants has revealed classes of molecules that have appreciable activity which is unlikely would have been predicted only from receptor studies. Regarding the treatment of the major diseases involved, it can be said that chemotherapy of AD with cholinesterase inhibitors is unlikely to develop very much further since effective agents such as galantamine have been introduced. Future trends could involve the use of a polyvalent ‘cocktail’ of drugs which act in different ways by mechanisms such as antioxidant and anti-inflammatory activity and the inhibition of the formation of fibrillary tangles and ß-amyloid plaques. Although the introduction of L-DOPA and other dopaminergic compounds over the last three decades has improved the condition of many sufferers of PD, the side effects and their unpredictability of occurrence highlight the fact that more work is needed. In this context also, the exploitation of compounds derived from plants with other mechanisms, such as monoamine oxidase inhibition, may provide a better treatment experience in due course. This may occur in extracts in any case, and clinical trials should be carried out to follow up preliminary results, such as those observed with Mucuna bean extract, which indicate an advantage in using an extract over a pure substance.

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8 McGuffey EC: Alzheimer’s disease: An overview for the pharmacist. JAMA 1997;NS37: 347–352. 9 Perry E, Tomlinson E, Blessed G, et al: Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. BMJ 1978;2:1457–1459. 10 Giacobini E: The cholinergic system in Alzheimer disease. Prog Brain Res 1990;84:321– 332. 11 Balfour DJK, Fagerström KO: Pharmacology of nicotine and its therapeutic use in smoking cessation and neurodegenerative disorders. Pharmacol Ther 1996;72:51–81. 12 Whitehouse PJ, Kalaria RN: Nicotinic receptors and neurodegenerative dementing diseases: Basic research and clinical implications. Alzheimer Dis Assoc Disord 1995;9(suppl 2):3–5. 13 Avery EE, Baker LD, Asthana S: Potential role of muscarinic agonists in Alzheimers disease. Drugs Aging 1997;11:450–459.

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67 Ryan MF, Byrne O: Plant-insect coevolution and inhibition of acetylcholinesterase. J Chem Ecol 1988;14:1965–1975. 68 Kuang P, Tao Y, Tian Y: Effect of radix Salviae miltiorrhizae on nitric oxide in cerebral ischemic-reperfusion injury. J Tradit Chin Med 1996a;16:224–227. 69 Ren Y, Houghton PJ, Hider RC, et al: Novel diterpenoid acetylcholinesterase inhibitors from Salvia miltiorrhiza. Planta Med 2004;70: 201–204. 70 Chung YK, Heo HJ, Kim EK, et al: Inhibitory effect of ursolic acid purified from Origanum majorana L. on the acetylcholinesterase. Mol Cells 2001;11:137–143. 71 Ghosal S, Lal R, Srivastava SK, et al: Immunomodulatory and CNS effects of sitoindosides IX and X, two new glycowithanolides from Withania somnifera. Phytother Res 1989;3: 201–206. 72 Schliebs R, Liebmann A, Bhattacharya SK, et al: administration of defined extracts from Withania somnifera (Indian ginseng) and shilajit differentially affects cholinergic but not glutamatergic markers in rat brain. Neurochem Int 1997;30:181–190. 73 Bhattacharya SK, Kumar A, Ghosal S: Effects of glycowithanolides from Withania somnifera on an animal model of Alzheimer’s disease and perturbed central cholinergic markers of cognition in rats. Phytother Res 1995;9:110–113. 74 Bhattacharya A, Ramanathan M, Ghosal S, et al: Effect of Withania somnifera glycowithanolides on iron-induced hepatotoxicity in rats. Phytother Res 2000;14:568–570. 75 Parihar MS, Hemnani T: Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity. J Biosci 2003;28:121–128. 76 Begum V, Sadique J: Long term effect of herbal drug Withania somnifera on adjuvant induced arthritis in rats. Ind J Exp Biol 1998;26:887– 882. 77 Dhuley JN: Effect of some Indian herbs on macrophage functions in ochratoxin A treated mice. J Ethnopharmacol 1997;58:15–20. 78 Hou YC, Chao PD, Chen SY: Honokiol and magnolol increased hippocampal acetylcholine release in freely-moving rats. Am J Chin Med 2000;28:379–384. 79 Kuribara H, Kishi E, Hattori N, et al: The anxiolytic effect of two oriental herbal drugs in Japan attributed to honokiol from Magnolia bark. J Pharm Pharmacol 2000;52:1425– 1429. 80 Squires RF, Ai J, Witt MR, et al: Honokiol and magnolol increase the number of [3H]muscimol binding sites three-fold in rat forebrain membranes in vitro using a filtration assay, by allosterically increasing the affinities of lowaffinity sites. Neurochem Res 1999;24:1593– 1602.

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Review Neurosignals 2005;14:23–33 DOI: 10.1159/000085383

Received: July 28, 2004 Accepted after revision: September 20, 2004

Regulation of Neuroinflammation by Herbal Medicine and Its Implications for Neurodegenerative Diseases A Focus on Traditional Medicines and Flavonoids

Kyoungho Suk Department of Pharmacology, Pain and Neural Injury Research Center, School of Medicine, Kyungpook National University, Daegu, Korea

Key Words Inflammation  Neuroglia  Neurodegenerative disease  Flavonoid  Neuroprotection  Microglia  Astrocyte  Central nervous system

Abstract Herbal medicine has long been used to treat neural symptoms. Although the precise mechanisms of action of herbal drugs have yet to be determined, some of them have been shown to exert anti-inflammatory and/or antioxidant effects in a variety of peripheral systems. Now, as increasing evidence indicates that neuroglia-derived chronic inflammatory responses play a pathological role in the central nervous system, anti-inflammatory herbal medicine and its constituents are being proved to be a potent neuroprotector against various brain pathologies. Structural diversity of medicinal herbs makes them valuable source of novel lead compounds against therapeutic targets that are newly discovered by genomics, proteomics, and high-throughput screening. Copyright © 2005 S. Karger AG, Basel

© 2005 S. Karger AG, Basel 1424–862X/05/0142–0023$22.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nsg

Introduction

In neuroinflammation, microglia and astrocytes play a critical role. Microglial cells are ubiquitously distributed in the central nervous system (CNS) and comprise up to 20% of the total glial cell population in brain [1, 2]. Although the ontogeny of microglial cells has long been debated, recent works using monoclonal antibodies specific for microglial cells indicated that these cells are closely related to monocytes and macrophages [3]. As the primary immune effector cells in the CNS, microglial cells migrate to the site of tissue injury or inflammation, where they respond to invading pathogens or other inflammatory signals [4, 5]. Like monocytes/macrophages, they also secrete inflammatory cytokines and toxic mediators which may amplify the neuroinflammatory responses [6, 7]. Astrocytes form an intimately connected network with neurons in the CNS, and they provide mechanical and metabolic support for neurons [8]. The critical role of these cells in ion buffering and clearance of neurotransmitters is also well established [9, 10]. Upon inflammatory stimulation, astrocytes proliferate and produce diverse intercellular mediators such as nitric oxide (NO) and tumor necrosis factor (TNF)- [11–13]. There is growing evidence that inflammatory mediators produced by activated astrocytes may be involved in the pathogenesis of various neurodegenerative diseases [10, 14]. Thus,

Kyoungho Suk Department of Pharmacology, Pain and Neural Injury Research Center Kyungpook National University School of Medicine 101 Dong-In, Joong-Gu, Daegu, 700-422 (Korea) Tel. +82 53 420 4835, Fax +82 53 256 1566, E-Mail [email protected]

Chemicals and drugs Physical factors

Infection

Autoimmunity Genetic disturbance

Nutritional imbalance Hypoxia Tissue damage

Reversible damage

Fig. 1. Relationship between inflammation and tissue injury. Tissue damage can be triggered by a number of genetic or environmental factors. While reversible tissue damages could be repaired for a functional recovery, irreversible damages associated with inappropriately controlled inflammation (chronic inflammation) could lead to diseases.

Tissue repair, functional recovery

the activation of astrocytes and ensuing production of toxic inflammatory mediators may need to be tightly regulated. Activation of inflammatory cells in CNS (microglia or astrocytes) may be intended to protect neurons at first. More frequently, however, activation of these neuroglial cells and inflammatory products derived from them have been implicated in neuronal destruction commonly observed in various neurodegenerative diseases [7]. Thus, our understanding of pathogenesis of neurodegenerative diseases may be enhanced by elucidation of the molecular mechanism underlying the regulation of neuroglial activation. Among many endogenous or exogenous factors that regulate neuroglial activation and resulting neuroinflammation [15], herbal medicine has recently drawn much attention due to its potent inhibitory effects on inflammatory responses and neuroprotective activity [16, 17]. A central role of microglia and astrocytes in neuroinflammation (and potentially neurodegeneration) and a regulatory effect of herbal medicine on the inflammatory activation of the neuroglia will be discussed in this review.

Inflammation and Tissue Injury

Injury, trauma or infection induce a series of complex and interconnected reaction sequences, initiated at the site of tissue damage [18, 19]. This sequence of reaction

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Irreversible damage

Inflammation Regulation

Diseases

No regulation, chronic inflammation

serves to contain and destroy the infection or damaging agents, and to prevent continued tissue damage and initiate repair processes to restore normal function. This rapid response is known as acute inflammation [20]. The toxic reactions, which are employed to destroy infectious organisms or protect host, also paradoxically have the capacity to injure host tissues. If these toxic responses are not tightly regulated, tissue injury may predominate over tissue protection and repair, thereby leading to inflammatory diseases (fig. 1). The characteristics of the inflammatory response include localized changes within the damaged tissue such as the followings: (1) the release of preformed inflammatory mediators from intracellular stores; (2) the initiation of reaction cascade through the activation of soluble plasma components; (3) the new synthesis of inflammatory mediators such as eicosanoids and cytokines, and (4) resolution of the inflammatory response. The acute inflammatory response is beneficial to the organism in that it helps to deal with potentially dangerous microorganisms. However, inflammation does cause some degree of damage to surrounding tissues. Reactive oxygen species (ROS), reactive nitrogen species (RNS), prostanoids, leukotrienes, and hydrolytic enzymes produced by neutrophils, macrophages, and monocytes may all play a role in mediating inflammation. Persistence of infection or defective resolution of inflammatory reaction results in chronic inflammation where severe tissue damage may occur. Although inflammation is normally a self-

Suk

Fig. 2. Neuroprotective versus neurotoxic ac-

tivities of neuroglia. Neuroglial activation is induced by a variety of intrinsic or extrinsic factors. Physiological neuroglial activation is beneficial and designed to ameliorate damage and stress. In contrast, pathological activation of neuroglia leads to neurotoxicity. Apoptotic elimination of overactivated neuroglia represents a self-regulatory mechanism. When the auto-regulation is at work, physiological neuroprotection may allow a neuroregeneration. On the contrary, a defective regulation may cause neurodegeneration.

Physiological neuroprotection

Intrinsic or extrinsic factors

Neuroregeneration

Apoptosis (self-regulation) Neuroglial activation Pathological neurotoxicity

Neurodegeneration

Apoptosis (self-regulation)

Microglia and astrocytes are essential for ensuring proper functioning of neurons. They are quick to intervene when neurons become injured or stressed. As they are sentinels of neuron well-being, pathological impairment of microglia or astrocytes could have devastating consequences for brain function. Nevertheless, there is still a debate over neuroprotective and neurotoxic functions of these neuroglial cells [22, 25] (fig. 2). It is assumed that neuroglial activation is largely determined by neuronal signals. Acute injury causes neurons to generate signals that inform neuroglia about the neuronal status. Depending on how severe a degree of neuronal injury, neu-

roglia will either nurse the injured neurons into regeneration or kill them if they are not viable. These types of neuroglial responses are considered to represent normal physiological and neuroprotective responses. In contrast, some processes that are chronic in nature persistently activate neuroglia eventually causing a failure in their physiological ability to maintain homeostasis. This could have detrimental consequences and may lead to bystander damage due to neuroglial dysfunction. In this scenario, neuroglia exert neurotoxic effects through the secretion of a variety of toxic inflammatory mediators. Thus, although activation of neuroglial cells may be intended to protect neurons, inflammatory products derived from activated neuroglia may also be implicated in neuronal injury, potentially leading to neurodegenerative diseases [7]. These deleterious effects of neuroglial activation may be exacerbated by the failure of auto-regulatory mechanisms of neuroglia. Recently, activated macrophages, whose functions are closely related to microglia, have been shown to undergo apoptosis [26–28]. It has been suggested that the apoptosis of activated macrophages is one mechanism whereby an organism may regulate immune and inflammatory responses involving macrophages [28]. It has been recently demonstrated that a similar regulatory mechanism exists for microglial cells [29, 30] and astrocytes [31] as well. Microglial cells and astrocytes underwent apoptosis upon inflammatory activation in a manner similar to activation-induced cell death (AICD) of lymphocytes [30, 31]. AICD is an active process. T and B lymphocytes undergo AICD as an autoregulatory mechanism for the body to remove unwanted activated cells after making appropriate use of them [32,

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Neurosignals 2005;14:23–33

limiting event and its benefit outweighs the minor tissue damage it causes, abnormal activation of the immune or inflammatory system has the potential to provoke a devastating response [21]. In gout, for example, elevated concentration of uric acid in the blood leads to precipitation of sodium urate crystal within joints which triggers inflammation by a variety of mechanisms. Another striking consequence of abnormal inflammatory response is autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune vasculitis, dermatomyositis, chronic autoimmune gastritis, and myasthenia gravis. Tissue-damaging chronic inflammatory response may also occur in CNS, where main inflammatory cells are microglia and astrocytes instead of monocytes/macrophages or neutrophils in periphery [22–24].

Neuroglia (Microglia, Astrocytes), Neuroinflammation, and Neurodegeneration

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33]. Compared to lymphocytes, neuroglial cells in CNS are not well studied in this respect. Now, as results in this and other laboratories indicated that neuroglial cells might be under the control of a similar regulatory mechanism [29–31, 34–37], further investigation is warranted to better understand the molecular mechanism(s) of neuroglial AICD and its physiological significance. Nevertheless, it has been shown that, in contrast to AICD of T lymphocytes where Fas-FasL interaction plays a central role, neither Fas-FasL interaction nor TNF- is important in AICD of microglial cells [30]. Instead, NO produced by activated neuroglial cells themselves was the major cytotoxic mediator [30, 31]. However, the presence of NO-independent cytotoxic mechanism has been also suggested [38, 39]. Elimination of activated neuroglial cells by apoptosis could be an important mechanism whereby undesirable effects of long-term neuroglial activation can be minimized. Inflammatory mediators that are produced by activated neuroglia in CNS may have harmful effects on neurons or other neuroglial cells that they originally intended to protect [6, 7]. Thus, in various neurodegenerative diseases involving chronic neuroglial activation, neuroglial functions seem to play a more significant role in mediating diseases than in the protection of neurons. According to the model of activation-induced apoptosis of neuroglial cells, inflammatory signals that activate neuroglia may also initiate internal death program [38, 40]. One interesting question that can be raised then is how neuroglial cells could survive the inflammatory activation. It should be kept in mind that neuroglial cells in vivo are heterogeneous and interact with other neuroglial cells as well as neurons. There is also growing evidence that activated neuroglial cells proliferate in vivo as one way of replenishment [1]. Thus, not all neuroglial cells may respond to the inflammatory signals in the same fashion. Upon inflammatory activation, individual neuroglial cells in heterogeneous population may either undergo AICD or return to the resting state via other regulatory mechanisms depending on the specific microenvironment under which they react to the signals. Although many of activated neuroglial cells may be eliminated, some would survive to be deactivated. Whatever the mechanism of down-regulation is, this may be an excellent auto-regulatory system for the neuroglial activation. One can easily imagine pathological situations where this type of auto-regulatory mechanism goes wrong. Failure of the auto-regulation of ‘over-activated’ neuroglial cells may result in pathological destruction of bystander cells (neurons and other neuroglial cells) exposed to toxic me-

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diators produced by activated neuroglia. Recently, upregulated Bcl-xL expression has been detected in reactive microglia of patients with neurodegenerative diseases [41]. Authors proposed that high level of Bcl-xL protein might render microglia more resistant to cytotoxic environment such as areas of neurodegeneration. Expression of anti-apoptotic Bcl-2 protein has been also associated with aged brain and neurodegenerative diseases [42]. An importance of the physiological regulation of neuroglial activation by AICD is supported by these previous reports. Recent studies focused on the possible role of neuroglia in causing neurodegeneration. Convincing evidence from in vitro studies pointed to the neurotoxic role of neuroglia during traumatic or ischemic brain injury [4] and AD pathogenesis [43]. Supernatants obtained from neuroglial cell cultures kill cultured neurons. Such supernatants contain various neurotoxic substances which include glutamate, NO, ROS, inflammatory cytokines, as well as yet unidentified neurotoxins [44, 45]. Production of these neurotoxins by neuroglia is enhanced by treatment with inflammatory stimuli such as lipopolysaccharide (LPS) and/or interferon (IFN)-. Paradoxically, other investigators have shown that neuroglia-conditioned media promote neuronal survival [46]. Thus, the balance of neurotoxic and neurotrophic effects of neuroglia appears to depend on the nature of the experimental paradigm used. In traumatic brain injury where neuronal regeneration may occur, neuroglial secretory products might help to promote regenerative efforts by injured, but surviving, neurons. However, a situation may be different in neurodegenerative diseases such as Alzheimer’s disease (AD) or human immunodeficiency virus (HIV)associated dementia, where functionally compromised neuroglia may produce neurotoxins, thereby resulting in neuronal damage. There is considerable evidence from postmortem examinations of AD brains that auto-destructive mechanisms are at work, which could in part be responsible for the neurodegeneration [47, 48].

Neuroglia as a Target of Pharmacological Intervention

Considering neuroglial activation as a common feature in many neuropathologies, and keeping in mind that overactivation of neuroglia can have neurotoxic outcomes, it is reasonable to assume that manipulation of neuroglial activation could serve future clinical approaches [49]. Although treatment of the primary events

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in neurodegenerative diseases would still be the preferred intervention, this may not always be possible. Brain or spinal cord injury is a sudden event that is followed by secondary cascades of destruction. Invading macrophages and intrinsic neuroglia in brain may carry a significant portion of these cascades of reaction. Now, there is growing evidence that toxic mediators produced by activated neuroglial cells might be involved in the pathogenesis of various neurodegenerative diseases such as Parkinson’s disease (PD), AD, and HIV-associated dementia [6, 7, 47]. Thus, it is of great interest to find a means to modulate neuroglial activation and CNS inflammatory responses for the therapeutic interventions against these neurodegenerative diseases. Based on understanding of intracellular signaling pathways that are specific for activated neuroglia, a temporary inhibition of signaling molecules or protein-protein interaction associated with signaling pathways would probably allow for a rather selective effect on activated neuroglia, while respective functions in other cell types are unaffected. Elucidation of the intracellular key events that drive neuroglial activation could provide new routes for drug development [49]. Alternatively, potentially harmful products of neuroglia could be neutralized to limit undesired consequences for CNS cells and tissues. Whether it is a direct inhibition of neuroglial activation or indirect suppression of neuroglia-derived toxic inflammatory mediators, a better understanding of neuroglial biology and selective manipulation of neuroglial activation processes represent a promising goal for developing novel neuroprotective strategies.

organic constituents. Similarly, a host of environmental factors, including soil, altitude, and seasonal variation in weather, may affect the levels of components in any given batch of an herb. Because of these multiple factors that affect concentration of active ingredients in the final herbal product, standardization is inevitable, in which certain unique chemical components of the herbs known as markers are identified and the production process is altered to achieve a consistent level of these markers in every final batch of the herbal product. The ten most commonly used herbs in United States are echinacea, garlic, Ginkgo biloba, saw palmetto, ginseng, grape seed extract, green tea, St. John’s wort, bilberry, and aloe [50]. The medical use of herbs in their natural and unprocessed form began when mankind first noticed that certain plants altered particular body functions. Now, much information exists about the historical use and effectiveness of botanical products. Unfortunately, however, the quality of this information is extremely variable. Necessary is evidence-based approach to the pharmacology and clinical efficacy of the herbal medicine.

Herbal Medicine against CNS Disorders

Herbs are generally defined as any form of plants or plant products, including leaves, stems, roots, and seeds [50, 51]. Herbal products may contain a single herb or combinations of several different herbs that are thought to have complementary effects. Herbal products are usually extracts of the plants, which are obtained by boiling or percolating the herbs in water, alcohol, or other organic solvents to release biologically active ingredients of the plants. Herbal products contain complicated mixtures of organic chemicals, which may include fatty acids, sterols, alkaloids, flavonoids, glycosides, saponins, tannins, terpenes and so forth. It is often difficult to determine which component(s) of the herb has biological activity. In addition, the processing of herbs, such as heating or boiling, may alter the pharmacological activity of the

In traditional practices of Chinese medicine, numerous plants have been used to treat stroke and cognitive disorders, including neurodegenerative diseases such as AD [52, 53]. Neurodegenerative diseases and brain injuries resulting from stroke are the major and increasing public health problem in both developed and developing countries worldwide [54]. It is believed that traditional Chinese medicines (TCM) are effective, with few or no side-effects. There are more than 120 traditional medicines in use for the therapy of CNS disorders in Asian countries. Some of their therapeutic effects have been confirmed by recent clinical studies. An ethnopharmacological approach has provided a potentially rich source for drug discovery and development [55]. Many drugs currently available in Western medicine were originally isolated from plants. Although a large number of compounds have been isolated from TCMs, most of these resources have not yet been fully characterized for pharmacological purposes. Some of the TCMs used in stroke therapy include Ledebouriella divaricata, Scutellaria baicalensis, Angelica pubescens, Morus alba, Salvia miltiorrhiza, Uncaria rhynchophylla, and Ligusticum chuanxiong [52]. Among these, S. baicalensis and U. rhynchophylla have been found to confer neuroprotection against transient global ischemia [56, 57]. S. baicalensis is one of

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Neurosignals 2005;14:23–33

Medical Use of Herbs

27

Inflammatory stimuli

Fig. 3. Herbal medicine as a neuroprotector

that targets neuroglial inflammation. Resting neuroglia can be activated by inflammatory stimuli such as LPS, IFN- and TNF-. Activated neuroglia secrete a variety of inflammatory mediators including ROS, nitric oxide, TNF-, and IL-1, which cause neuronal injury (thereby resulting in neurodegeneration). Herbal medicine may be neuroprotective by blocking the inflammatory activation of neuroglia.

Resting neuroglia

the most widely used herbal medicines against bacterial infections of the respiratory and gastrointestinal tract, and various inflammatory diseases. The herb has antipyretic, antibacterial, and antihypertensive properties. The main components of S. baicalensis – baicalin, baicalein, and wogonin – have been previously shown to exert anti-inflammatory effects [58–60]. Based on the use of S. baicalensis for the treatment of stroke in traditional oriental medicine, neuroprotective effects of S. baicalensis have been evaluated after transient global ischemia using rat 4-vessel occlusion model [56]. Methanol extracts from the dried roots of S. baicalensis (0.1–10 mg/kg) administered intraperitoneally significantly protected CA1 neurons against 10 min transient forebrain ischemia as demonstrated by measuring the density of neuronal cells stained with cresyl violet. The neuroprotective effects of S. baicalensis observed in vivo was explained in part by its inhibitory effects on microglial TNF- and NO production as well as protection of nerve growth factor (NGF)-differentiated PC12 cells from hydrogen peroxide toxicity in vitro. U. rhynchophylla also exerted neuroprotective effects against transient global ischemia [57]. In traditional Oriental medicine, U. rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Methanol extract of U. rhynchophylla administered intraperitoneally (100–1,000 mg/kg at 0 and 90 min after reperfusion) significantly reduced the death of hippocampal CA1 neurons following the transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxy-

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Activated neuroglia

Herbal medicine

Neurotoxic inflammatory mediators

Neurodegeneration

genase-2 in hippocampus at 24 h after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF- and NO production in BV-2 mouse microglial cells in vitro in a manner similar to what has been observed with S. baicalensis extract. These anti-inflammatory actions of U. rhynchophylla extract (and other herbal medicines) may contribute to its neuroprotective effects (fig. 3). Ginkgo leaf extracts have been primarily used for the treatment of dementia and neurosensory problems [51, 53, 61]. They contain terpenoids (ginkgolides and bilobalides) and flavonoids. Administration of ginkgo extracts (EGb 761) has shown biological activities relevant to the treatment of CNS disorders [61]. Favorable effects have been observed on cerebral circulation and neuronal cell metabolism. The extract was also neuroprotective against -amyloid- and NO-induced toxicity [62, 63]. These effects have been attributed, in part, to platelet-activating factor antagonism of the ginkgolides [64] and the free radical scavenging and anti-oxidant properties of the flavonoids [65]. In addition to Ginkgo biloba, Huperzia serrata, Lycoris radiata, Magnolia officinalis, and Polygala tenuifolia have been used for improvement of memory and cognitive function.

Plant Flavonoids as a Neuroprotector: Inhibition of Neuroinflammation

Flavonoids are a group of low molecular weight polyphenolic compounds of plant origin, many of which alter metabolic processes and have a positive impact on health [66]. They exhibit a variety of biological activities such as anti-inflammatory, anti-oxidant, anti-viral, and antitumor actions [67, 68]. Wogonin (5,7-dihydroxy-8-me-

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Ischemia

Neuronal density (cell number/mm2)

Sham

No ischemia Ischemia

300

Wogonin

Ischemia Wog.

Ischemia

Ischemia

Sham

200

iNOS

1.0

100

4.5

1.2

TNF 0 Sham

Saline

g

0.5

1

10

Wogonin (mg/kg)

Fig. 4. Neuroprotective effects of wogonin against experimental brain injury. a–f Treatment of experimental animals with wogonin

h

1.0

3.3

1.8

(10 mg/kg i.p., 0 and 90 min right after 10 min ischemia and reperfusion) conferred neuroprotection by markedly reducing the number of damaged pyramidal cells in the CA1 subfield. Representative photomicrographs of cresyl violet-stained hippocampal regions of sham-operated animals (a, d) or animals that had been subjected to 10 min ischemia followed by treatment with either saline (b, e) or 10 mg/kg of wogonin (c, f). Boxed regions in a, b, and c (!40) are shown in d, e, and f (!200), respectively. Scale bar is 100 m. g The neuroprotective effect of wogonin was dose dependent. Either saline or wogonin (0.5, 1 and 10 mg/kg) was intraperitoneally administered into animals following 10 min ischemia. Seven days later, neuronal cell density in CA1 region was measured by Nissl

staining and cell counting. Asterisks indicate statistically significant differences from saline-treated ischemic group (p ! 0.05). Sham = Sham-operated animals (n = 7); saline = saline-treated animals following ischemia (n = 7); wogonin = wogonin-treated animals following ischemia (n = 3). h Wogonin inhibited expression of inflammatory mediators following the ischemic brain injury. At 4 days after forebrain ischemia, the expression of iNOS and TNF- was assessed by RT-PCR analysis of hippocampal tissue followed by Southern blot analysis using sequence-specific oligonucleotide probes. Wogonin (10 mg/kg) markedly reduced the ischemic induction of iNOS and TNF- . Results are representative of three independent experiments. The numbers indicate a fold induction of the gene expression normalized to GAPDH as determined by densitometric analysis of Southern blot of RT-PCR products.

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Table 1. Effect of flavonoid wogonin on the histological changes in hippocampus after kainate injection

Experimental animal groups

Histology scores1

Saline (n = 3) Kainate (n = 5) Kainate + wogonin, 1 mg/kg (n = 5) Kainate + wogonin, 10 mg/kg (n = 6)

0 3.1180.452 2.6780.37 2.2580.322

To examine the neuroprotective effect of wogonin in vivo, excitotoxic neuronal injury was induced by systemic administration of kainate (30 mg/kg i.p.). Injection of kainate induced a severe neuronal cell death in CA1 and CA3 of hippocampus. Pretreatment with wogonin (10 mg/kg i.p., 60 min prior to kainate injection) significantly attenuated the hippocampal cell death both in CA1 and CA3 as determined by histological scoring. Values represent mean 8 SEM. 1 Damage or loss of hippocampal neurons was assessed by Nissl staining at 2 days after kainate administration with or without wogonin pretreatment. Histological damage was scored as follows: 0 = no damage; 1 = occasional injured neurons in CA1 or CA3; 2 = small area (!10%) with neuronal damage or loss in CA1 or CA3; 3 = greater area (10–50%) with neuronal damage of loss in CA1 or CA3; 4 = extended (150%) neuronal damage of loss in both in CA1 and CA3. 2 Statistically significant differences among each other (p < 0.05).

thoxyflavone) and baicalein (5,6,7-trihydroxyflavone) are flavonoids derived from the root of S. baicalensis. These flavonoids have been shown to exert various anti-inflammatory activities in vitro as well as in vivo. Wogonin inhibited LPS-induced production of NO [60, 69] and prostaglandin E2 [70] in macrophages. Wogonin inhibited monocyte chemotactic protein-1 gene expression in human endothelial cells [71]. It also inhibited TPA-induced cyclooxygenase-2 expression and skin inflammation in mice [72]. Moreover, wogonin showed free radical scavenging and anti-oxidant activities [73–75]. In the CNS, however, little information is available about its effects on glial cells and neurons. Gao et al. [73, 76] demonstrated neuroprotective effects of four flavonoids from S. baicalensis, including wogonin, in cultured human neuroblastoma cells. Recently, it has been demonstrated that wogonin inhibits NO production and inducible NO synthase (iNOS) induction in cultured rat astrocytes [77], suggesting that the flavonoid may act as an anti-inflammatory agent in CNS as well. This was confirmed by a recent study where wogonin has been shown to be neuroprotective against experimental brain injury by inhibiting inflammatory activation of microglia [78]. Wogonin in-

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hibited inflammatory activation of cultured brain microglia by diminishing LPS-induced TNF-, IL-1, and NO production. Wogonin inhibited NO production by suppressing iNOS induction and NF-B activation in microglia. Inhibition of inflammatory activation of microglia by wogonin led to the reduction in microglial cytotoxicity toward co-cultured PC12 cells, supporting a neuroprotective role for wogonin in vitro. The neuroprotective effect of wogonin was further demonstrated in vivo using two experimental brain injury models; transient global ischemia by 4-vessel occlusion (fig. 4) and excitotoxic injury by systemic kainate injection (table 1). In both animal models, wogonin conferred neuroprotection by attenuating the death of hippocampal neurons, and the neuroprotective effect was associated with inhibition of the inflammatory activation of microglia. Hippocampal induction of inflammatory mediators such as iNOS and TNF- was reduced by wogonin in the global ischemia model (fig. 4), and microglial activation was markedly down-regulated by wogonin in the kainate injection model as judged by microglia-specific isolectin B4 staining. A similar neuroprotective activity has been demonstrated with baicalein in rats [Kim et al., unpublished results] as well as in gerbils [79]. Baicalein attenuated NO production and apoptosis of LPS-activated, but not IFN--activated, BV-2 mouse microglial cells as well as rat primary microglia cultures [80]. The inhibition of NO production by baicalein was due to the suppression of iNOS induction. Moreover, baicalein inhibited LPSinduced NF-B activity in BV-2 cells without affecting caspase-11 activation, interferon regulatory factor (IRF)-1 induction, or signal transducer and activator of transcription (STAT)-1 phosphorylation. IRF-1 and STAT-1 are central components of IFN- signaling [81]. Taken together, flavonoids such as wogonin and baicalein seem to exert their neuroprotective effects by inhibiting microglial activation, which is a critical component of pathogenic inflammatory responses in neurodegenerative diseases. These findings emphasize the importance of herbal medicines and their constituents as an invaluable source for the development of novel neuroprotective drugs. In neurodegenerative diseases, a pathogenic role of uncontrolled microglial activation is widely accepted at present [7]. In search of neuroprotective agents, now it is time to focus on killer cells (microglia and astrocytes) instead of killed cells (neurons); eliminating or at least suppressing killer microglial activation will provide a better chance for neuroprotection compared to just salvaging dying neurons. Identification of a potent neuroprotector from natural source that inhibits the killer cell activity

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will certainly instigate further investigations in the related areas, which will ultimately lead to the successful development of novel neuroprotective drugs based on flavonoids or other constituents of the medicinal herbs.

Other Components of Herbal Medicine and Other Mechanisms of Neuroprotection

As mentioned above, herbal extracts contain complicated mixtures of organic chemicals, including fatty acids, sterols, alkaloids, flavonoids, glycosides, saponins, tannins, and terpenes. Flavonoids are not the only component possessing neuroprotective effects [82]. Anti-dementia effects of galanthamine, an alkaloid widely occurring in Amaryllidaceous plants, have been well demonstrated in a variety of animal models [83–85]. Acute and chronic treatment with galanthamine significantly improved the impairment of learning, short-term and spatial memory. The p-hydroxybenzyl alcohol and gastrodin are the active ingredients isolated from Gastrodia elata roots, and they have been shown to possess anti-amnesic activities in experimental animals [86–88]. Huperzine A, a sesquiterpene alkaloid purified from the Chinese medicinal herb Huperia serrata, exhibits a broad range of neuroprotective actions [89]. Huperzine A ameliorated learning and memory impairments and improved spatial working memory. Ginsenosides (ginseng saponins), as the major active constituents of ginseng, are another example of herb components with potent neuroprotective effects [90–92]. The cellular and molecular mechanisms underlying the neuroprotective effects of various components of herbal extracts may be as diverse as the plants which these components were isolated from. Although this review has been focused on the role of neuroinflammation in neurodegenerative diseases and its inhibition by neuroprotective herbs, the antioxidant activity of herbal extracts is certainly another important aspect of neuroprotection [93, 94]. A variety of herbal extracts and their components have been demonstrated to exert neuroprotective effects associated with antioxidant activities, either by directly stimulating antioxidant response genes or by potentiating the bodies’ own natural antioxidant defense systems. Modulation of neuroinflammation and the antioxidant activity are not mutually exclusive mechanisms of action. ROS and RNS can be generated during inflammatory responses. These compounds function as important signal-transducing messengers or as an effector to kill invading microorganisms. High concentrations of ROS or RNS, however, may cause tissue injury,

Herbal Medicine and Neuroinflammation

which in turn leads to further inflammation. The beneficial versus detrimental effect of ROS and RNS is tightly regulated by antioxidant defense systems, whereas neuroinflammation is controlled by various endogenous mediators as well as by auto-regulatory apoptosis of inflammatory cells [30, 31]. Inflammation and generation of ROS or RNS seem to be interconnected physiological responses, which may also have pathological implications if left uncontrolled. This is supported by the findings that many herbal extracts and their components with neuroprotective activities exert both anti-inflammatory and antioxidant effects at the same time [56, 65, 95, 96].

Conclusions

Activation of microglia and astrocytes plays a pivotal role in the initiation and progression of various neurodegenerative diseases. Inhibition of the neuroglial activation may provide an effective therapeutic intervention that alleviates the progression of the neurodegenerative diseases. Herbal medicine, especially their flavonoid constituents, may be a useful candidate for such a therapeutic approach. Continual investigation of the mechanisms underlying neuroglial activation, regulation of neuroinflammation, modulatory role of herbal medicine in these processes would not only lead to the discovery of novel neuroprotective agents based on medicinal herbs, but also help to understand complex pathophysiology of neurodegenerative diseases.

Acknowledgement This work was supported by the Korea Research Foundation Grant (KRF-2004-000-E00058). This work was also supported by the Neurobiology Research Program from the Korea Ministry of Science and Technology (2004-01323).

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68 Middleton E Jr, Kandaswami C: Effects of flavonoids on immune and inflammatory cell functions. Biochem Pharmacol 1992;43:1167– 1179. 69 Kim HK, Cheon BS, Kim YH, Kim SY, Kim HP: Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships. Biochem Pharmacol 1999; 58:759–765. 70 Wakabayashi I, Yasui K: Wogonin inhibits inducible prostaglandin E(2) production in macrophages [In Process Citation]. Eur J Pharmacol 2000;406:477–481. 71 Chang YL, Shen JJ, Wung BS, Cheng JJ, Wang DL: Chinese herbal remedy wogonin inhibits monocyte chemotactic protein-1 gene expression in human endothelial cells. Mol Pharmacol 2001;60:507–513. 72 Park BK, Heo MY, Park H, Kim HP: Inhibition of TPA-induced cyclooxygenase-2 expression and skin inflammation in mice by wogonin, a plant flavone from Scutellaria radix. Eur J Pharmacol 2001;425:153–157. 73 Gao Z, Huang K, Yang X, Xu H: Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1999;1472:643–650. 74 Shieh DE, Liu LT, Lin CC: Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin [In Process Citation]. Anticancer Res 2000;20:2861–2865. 75 Kandaswami C, Middleton E Jr: Free radical scavenging and antioxidant activity of plant flavonoids. Adv Exp Med Biol 1994;366:351– 376. 76 Gao Z, Huang K, Xu H: Protective effects of flavonoids in the roots of Scutellaria baicalensis Georgi against hydrogen peroxide-induced oxidative stress in HS-SY5Y cells. Pharmacol Res 2001;43:173–178. 77 Kim H, Kim YS, Kim SY, Suk K: The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production. Neurosci Lett 2001;309:67–71. 78 Lee H, Kim YO, Kim SY, Kim H, Noh HS, Kang SS, Cho GJ, Choi WS, Suk K: Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia. FASEB J 2003;17:1943–1944. 79 Hamada H, Hiramatsu M, Edamatsu R, Mori A: Free radical scavenging action of baicalein. Arch Biochem Biophys 1993;306:261–266. 80 Suk K, Lee H, Kang SS, Cho GJ, Choi WS: Flavonoid baicalein attenuates activation-induced cell death of brain microglia. J Pharmacol Exp Ther 2003;305:638–645. 81 Boehm U, Klamp T, Groot M, Howard JC: Cellular responses to interferon-gamma. Annu Rev Immunol 1997;15:749–795. 82 Zhang ZJ: Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders. Life Sci 2004; 75: 1659– 1699.

83 Chopin P, Briley M: Effects of four non-cholinergic cognitive enhancers in comparison with tacrine and galanthamine on scopolamine-induced amnesia in rats. Psychopharmacology (Berl) 1992;106:26–30. 84 Woodruff-Pak DS, Vogel RW 3rd, Wenk GL: Galantamine: Effect on nicotinic receptor binding, acetylcholinesterase inhibition, and learning. Proc Natl Acad Sci USA 2001; 98: 2089–2094. 85 Sweeney JE, Bachman ES, Coyle JT: Effects of different doses of galanthamine, a long-acting acetylcholinesterase inhibitor, on memory in mice. Psychopharmacology (Berl) 1990; 102: 191–200. 86 Ha JH, Shin SM, Lee SK, Kim JS, Shin US, Huh K, Kim JA, Yong CS, Lee NJ, Lee DU: In vitro effects of hydroxybenzaldehydes from Gastrodia elata and their analogues on GABAergic neurotransmission, and a structure-activity correlation. Planta Med 2001;67: 877–880. 87 Kim HJ, Moon KD, Lee DS, Lee SH: Ethyl ether fraction of Gastrodia elata Blume protects amyloid beta peptide-induced cell death. J Ethnopharmacol 2003;84:95–98. 88 Kim HJ, Lee SR, Moon KD: Ether fraction of methanol extracts of Gastrodia elata, medicinal herb protects against neuronal cell damage after transient global ischemia in gerbils. Phytother Res 2003;17:909–912. 89 Zangara A: The psychopharmacology of huperzine A: An alkaloid with cognitive enhancing and neuroprotective properties of interest in the treatment of Alzheimer’s disease. Pharmacol Biochem Behav 2003;75:675–686. 90 Wen TC, Yoshimura H, Matsuda S, Lim JH, Sakanaka M: Ginseng root prevents learning disability and neuronal loss in gerbils with 5minute forebrain ischemia. Acta Neuropathol (Berl) 1996;91:15–22. 91 Lim JH, Wen TC, Matsuda S, Tanaka J, Maeda N, Peng H, Aburaya J, Ishihara K, Sakanaka M: Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root. Neurosci Res 1997; 28: 191– 200. 92 Kennedy DO, Scholey AB: Ginseng: Potential for the enhancement of cognitive performance and mood. Pharmacol Biochem Behav 2003; 75:687–700. 93 Fahn S, Cohen G: The oxidant stress hypothesis in Parkinson’s disease: Evidence supporting it. Ann Neurol 1992;32:804–812. 94 Metodiewa D, Koska C: Reactive oxygen species and reactive nitrogen species: Relevance to cyto(neuro)toxic events and neurologic disorders: An overview. Neurotox Res 2000; 1: 197–233. 95 Ahlemeyer B, Krieglstein J: Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci 2003;60:1779–1792. 96 Rahman K: Garlic and aging: New insights into an old remedy. Ageing Res Rev 2003; 2: 39–56.

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33

Review Neurosignals 2005;14:34–45 DOI: 10.1159/000085384

Received: October 11, 2004 Accepted after revision: November 16, 2004

Search for Natural Products Related to Regeneration of the Neuronal Network Chihiro Tohda a Tomoharu Kuboyama a, b Katsuko Komatsu a, b a Research

Center for Ethnomedicines, Institute of Natural Medicine, and b 21st Century COE Program, Toyama Medical and Pharmaceutical University, Toyama, Japan

Key Words Neuritic atrophy W Synaptic loss W Dendrite W Axon W Alzheimer’s disease W Amyloid-beta W Ginseng W Withania somnifera W Ashwagandha W Coffee bean

Abstract The reconstruction of neuronal networks in the damaged brain is necessary for the therapeutic treatment of neurodegenerative diseases. We have screened the neurite outgrowth activity of herbal drugs, and identified several active constituents. In each compound, neurite outgrowth activity was investigated under amyloid-ß-induced neuritic atrophy. Most of the compounds with neurite regenerative activity also demonstrated memory improvement activity in Alzheimer’s disease-model mice. Protopanaxadiol-type saponins in Ginseng drugs and their metabolite, M1 (20-O-ß-D-glucopyranosyl(20S)-protopanaxadiol), showed potent regeneration activity for axons and synapses, and amelioration of memory impairment. Withanolide derivatives (withanolide A, withanoside IV, and withanoside VI) isolated from the Indian herbal drug Ashwagandha, also showed neurite extension in normal and damaged cortical neurons. Trigonelline, a constituent of coffee beans, demonstrated the regeneration of dendrites and axons, in addition to memory improvement.

Introduction

Despite the great number of ongoing investigations, neurodegenerative diseases remain incurable. The drugs currently available for dementia, such as donepezil, an acetylcholinesterase inhibitor, are efficacious in the temporary treatment of memory dysfunction, but do not prevent or reverse the underlying neurodegeneration [1]. In patients with Alzheimer’s disease, neuritic atrophy and synaptic loss are considered the major causes of cognitive impairment, based on the results of neuropathological postmortem studies of the brain [2–4]. In the brains of patients suffering from other neurodegenerative diseases, such as Parkinson’s disease, Huntington’s disease, and Creutzfeldt-Jakob disease, neurite atrophy has also been observed [5–7]. Such atrophy leads to the destruction of neuronal networks, and subsequently to the fatal dysfunction of brain systems in these patients. The exclusion of, or at least a decrease in the magnitude of, the causes of each disease may prevent the progression of symptoms, but such inhibition is not associated with the repair of already severely damaged brain function. We hypothesized that the reconstruction of neuronal networks in the injured brain would be the most necessary step in the fundamental recovery of brain function, requiring neuritic regeneration and synaptic reconstruction.

Copyright © 2005 S. Karger AG, Basel

ABC

© 2005 S. Karger AG, Basel 1424–862X/05/0142–0034$22.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nsg

Katsuko Komatsu, PhD Research Center for Ethnomedicines, Institute of Natural Medicine Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama 930-0194 (Japan) Tel. +81 76 434 7645, Fax +81 76 434 5064, E-Mail [email protected]

Table 1. Natural medicine-oriented compounds which enhance neurite outgrowth

Compound

Main botanical source

Cell used

Effective dose Function

Reference

Ginsenoside Rb1

Panax ginseng Panax notoginseng

rat cortical neuron

0.1–100 ÌM

axon extension synaptogenesis memory improvement

14, 21

Metabolite 1*

(protopanaxadiol-type saponins)

rat cortical neuron

0.01–1 ÌM

axon extension synaptogenesis memory improvement

21

Withanolide A

Withania somnifera

rat cortical neuron

1 ÌM

axon extension dendrite extension synaptogenesis memory improvement

36 37

Withanoside IV

Withania somnifera

rat cortical neuron

1 ÌM

axon extension dendrite extension synaptogenesis memory improvement

36

Withanoside VI

Withania somnifera

rat cortical neuron

1 ÌM

axon extension dendrite extension synaptogenesis memory improvement

36

Trigonelline

coffee bean

rat cortical neuron

30–100 ÌM

axon extension dendrite extension memory improvement

41

Honokiol

Magnolia obovata Magnolia officinalis

rat cortical neuron

0.1–10 ÌM

neurite outgrowth

43

(–)-3,5-Dicaffeoylmuco-quinic acid

Aster scaber

PC12

1–10 ÌM

neurite outgrowth

44

Catalpol

Rehmannia glutinosa

PC12h

0.1–1 Ìg/ml

neurite outgrowth

45

Geniposide

Gardenia jasminoides

PC12h

0.1–10 Ìg/ml

neurite outgrowth

45

Gardenoside

Gardenia jasminoides

PC12h

0.1–10 Ìg/ml

neurite outgrowth

45

Picroside I

Picrorhiza scrophulariiflora

PC12D

10–100 ÌM

potentiating NGF-induced neurite outgrowth

46

Picroside II

Picrorhiza scrophulariiflora

PC12D

0.1–100 ÌM

potentiating NGF-induced neurite outgrowth

46

Nardosinone

Nardostachys chinensis

PC12D

0.1–100 ÌM

potentiating NGF-induced neurite outgrowth

47

* 20-O-ß-D-Glucopyranosyl-(20S)-protopanaxadiol.

Natural Products Enhancing Neurite Outgrowth

Neurite outgrowth is the first step in the construction of the neuronal network, and neurite outgrowth activity has been investigated in many crude drugs. Of these

Natural Products Related to Regeneration of the Neuronal Network

extracts, several constituents have been identified as active compounds (table 1). It is critical that extended neurites have specific functions, such as axons and dendrites, and can make circuits by synaptic connections. However, the identification of axons and dendrites and the mea-

Neurosignals 2005;14:34–45

35

surement of synaptogenesis have not been undertaken in studies of natural products, apart from in our research. Ginseng drugs, Ashwagandha and coffee beans contain interesting compounds with potent neurite regeneration, synaptic reconstruction and memory improvement activities.

Ginseng Drugs

Neurite Outgrowth Using Methanol Extracts and Isolated Saponins in SK-N-SH Cells Ginseng, the root of Panax ginseng, is widely used as a tonic throughout the world, and is efficacious in the treatment of amnesia. In addition, significant improvement in learning and memory has been observed in brain-damaged [8, 9] and aged rats [9] after the oral administration of Ginseng powder, and the major Ginseng saponins, ginsenoside Rb1 and Rg1, are known to improve spatial learning in normal mice [10]. Regarding the effects on neuronal cells, it has been shown that neurite outgrowth of cultured rat cerebral cortical neurons is enhanced by crude Ginseng saponins [11], and that ginsenoside Rb1 potentiates the nerve growth factor (NGF)-mediated neurite outgrowth of chick dorsal root ganglia [12, 13]. We tested the neurite outgrowth activity of methanol extracts of 6 types of Ginseng drugs and P. stipuleanatus plant material in SK-N-SH cells [14]. The methanol extracts of Ginseng (dried root of P. ginseng), Red Ginseng (steamed and dried root of P. ginseng), Notoginseng (dried root of P. notoginseng) and Ye-Sanchi (dried rhizome and root of P. vietnamensis var. fuscidiscus) increased neurite outgrowth, with the effects of Red Ginseng and Ye-Sanchi being particularly significant. Thirty saponins were isolated from Ye-Sanchi and structurally elucidated [15, 16]. Oleanolic acid-type saponins were also isolated from Kouzichi (dried rhizome of P. japonicus var. major from Hubei province) [17], and 19 saponins (ginsenosides Rb1, Rb3, Rg1 and Re, notoginsenosides R4, Fa and R1, Yesanchinoside J, 20-O-glc-ginsenoside Rf, majonoside R2, (24S)-pseudoginsenoside RT4 and F11, vina-ginsenoside R1, R2 and R6 from YeSanchi, notoginsenoside R2, ginsenoside Rg2 and Ro, chikusetsusaponin IVa from Kouzichi) were tested. Protopanaxadiol (ppd)-type saponins, ginsenosides Rb1 and Rb3, and notoginsenosides R4 and Fa significantly extended the neurites in SK-N-SH cells at a concentration of 100 ÌM, and their activity increased dose-dependently. On the other hand, protopanaxatriol, ocotillol and oleanolic acid-type saponins showed no effect [14]. This sug-

36

Neurosignals 2005;14:34–45

gests that ppd-type saponins are active compounds. Ginseng, Red Ginseng, Notoginseng and Ye-Sanchi, which showed neurite outgrowth activity, have been demonstrated to contain comparatively rich ppd-type saponins in our quantitative study [18]. This suggested that the effects of these drugs could be mainly attributed to ppdtype saponins. However, Zhuzishen (dried rhizome of P. japonicus var. major from Yunnan province) and a rhizome of P. stipuleanatus, which inhibited cell viability, may contain some cytotoxic compounds. Effect of M1, a Metabolite of Protopanaxadiol-Type Saponins, on Aß(25–35)-Induced Memory Impairment, Axonal Atrophy and Synaptic Loss in Mice When taken orally, ppd-type saponins are mostly metabolized by intestinal bacteria to ppd monoglucoside, 20O-ß-D-glucopyranosyl-(20S)-protopanaxadiol (M1) [19, 20] (fig. 1). As Ginseng is generally taken orally, a metabolite of ppd-type saponins, M1, should be investigated to determine the active constituent of Ginseng responsible for its major effects. We therefore conducted experiments to determine whether treatment with ginsenoside Rb1, as a representative of ppd-type saponins, and its metabolite, M1, can induce recovery from memory disorder, axonal atrophy, and synaptic loss induced by the active fragment of the amyloid-ß peptide (Aß(25–35)) [21]. Male ddY mice (6 weeks old) were prepared to create a mouse model of Alzheimer’s disease (AD). Seven days after an i.c.v. injection of Aß(25–35), ginsenoside Rb1 (10 Ìmol/kg), M1 (10 Ìmol/kg), donepezil hydrochloride (DNP, 0.5 mg/kg), or the vehicle (tap water) was administered orally once daily for 14 days. Mice were trained in the water maze for 7 days starting 14 days after the i.c.v. administration of Aß(25–35) (fig. 2a). The escape latency to find the platform in the Aß(25–35)-injected group significantly increased compared with the saline-injected group, whereas the escape latencies of the groups administered ginsenoside Rb1 and M1 p.o. significantly decreased as compared with the vehicle-administered group. The donepezil-administered group showed no significant shortening of the escape latency. In the retention test (fig. 2b), the number of crossings over a previous platform position was significantly decreased in the Aß(25–35)-injected group compared with the saline-injected group. The number of crossings recovered after treatment with ginsenoside Rb1 and M1. Treatment with donepezil showed the smallest effect in the retention test. All mice showed normal swimming performance and a constant increase in body weight. Locomotor activity did not differ among groups.

Tohda/Kuboyama/Komatsu

Glc1-O OH

COO⫺

N⫹ HO CH3 20-O-␤ ␤-D-glucopyranosyl-(20S)-protopanaxadiol (M1)

Trigonelline

CH3

CH2R2

OH

R1 O

O

O

H H

O

OH

H

OH

H H

O

H H

H O

H

H

Glc1-6Glc1–O

Withanolide A (WL-A)

Withanoside IV

(WS-IV)

Withanoside VI

(WS-VI)

R1

R2

H OH

OH H

Fig. 1. Chemical structures of compounds that regenerate the neuronal network.

After the retention test, the expression levels of phosphorylated NF-H (axonal marker), synaptophysin (synaptic marker) and MAP2 (dendritic marker) were measured in mouse brains. We observed two cortical areas (parietal cortex and temporal cortex) and three hippocampal areas (CA1, CA3, and the dentate gyrus), as it is known that synaptic loss occurs primarily in the cerebral cortex and hippocampus in AD patients [22, 23] and in AD model mice [24]. The phosphorylated NF-H levels were remarkably reduced in these five areas of the brain in Aß(25– 35)-injected compared with saline-injected mice (fig. 3a). Significant decreases were seen in the parietal cortex, CA1 and CA3; however, the expression levels of phosphorylated NF-H were nearly equal to those of the control in ginsenoside Rb1- and M1-treated mice. Donepezil treatment had no effect on the levels of phosphorylated NF-H. The synaptophysin levels were also reduced in these five areas of the brain in Aß(25–35)-injected compared with salineinjected mice (fig. 3b). Significant decreases were seen in

the temporal cortex and CA1. In all areas, the synaptophysin levels were almost equal to or higher than control levels in ginsenoside Rb1- and M1-treated mice. Donepezil treatment had no effect on the synaptophysin levels. The MAP2 levels were also reduced in the cerebral cortex and CA1 of the brain in Aß(25–35)-injected compared with saline-injected mice (fig. 3c). Significant decreases were seen in the temporal cortex; however, these decreases in the expression levels of MAP2 were not clearly recovered by ginsenoside Rb1, M1 or donepezil. Although treatment with M1 tended to increase the MAP2 level in the temporal cortex, the effect was weak. No differences in neuronal density were observed among the groups in any brain areas. Treatment with M1, a metabolite of ginsenoside Rb1, results in the recovery of impaired learning and memory in Aß(25–35)-injected mice with degenerated axons and synapses. The maintained retention of spatial memory was also seen after the discontinuation of ginsenoside Rb1 and M1 administration. These results

Natural Products Related to Regeneration of the Neuronal Network

Neurosignals 2005;14:34–45

37

60

]

Sal/Veh A␤/Veh

Escape latencies (s)

50

]

A␤/GRb1

]

A␤/M1

40

A␤/DNP

30

20

10

0 1

2

a

3 4 5 Days of acquisition test

6

7

Effect of M1 on Aß(25–35)-Induced Axonal Atrophy in Rat Cortical Neurons In in vitro experiments, M1 demonstrated an axonal regeneration effect. To investigate the Aß(25–35)-induced damage to the neuronal network and the reconstructive activity of drugs, 10 ÌM Aß(25–35) was added to the cortical neurons on day 7, and after 3 days the medium was replaced by fresh medium, including drugs. Although the cortical neurons connected with each other during the 7day culture, some of the connections were lost 3 days after Aß(25–35) treatment. At 4 days, both phosphorylated NF-H-positive (fig. 4a) and MAP2-positive (fig. 4b) neurites were significantly shortened by Aß(25–35) treatment. Treatment with 0.01 ÌM M1 (to 78.5% of the control) significantly increased the recovery of the length of phosphorylated NF-H-positive neurites (fig. 4a), while MAP2-positive neurites were not extended (fig. 4b). NGF significantly enhanced the lengths of phosphorylated NF-

Number of crossings over a platform position for 60 s

8 ] 7

6

5

4

3

2

1

Veh

b

Saline

Veh

GRb 1

M1

DNP

A␤(25–35)

Fig. 2. Effects of ginsenoside Rb1 and M1 on the impairment of spatial memory induced by Aß(25–35) injection. a Escape latencies per

group in four trials were tested in a Morris water maze over 7 days. Vehicle was administered p.o. to saline-i.c.v.-injected mice. To Aß(25–35)-i.c.v.-injected mice (5 nmol), the vehicle, ginsenoside Rb1 (10 Ìmol/kg), M1 (10 Ìmol/kg), or donepezil (0.5 mg/kg) was administered p.o. for 14 days. Values represent the means and SEM of 9 mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicletreated group. Two-way repeated measure analysis of variance was carried out, followed by Dunnett’s post hoc test. b The number of crossings over the previous position of a platform had previously

38

suggest that ginsenoside Rb1 and M1 may induce the structural repair of neuronal connections. In the rat large intestine, ginsenoside Rb1 is completely metabolized to M1 3 h after administration [25]. In mice, only M1 is continuously detected in the blood from 30 min to 16 h after oral administration of ginsenoside Rb1 [26]. In humans, M1 is detected in plasma from 7 h after the ingestion of Ginseng, and in urine from 12 h after intake, and aglycone is not detected in either plasma or urine [20]. These results suggest that M1 is the final metabolite of ppd-type saponins. The recovery potency in Aß(25–35)-injected mice by p.o.-administered ginsenoside Rb1 and M1 was almost identical, indicating that the majority of orally administered ginsenoside Rb1 was metabolized into M1. Considering that most ppd-type saponins are metabolized to M1, which is the active principal, the total content of ppd-type saponins is possibly an important index of the anti-AD activity of Ginseng.

Neurosignals 2005;14:34–45

been measured over 60 s, 6 days after the last acquisition test. This was also 6 days after the discontinuance of drug treatment. Vehicle was administered p.o. to saline-i.c.v.-injected mice. To Aß(25–35)i.c.v.-injected mice, vehicle (Veh), ginsenoside Rb1 (GRb1), M1, or donepezil (DNP) was administered p.o. Values represent the means and SEM of 9 mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicle-treated group. One-way analysis of variance was carried out, followed by Dunnett’s post hoc test.

Tohda/Kuboyama/Komatsu

Fig. 3. Effects of ginsenoside Rb1 and M1 on axonal atrophy and synaptic loss induced by Aß(25–35) injection. Expression levels of phosphorylated NF-H (a), synaptophysin (b) and MAP2 (c) in brain slices were quantified. Vehicle was administered p.o. to saline-i.c.v.-injected mice. To Aß(25–35)i.c.v.-injected mice, vehicle, ginsenoside Rb1 (10 Ìmol/kg), M1 (10 Ìmol/kg), or donepezil (0.5 mg/kg) was administered p.o. for 14 days. The parietal cortex (PC), temporal cortex (TC), hippocampal CA1 and CA3, and dentate gyrus (DG) were observed. The fluorescence intensities of six areas in each slice were measured. Values represent the means and SEM of three mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicletreated group. One-way analysis of variance was carried out, followed by Dunnett’s post hoc test.

Natural Products Related to Regeneration of the Neuronal Network

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39

Length of NF-H-positive neurites per cell (␮m)

500

]

400

]

]

300

200

100

0

Veh

M1

Veh

a

NGF

A␤(25–35)

Length of MAP2-positive neurites per cell (␮m)

500

]

]

400

Ashwagandha 300

200

100

0 Veh

Veh

M1

NGF

A␤(25–35) Immunostaining

Culture

7d

b

4d

3d A␤

Drugs

Fig. 4. Effect of post-treatment with M1 on Aß(25–35)-induced axonal and dendritic atrophy. Aß(25–35) (10 ÌM ) was added to rat cortical neurons at 7 days in vitro. Three days later, the medium was replaced by new medium containing M1 (0.01 ÌM ), NGF (100 ng/ ml) or the vehicle (Veh, DMSO). Four days later, the cells were fixed and immunostained for phosphorylated NF-H (a) and MAP2 (b). The lengths of neurites positive for phosphorylated NF-H or MAP2 per cell were measured. Values represent the means and SEM of 30 cells. * p ! 0.05 when compared with the Aß(25–35) plus vehicletreated group. One-way analysis of variance was carried out, followed by Dunnett’s post hoc test.

40

H-positive (to 82.0% of the control) and MAP2-positive (to 95.3% of the control) neurites. In addition, M1 increased in pre-synaptic density to the control level after Aß(25–35)-induced synaptic loss occurred [our unpublished data]. Neuritic atrophy by Aß(1–40) and Aß(25–35) has been reported in chick sympathetic neurons [27] and rat cortical neurons [28]. As neurite atrophy is thought to be due to unusual cell adhesion [27, 29], M1 may be capable of normalizing the adhesive mechanism. Although Aß is known to cause neuronal death through increased [Ca2+] neurons [30], increased peroxynitrites in microglias [31], and mitochondrial dysfunction in neurons [32], the death pathway has been shown to be mediated by separate molecular mechanisms of a neuritic dystrophy event [27– 29]. Since ginsenoside Rb1 did not inhibit neuronal death induced by Aß(25–35), the mechanism of rescuing axonal atrophy may not be identical to that for recovery from Aß-induced neuronal death.

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Neurite Outgrowth with Methanol Extract and Isolated Withanolides Ashwagandha (root of Withania somnifera Dunal) is the most popular herbal drug in Ayurvedic medicine, and has been used traditionally and commonly as a tonic and nootropic agent. It has also been reported as associated with improvements in scopolamine-induced memory deficits in mice [33]. Treatment with a methanol extract of Ashwagandha induced neurite extension [34]. We further identified 6 withanolide derivatives from methanol extract (withanolide A, withanoside IV, withanoside VI, etc.; fig. 1), which induced neurite outgrowth in human neuroblastoma SH-SY5Y cells [35]. In normal cortical neurons, the predominant dendritic outgrowth was induced by treatment with withanoside IV or withanoside VI, whereas predominant axonal outgrowth was observed in treatment with withanolide A in normal cortical neurons [36]. Effect of Withanolides on Aß(25–35)-Induced Neuritic Atrophy and Synaptic Loss In Aß(25–35)-induced damaged cortical neurons, withanolide A, withanoside IV, and withanoside VI showed neuritic regeneration and synaptic reconstruction. 24 h after culture initiation, 10 ÌM Aß(25–35) was added to the culture medium simultaneously with the drugs. Four days later, Aß(25–35) treatment significantly

Tohda/Kuboyama/Komatsu

Length of MAP2-positive neurites per cell (␮m)

] ] 150

] ]

]

100

0

Cont

Veh

WL-A

a

WS-IV

WS-VI

NGF

A␤(25–35) ]

Fig. 5. Effects of withanolide A, withanoside IV, and withanoside VI on the prevention of Aß(25–35)-induced dendritic and axonal atrophy. Cortical neurons were cultured for 24 h, and then the cells were treated simultaneously with 10 ÌM Aß(25–35), and withanolide A (WL-A), withanoside IV (WS-IV), or withanoside VI (WS-VI) at a concentration of 1 ÌM; or NGF or BDNF at a concentration of 100 ng/ml; or vehicle (Veh); or with vehicle alone (Cont). Four days after treatment, the cells were fixed and immunostained for MAP2 or phosphorylated NFH. Lengths of MAP2-positive neurites (a) and phosphorylated NF-H-positive neurites (b) were measured in each treatment. The values represent the means and SEM of 30 cells. * p ! 0.05 when compared with the Aß(25–35) plus vehicle-treated group. Oneway analysis of variance was carried out, followed by Dunnett’s post hoc test.

Length of NF-H-positive neurites per cell (␮m)

] 150

100

0

Cont

Veh

WL-A

WS-IV

WS-VI

NGF

A␤(25–35) Immunostaining

Culture A␤

1d

b

4d

Drug

inhibited the outgrowth of both MAP2-positive neurites and phosphorylated NF-H-positive neurites, showing that Aß(25–35) induced both dendritic and axonal atrophy in rat cortical neurons. Simultaneous treatment with Aß(25– 35) and withanolide A, withanoside IV, or withanoside VI at a concentration of 1 ÌM prevented both dendritic and axonal atrophy induced by Aß(25–35). Dendritic atrophy

was completely prevented by treatment with withanolide A (97.0% of the control), withanoside IV (106.3% of the control), or withanoside VI (117.4% of the control) (fig. 5a). In particular, treatment with withanosides IV and VI tended to induce the growth of longer dendrites than treatment with withanolide A.

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Axonal atrophy was partially prevented by treatment with withanoside IV (88.0% of the control) and withanoside VI (90.0% of the control), whereas treatment with withanolide A (98.6% of the control) completely prevented axonal atrophy (fig. 5b). To determine whether regenerated neurites are able to reconstruct synapses, the expressions of synaptic markers were investigated. Rat cortical neurons were cultured for 21 days to construct mature synapses in vitro, and after the culture period, Aß(25–35) was added to the samples. Four days later, the cells were immunostained with an antibody for post-synaptic density, (PSD)-95 (post-synaptic marker), or with synaptophysin (pre-synaptic marker). PSD-95- and synaptophysin-positive puncta were significantly decreased by treatment with Aß(25–35) [37]. Withanolide A, withanoside IV, withanoside VI, or NGF was added to the culture medium after 4 days of treatment with Aß(25–35) after synaptic loss had occurred. Seven days after the addition of the drug, the cells were fixed and immunostained for PSD-95 or synaptophysin. Treatment with withanolide A, withanoside IV, or withanoside VI significantly induced both PSD-95 and synaptophysin expression, as compared with treatment with the vehicle. These results indicate that withanolide A, withanoside IV, and withanoside VI facilitated the reconstruction of both post-synaptic and pre-synaptic regions in neurons in which severe synaptic loss had already occurred. This increase in post-synaptic structures tended to be significant following treatment with withanoside IV (86.0% of the control) and withanoside VI (83.6% of the control), as compared with withanolide A treatment (68.0% of the control). However, reconstruction of the pre-synaptic region was induced significantly and markedly by treatment with withanolide A (108.1% of the control), as compared with withanoside IV (81.3% of the control) and withanoside VI (75.8% of the control) treatments. Treatment with NGF did not lead to an increase in the development of either the post-synapses (57.7% of the control) or the presynapses (54.4% of the control).

Fig. 6. The effect of trigonelline on the prevention of Aß(25–35)-

induced dendritic and axonal atrophy. Cortical neurons were cultured for 3 days, and then the cells were treated simultaneously with 10 ÌM Aß(25–35), and trigonelline at a concentration of 30 or 100 ÌM, or vehicle (Veh), or with the vehicle alone (Cont). Five days after treatment, the cells were fixed and immunostained for MAP2 or

42

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phosphorylated NF-H. Lengths of MAP2-positive neurites (a) and phosphorylated NF-H-positive neurites (b) were measured in each treatment. The values represent the means and SEM of 12–20 cells (a) or 14–22 cells (b). * p ! 0.05 when compared with the Aß(25–35) plus vehicle-treated group. One-way analysis of variance was carried out, followed by Dunnett’s post hoc test.

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Although NGF extended both axons and dendrites (fig. 4, 5), it has no effect on synaptogenesis. Since NGF itself is not able to pass through the blood-brain barrier, low-molecular-weight substances that mimic NGF action have been developed as anti-dementia drugs. However, such NGF-like drugs are not expected to cure dementia because of a lack of synaptogenesis activity.

Coffee Beans

Neurite Outgrowth with Trigonelline Coffee is consumed as a drink, and is known to stimulate the central nervous system as well as the heart and circulation [38]. It is thought that these effects are mainly caused by caffeine [39] but the effects of other coffee constituents on the central nervous system have hardly been reported. Coffee beans are crude drugs, used in the traditional system of Unani medicine [40]. Among the extracts of raw and roasted coffee beans, a methanol-soluble fraction of the ethanol extract (1 Ìg/ml) of raw beans significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells [41]. It was demonstrated that the neurite outgrowth activity of the methanol fraction decreased depending on the extent of roasting. Among subfractions of this methanol fraction, the basic fraction had significant neurite outgrowth activity. In this basic fraction, trigonelline was identified as an active constituent (fig. 3). It is known that a decrease in trigonelline is related to the degree of roasting [42]. In rat cortical neurons, trigonelline showed dendritic and axonal regeneration. Three days after initiation of the culture, Aß(25–35) was added to the culture medium with trigonelline. Trigonelline (30 and 100 ÌM) treatment dose-dependently prevented both dendritic (fig. 6a) and axonal (fig. 6b) atrophy induced by Aß(25–35).

Fig. 7. Effect of trigonelline on the impairment of spatial memory induced by Aß(25–35) injection. The number of crossings over the previous position of a platform was measured over 60 s, 6 days after the last acquisition test in a Morris water maze. This was also 6 days after the discontinuance of drug treatment. Vehicle was administered p.o. to saline-i.c.v.-injected mice. To Aß(25–35)-i.c.v.-injected mice (4.7 nmol), the vehicle (Veh), 500 mg/kg trigonelline (TGN), or 0.5 mg/kg donepezil (DNP) was administered p.o. Values represent the means and SEM of 9 mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicle-treated group. One-way analysis of variance was carried out, followed by Dunnett’s post hoc test.

jected group. The number of crossings was recovered by treatment with trigonelline, suggesting that memory retention is improved by trigonelline.

Conclusions

Effect of Trigonelline on Aß(25–35)-Induced Memory Impairment Fourteen days after the i.c.v. injection of Aß(25–35) in male ddY mice (6 weeks old), trigonelline (500 mg/kg), donepezil hydrochloride (0.5 mg/kg), or the vehicle (tap water) was administered orally once daily for 15 days. Mice were trained in the water maze for 5 days, starting 21 days after the i.c.v. administration of Aß(25–35). Six days after the last acquisition test, the retention test was performed (fig. 7). The number of crossings over a previous platform position was significantly decreased in the Aß(25–35)-injected group compared with the saline-in-

The ppd-type saponins of Ginseng drugs and M1 (a metabolite of ppd-type saponins by intestinal bacteria) induced significant recovery from memory impairment, axonal atrophy and synaptic loss in mice. The effect of M1 on axonal reconstruction was further confirmed in cultured cortical neurons. These results suggest that orally administered ppd-type saponins potentially ameliorate dementia by reconstructing the neuronal network. Withanolide A, withanoside IV, and withanoside VI, which were isolated from Ashwagandha, facilitated the regeneration of dendrites and axons, and led to the dramatic construction of synapses, although the neuron damage was

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profound and severe. Trigonelline also had dendritic and axonal regeneration activity, and improved memory retention. These compounds, sourced from natural products, and used with treatments preventing pathogenesis and neuronal death, are expected to play an important role as new categorized drugs in curing neurodegenerative diseases in the near future. Although we have shown the high potential of neuronal regeneration from compounds isolated from Ginseng drugs, Ashwagandha and coffee beans, it is dangerous to simply imply that these herbal drugs are expected to be excellent anti-dementia drugs. When taking herbal drugs, the risk of side effects brought by other constituents, and sufficient efficacy compared with isolated compounds should be investigated and carefully considered. However, drugs used in traditional med-

icine may offer a treasury of new medicines to treat intractable diseases with the use of novel study concepts and the application of objective scientific analyses.

Acknowledgments We thank Prof. M. Hattori, Dr. K. Zou, Mr. N. Matsumoto, Dr. M. R. Meselhy, Dr. N. Nakamura and Dr. J. Zhao of the Institute of Natural Medicine, Toyama Medical and Pharmaceutical University for their extensive contribution to this study. This work was supported by Kampou Science Foundation, Uehara Memorial Foundation, Grants-in-Aid for Scientific Research (B), No. 11695086 in 1999– 2001 and No. 14406030 in 2002–2004 from the Japan Society for the Promotion of Science, and the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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10 Mook-Jung I, Hong HS, Boo JH, Lee KH, Yun SH, Cheong MY, Joo I, Huh K, Jung MW: Ginsenoside Rb1 and Rg1 improve spatial learning and increase hippocampal synaptophysin level in mice. J Neurosci Res 2001;63:509–515. 11 Sugaya A, Yuzurihara M, Tsuda T, Yasuda K, Kajiwara K, Sugaya E: Proliferative effect of Ginseng saponin on neurite extension of primary cultured neurons of the rat cerebral cortex. J Ethnopharmacol 1988;22:173–181. 12 Saito H, Suda K, Schwab M, Thoenen H: Potentiation of the NGF-mediated nerve fiber outgrowth by ginsenoside Rb1 in organ cultures of chicken dorsal root ganglia. Jpn J Pharmacol 1977;27:445–451. 13 Nishiyama N, Cho SI, Kitagawa I, Saito H: Malonylginsenoside Rb1 potentiated nerve growth factor (NGF)-induced neurite outgrowth of cultured chick embryonic dorsal root ganglia. Biol Pharm Bull 1994;17:509–513. 14 Tohda C, Matsumoto N, Zou K, Meselhy RM, Komatsu K: Axonal and dendritic extension by protopanaxadiol-type saponins from Ginseng drugs in SK-N-SH cells. Jpn J Pharmacol 2002; 90:254–262. 15 Zou K, Zhu S, Tohda C, Cai SQ, Komatsu K: Dammarane-type triterpene saponins from Panax japonicus. J Natural Products 2002;65: 346–351. 16 Zou K, Zhu S, Meselhy RM, Tohda C, Cai SQ, Komatsu K: Dammarane-type saponins from Panax japonicus and their neurite outgrowth activity in SK-N-SH cells. J Natural Products 2002;65:1288–1292. 17 Zou K, Zhu S, Cai SQ, Komatsu K: In Ye San Qi, Kou Zi Qi: Constituents from the underground part of Panax plants. Abstract 121th Ann Meet Pharmaceutical Soc Japan, 2001, p 110.

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18 Zhu S, Zou K, Fushimi H, Cai SQ, Komatsu K: Comparative study on triterpene saponins of Ginseng drugs. Planta Medica 2004;70:666– 677. 19 Odani T, Tanizawa H, Takino Y: Studies on the absorption, distribution, excretion and metabolism of Ginseng saponins. III. The absorption, distribution and excretion of ginsenoside Rb1 in the rat. Chem Pharm Bull 1983;31: 1059–1066. 20 Tawab MA, Bahr U, Karas M, Wurglics M, Schubert-Zsilavecz M: Degeneration of ginsenosides in humans after oral administration. Drug Metab Dispos 2003;31:1065–1071. 21 Tohda C, Matsumoto N, Zou K, Meselhy RM, Komatsu K: Aß(25–35)-induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by M1, a metabolite of protopanaxadiol-type saponins. Neuropsychopharmacology 2004;29:860–868. 22 DeKosky ST, Scheff SW: Synapse loss in frontal cortex biopsies in Alzheimer’s disease: Correlation with cognitive severity. Ann Neurol 1990;27:457–464. 23 Heinonen O, Soininen H, Sorvari H, Kosunen O, Paljarvi L, Koivisto E, Riekkinen PJ Sr: Loss of synaptophysin-like immunoreactivity in the hippocampal formation is an early phenomenon in Alzheimer’s disease. Neuroscience 1995;64:375–384. 24 Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowitz P, Lieberburg I, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, Vitale J, Wadsworth S, Wolozin B, Zhao J: Alzheimer-type neuropathology in transgenic mice overexpressing V717Fß-amyloid precursor protein. Nature 1995;373:523–527.

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25 Karikura M, Miyase T, Tanizawa H, Taniyama T, Takino Y: Studies on absorption, excretion and metabolism of Ginseng saponons. VII. Comparison of the decomposition of ginsenoside-Rb1 and -Rb2 in the digestive tract of rats. Chem Pharm Bull 1991;39:2357–2361. 26 Hasegawa H, Sung J-H, Benno Y: Role of human intestinal Prevotella oris in hydrolyzing Ginseng saponins. Planta Medica 1997;63: 436–440. 27 Postuma RB. He W, Nunan J, Beyreuther K, Masters CL, Barrow CJ, Small DH: Substratebound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures. J Neurochem 2000;74:1122– 1130. 28 Grace EA, Rabiner CA, Busciglio: Characterization of neuronal dystrophy induced by fibrillar amyloid beta: Implications for Alzheimer’s disease. J Neurosci 2002;114:265–273. 29 Grace EA, Busciglio J: Aberrant activation of focal adhesion proteins mediates fibrillar amyloid ß-induced neuronal dystrophy. J Neurosci 2003;23:493–502. 30 Lin H, Bhatia R, Lal R: Amyloid ß protein forms ion channels: implications for Alzheimer’s disease pathophysiology. FASEB J 2001; 15:2433–2444. 31 Xie Z, Wei M, Morgan TE, Fabrizio P, Han D, Finch CE, Longo VD: Peroxynitrite mediates neurotoxicity of amyloid beta-peptide1–42and lipopolysaccharide-activated microglia. J Neurosci 2002;22:3484–3492.

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32 Casley CS, Land JM, Sharpe MA, Clark JB, Duchen MR, Canevari L: ß-Amyloid fragment 25–35 causes mitochondrial dysfunction in primary cortical neurons. Neurobiol Dis 2002;10: 258–267. 33 Dhuley JN: Nootropic-like effect of Ashwagandha (Withania somnifera L.) in mice. Phytother Res 2001;15:524–528. 34 Tohda C, Kuboyama T, Komatsu K: Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells. NeuroReport 2000;11:1981–1985. 35 Zhao J, Nakamura N, Hattori M, Kuboyama T, Tohda C, Komatsu K: Withanolide derivatives from the roots of Withania somnifera and their neurite outgrowth activities. Chem Pharm Bull 2002;50:760–765. 36 Kuboyama T, Tohda C, Zhao J, Nakamura N, Hattori M, Komatsu K: Axon- or dendrite-predominant outgrowth induced by constituents from Ashwagandha. NeuroReport 2002;13: 1715–1720. 37 Kuboyama T, Tohda C, Komatsu K: Neuritic regeneration and synaptic reconstruction induced by withanolide A. Br J Pharmacol 2005;144:961–971. 38 Czok G: Coffee and health (abstract in English). Z Ernährungswiss 1977;16:248–255. 39 Riedel W, Hogervorst E, Leboux R, Verhey F, van Praag H, Jolles J: Caffeine attenuates scopolamine-induced memory impairment in humans. Psychopharmacology 1995;122:158– 168. 40 Liu YM, Shawuti Y: Pharmacography of Uighur. Wulumuqi, Xinjiang People’s Publishing House, 1985, p 267.

41 Tohda C, Nakamura N, Komatsu K, Hattori M: Trigonelline-induced neurite outgrowth in human neuroblastoma SK-N-SH cells. Biol Pharm Bull 1999;22:679–682. 42 Taguchi H: Biosynthesis and metabolism of trigonelline, and physiological action of the compound (abstract in English). Vitamins 1988;62: 549–557. 43 Fukuyama Y, Nakade K, Minoshima Y, Yokoyama R, Zhai H, Mitsumoto Y: Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons. Bioorg Med Chem Lett 2002;12:1163–1166. 44 Hur JY, Lee P, Kim H, Kang I, Lee KR, Kim SY: (–)-3,5-Dicaffeoyl-muco-quinic acid isolated from Aster scaber contributes to the differentiation of PC12 cells: Through tyrosine kinase cascade signaling. Biochem Biophys Res Commun 2004;313:948–953. 45 Yamazaki M, Chiba K, Mohri T: Neuritogenesis effect of natural iridoid compounds on PC12h cells and its possible relation to signaling protein kinases. Biol Pharm Bull 1996;19: 791–795. 46 Li P, Matsunaga K, Yamakuni T, Ohizumi Y: Potentiation of nerve growth factor-action by picrosides I and II, natural iridoids, in PC12D cells. Eur J Pharmacol 2000;406:203–208. 47 Li P, Matsunaga K, Yamamoto K, Yoshikawa R, Kawashima K, Ohizumi Y: Nardosinone, a novel enhancer of nerve growth factor in neurite outgrowth from PC12D cells. Neurosci Lett 1999;273:53–56.

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Review Neurosignals 2005;14:46–60 DOI: 10.1159/000085385

Received: January 28, 2005 Accepted after revision: March 1, 2005

Multifunctional Activities of Green Tea Catechins in Neuroprotection Modulation of Cell Survival Genes, Iron-Dependent Oxidative Stress and PKC Signaling Pathway

Silvia A. Mandel Yael Avramovich-Tirosh Lydia Reznichenko Hailin Zheng Orly Weinreb Tamar Amit Moussa B.H. Youdim Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, Technion-Faculty of Medicine, Haifa, Israel

Key Words (–)-Epigallocatechin-3-gallate  Neurorescue  Neurodegeneration  Neuroprotection  Parkinson’s disease  Neurite outgrowth  Green tea catechins  Iron chelation  Cell signaling  Protein kinase C

Abstract Many lines of evidence suggest that oxidative stress resulting in reactive oxygen species (ROS) generation and inflammation play a pivotal role in the age-associated cognitive decline and neuronal loss in neurodegenerative diseases including Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s diseases. One cardinal chemical pathology observed in these disorders is the accumulation of iron at sites where the neurons die. The buildup of an iron gradient in conjunction with ROS (superoxide, hydroxyl radical and nitric oxide) are thought to constitute a major trigger in neuronal toxicity and demise in all these diseases. Thus, promising future treatment of neurodegenerative diseases and aging depends on availability of effective brain permeable, iron-chelatable/radical scavenger neuroprotective drugs that would prevent the progression of neurodegeneration. Tea flavonoids (catechins) have been reported to possess potent ironchelating, radical-scavenging and anti-inflammatory ac-

© 2005 S. Karger AG, Basel 1424–862X/05/0142–0046$22.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nsg

tivities and to protect neuronal death in a wide array of cellular and animal models of neurological diseases. Recent studies have indicated that in addition to the known antioxidant activity of catechins, other mechanisms such as modulation of signal transduction pathways, cell survival/death genes and mitochondrial function, contribute significantly to the induction of cell viability. This review will focus on the multifunctional properties of green tea and its major component (–)-epigallocatechin3-gallate (EGCG) and their ability to induce neuroprotection and neurorescue in vitro and in vivo. In particular, their transitional metal (iron and copper) chelating property and inhibition of oxidative stress. Copyright © 2005 S. Karger AG, Basel

Introduction

Polyphenols are natural substances present in beverages obtained from plants, fruits and vegetables such as olive oil, red wine and tea. Flavonoids are the largest group of polyphenols, which include the subclasses of flavones, isoflavones, flavanols, flavans and flavonols [1]. Several prototypes of these groups have been shown to promote a number of physiological benefits, especially in cognitive function and memory impairment. Fresh tea

Prof. M.B.H. Youdim Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, Technion-Faculty of Medicine POB 9697, 31096 Haifa (Israel) Tel. +972 4 8295290, Fax +972 4 8513145, E-Mail [email protected]

(Camellia sinensis) leaves contain a high amount of catechins, a group of flavonoids or flavanols, known to constitute 30–45% of the solid green tea extract [2, 3]. The favorable properties ascribed to tea consumption are believed to rely on its bioactive components, catechins and their derivatives, demonstrated to act directly as radical scavengers and exert indirect antioxidant effects through activation of transcription factors and antioxidant enzymes [for reviews, see 4, 5]. The most abundant polyphenolic compound is EGCG, thought to contribute to the beneficial effects attributed to green tea, such as its anticancer, cardiovascular function improvement and antioxidant anti-inflammatory properties. Indeed, a number of epidemiological studies have shown that phenolic compounds reduce the risk of coronary heart disease, possibly via their anti-inflammatory effects, including inhibition of adhesion molecule and cytokine expression and augmentation of endothelial nitric oxide release [6]. Relative antioxidant activities among tea catechins have been found to be EGCG = (–)-epicatechin-3-gallate (ECG) 1 (–)-epigallocatechin (EGC) 1 (–)-epicatechin (EC) [7]. EGCG accounts for more than 10% of the extract dry weight (30–130 mg per cup of tea) followed by EGC 1 EC 6 ECG [2]. In addition to their radical scavenging action, green tea catechins possess well-established metal-chelating properties. Structurally important features defining their chelating potential are the 3,4-dihydroxyl group in the B ring [8] as well as the gallate group [9, 10], which may neutralize ferric iron to form redoxinactive iron, thereby protecting cells against oxidative damage [11]. However, a wealth of data is accumulating and indicating that the antioxidant/metal chelating attributes of the catechin polyphenols are unlikely to be the sole explanation for their neuroprotective and neurorescue capacity. Thus, catechin polyphenols were found to invoke a wide spectrum of different mechanisms of action responsible for cell survival [for our recent reviews, see 12, 13]. There is evidence that polyphenol metabolites and their parent compounds have access to the brain. Studies with radioactively labeled EGCG in mouse or chemiluminescence-based detection of EGCG in rats demonstrated its incorporation into brain, as well as in various organs including kidney, heart, liver, spleen and pancreas [14, 15]. Furthermore, it has been shown that the methylated and glucuronidated derivatives of epicatechin are both detected in rat brain following oral administration [16]. Significant body of evidence support the hypothesis that brain iron dysregulation and oxidative stress (OS),

resulting in ROS generation from H2O2 and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinson’s (PD), Alzheimer’s (AD) and Huntington’s diseases and amyotrophic lateral sclerosis (ALS) [17]. There is also evidence for increased expression of apoptotic proteins (for review see [18]), as well as mitochondria (complex I) and ubiquitin-proteasome system (UPS) dysfunction, which may lead to breakdown of energy metabolism and consecutive intraneuronal calcium overload [19–22]. Thus, neurodegeneration appears to be multifactorial, where a complex set of reactions lead to the demise of neurons. This assumption receives support from the familial (genetic) forms of neurodegenerative diseases identified in the last years, where mutations in genes, such as -synuclein, parkin and ubiquitin C-terminal hydrolase-L1 (UCHL-1) described in rare forms of hereditary PD [23], may lead to impairment in the activity of the UPS. More recently, recessive mutations in DJ-1 [24] and PINK1 (PTENinduced kinase 1) [25] were proposed to play a role in cellular response to OS, supporting a pathogenic role of ROS in the etiology of neurodegenerative diseases. Therefore, it is not surprising that antioxidants were the first drugs to be studied in an attempt to retard the progress of PD. Recently, coenzyme Q10, an intrinsic component of the mitochondrial respiratory chain acting as a bioenergizer and an antioxidant, was studied as a putative neuroprotective agent in PD. This double-blind, placebo-controlled pilot study demonstrated that high doses of coenzyme Q10 (1,200 mg/d) were associated with a reduced rate of deterioration in motor function from baseline over the 16-month course of this trial [26]. Neuropathological and neurochemical studies on substantia nigra (SN) from PD brains and its animal models [23, 27, 28] and our recent gene expression profiling of human SN pars compacta (pc) from PD patients [29] demonstrate the existence of a ‘domino’ cascade of neurotoxic events, which can be initiated at any point in the cascade. These series of events may act independently or cooperatively during the course of the disease, leading eventually to the demise of dopaminergic neurons. This has led to the current notion that drugs directed against a single target will be ineffective and rather a single drug or cocktail of drugs with pluripharmacological properties may be more suitable to be employed. According to this belief, green tea catechins well fulfill the requirements for a putative neuroprotective drug having diverse pharmacological activities. Thus, it is not surprising that they

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have attracted increasing interest as therapeutic cytoprotective agents for the treatment of neurodegenerative and other diseases. In this article the state of the art of the diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective action of green tea catechin polyphenols is reviewed. Particular attention has been paid to their iron-chelating properties with respect to the potential promise for iron chelation therapy, as a novel treatment for neurodegenerative diseases.

Neuroprotective Effects of Catechins: Insights from in vivo and in vitro Studies

There is a growing recognition that polyphenolic catechins exert a protective role in neurodegeneration. An experimental study conducted in N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) model of PD has shown that both green tea extract and EGCG effectively prevent mice striatal dopamine (DA) depletion and SN dopaminergic neuron loss [30]. The protection exerted by green tea polyphenols in vivo may involve direct scavenging of ROS and regulation of antioxidant protective enzymes. EGCG was found to elevate the activity of two major oxygen-radical species metabolizing enzymes, superoxide dismutase (SOD) and catalase in mice striatum [30]. This is supported by a previous finding where 1 month’s administration of a catechin-containing antioxidant preparation increased SOD activity in the mitochondria fraction of striatum and midbrain and decreased thiobarbiturate reactive substance formation in the cortex and cerebellum of aged rats [31]. The structural catechol resemblance of EGCG may explain a recently reported inhibitory effect of green tea polyphenols on the DA presynaptic transporters. This inhibition lead to 1methyl-4-phenylpyridinium (MPP+) uptake blockade (because of competition for the vesicular transporter) thereby protecting DA-containing neurons against MPP(+)-induced injury [32]. In addition, EGCG greatly inhibited catechol-O-methyltransferase (COMT) activity in rat liver cytosol at a low IC50 concentration (0.2 M) [33]. This action may be of particular significance for PD patients, given that DA and related catecholamines are physiological substrates of COMT, thus its inhibition will result in increased DA in the synapse. Green tea polyphenols have been also shown beneficial in animal models of cerebral ischemia: intraperitoneal injection of EGCG reduced hippocampal neuronal damage and brain edema caused by global [34] or unilateral

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[35] cerebral ischemia in gerbils. Insights into the possible mechanism of neuroprotection by EGCG in the infarct area of ischemic rats, revealed that it acts by reducing iNOS expression, infiltration and peroxynitrite formation [36], by increasing endothelial and neuronal NOS and preservation of mitochondrial complex activity and integrity [37]. In this context, the decrease in the activity of the transcription factor signal transducer and activator of transcription-1alpha (STAT-1alpha) by EGCG in ischemic rat cardiac myocytes may well account for the reduced mRNA levels of iNOS, a target of STAT-1 [38]. Other investigators have recently shown that EGCG reduced brain inflammation and neuronal damage in experimental autoimmune encephalomyelitis (EAE), when given at initiation or after the onset of EAE [39]. An extensive number of studies regarding neuroprotection by green tea flavonoids in cellular and animal models of neurodegenerative diseases are starting to accumulate. Hence, the flavonoid epicatechin was shown to attenuate the toxicity induced by oxidized low-density lipoprotein in mouse-derived striatal neurons [40] or fibroblasts [41] and to confer protection to primary culture of mesencephalic neurons challenged with 6-hydroxydopamine (6-OHDA) [42]. Recently, catechin was shown to reduce injury produced by hydrogen peroxide, 4-hydroxynonenal, rotenone and 6-OHDA in primary rat mesencephalic cultures, as shown by increases in cellular viability and [3H]DA uptake [43]. Similarly, EGCG was reported to protect human neuroblastoma cells from damage induced by 6-OHDA and MPP+ [44]. EGCG also protects primary hippocampal neurons [45] and rescues rat pheochromocytoma (PC12) cells from amyloid- peptide (A)-induced toxicity [46]. More recently, EGCG was reported to exert a neurorescue activity in long-term serum-deprived PC12 cells and to promote neurite outgrowth, as manifested by the expression of a surrogate marker of cell differentiation, growth-associated protein GAP-43 (GAP-43) [47]. This could have important implications with regard to aging, PD and AD, suggesting a potential therapeutic use of EGCG in regenerating injured neuronal cells.

Neuroprotection and Neurotoxicity by Low and High Concentrations of Catechins and Other Flavonoids

Studies from our and other laboratories have shown that green tea polyphenols display a concentration-dependent window of neuroprotective action: They protect

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at low micromolar concentrations, whereas they become pro-oxidant and pro-apoptotic when increasing the concentrations over 10–20 M [44, 48]. This bell-shaped pattern is typical of antioxidative drugs, such as vitamin C [49], R-apomorphine [50] and DA [51], being neuroprotective at low (1–10 M) concentrations, while having pro-oxidant/pro-toxic activity at higher (10–50 M) concentrations. The toxicity of not only green tea polyphenols, but also of several other flavonoids, is responsible for their antiproliferative and chemopreventive actions. The anticancer properties of polyphenols is attributed to the ability to inhibit phase I and induce phase II carcinogen metabolizing enzymes (in animals) that initiate carcinogenesis, inhibition of cell cycle progression effectors, promotion of ROS and nitrogen species (thereby collapsing the mitochondrial membrane potential), induction of p53 and apoptogenic factors and inactivation of protein kinases that contribute to survival-associated signal transduction [for extensive reviews see 5, 52, 53]. Since the present article applies to the mechanisms of neuroprotection by tea catechins, the literature regarding their anticarcinogenic or pro-apoptotic properties will not be reviewed.

Mechanism of Neuroprotection by Green Tea Catechins

Cell Signaling Pathways Selective Activation of Protein Kinase C (PKC) in Brain Neurons PKC expression has been previously coupled with the preservation of cell survival and the formation and consolidation of different types of memory [54–56]. The induction of PKC activity in neurons is thought to be a prerequisite for neuroprotection against several exogenous insults. Indeed, PKC activation after ischemic preconditioning or pharmacologic preconditioning (with PKC, NMDA, or A1AR agonists) was shown essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures [57]. In accordance, activation of PKC by estrogen or by the grape flavonoid resveratrol, in rat cortical or hippocampal neurons, respectively, protects against A toxicity [58, 59]. Also, we have recently shown that the anti-Parkinson/monoamine oxidase-B (MAO-B) inhibitor drug, rasagiline (Teva Pharmaceutical Industries) [60], prevented PC12 cell death induced by serum deprivation via PKC signaling cascade [61]. Similarly, we have reported that phosphorylative activation of PKC by EGCG is responsible for the protec-

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tive effects against 6-OHDA- and A-induced cell death in SH-SY5Y and PC12 cells [44, 46] and for the neurorescue effect against long-term growth factors withdrawal in PC12 cells [47]. This is supported by the observation that EGCG could not overcome cell death under PKC pathway blockade, as determined both morphologically and by monitoring various apoptotic markers, suggesting that this cascade is essential for the neuronal protection and rescue effects of EGCG. Consistent with these findings, recent animal studies have shown that two weeks consumption of EGCG (2 mg/kg) led to a highly significant up-regulation of PKC isoform in mice striatum [62] and to a significant increase in PKC isoenzymes  and  in the membrane and cytosolic fractions of mice hippocampus [46]. The implication of PKC in neuronal survival by EGCG is further demonstrated in vitro by the rapid translocation of PKC to the membrane compartment in PC12 cells, in response to EGCG (fig. 1). PKC is a well-established neuron cell survival factor participating also in cell growth and differentiation [63, 64]. In support, a recent report shows that treatment of human cells with EGCG induces a specific translocation of PKC to the membrane [65]. More direct evidence implicating PKC in EGCG mechanistic action has come from a recent study employing solid-state nuclear magnetic resonance, showing that EGCG interacts with the head group region of the phospholipids within lipid bilayers from liposomes [66]. The interaction pattern of EGCG in terms of rotational motion within the lipid bilayers was similar to that described for 12-O-tetradecanoylphorbol-13-acetate (TPA) [67], a phorbol ester. Phorbol esters are prototype activators of PKC, suggesting that direct interaction of green tea catechins with cell membranes may be sufficient for the rapid activation of PKC by EGCG previously reported by us [44]. The impact of EGCG on membrane fluidity may give rise to activation of other membrane-associated signaling pathways (e.g. G proteins), which can contribute as well to its protective action. This clearly needs to be examined. Modulation of Other Signal Transduction Pathways and Intracellular Transducers In addition to PKC, other cell signaling pathways have been also implicated in the action of green tea catechins such as the mitogen-activated protein kinases (MAPKs) and phosphatidylinositide 3-OH kinase (PI3K)/AKT signaling cascades. These cascades have been shown to play central functions in neuronal protection against a variety of extracellular insults and to be essential for neu-

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Fig. 1. EGCG activates p-PKC (pan) and induces a rapid activation of PKC and translocation to the membrane. A Cell lysates from PC12 cells deprived of serum for 24 h before short-term (15 min) exposure to EGCG (1–10 M) were subjected to SDS-PAGE and Western blot, with p-PKC (pan) antibody. B Cultured PC12 cells grown with full serum (FS) support (control) were deprived of serum (SF) for 24 h before short-term (15 min) exposure to EGCG (1–10 M). The cells were fixed and permeabilized for subcellular localization of PKC by confocal microscopy, using an isoenzymespecific antibody and FITC-conjugated secondary antibody (light areas). DRAQ5 stains nuclei (dark areas). Under FS conditions (control) PKC is evenly confined to both plasma membrane (grey arrow) and cytosol (white arrow) (a). Upon serum withdrawal, PKC immunostaining is mosly cytosolic (b). In cells treated with EGCG PKC is, in its majority (1 M), or entirely (10 M) localized to the cell membrane (arrows) (c, d). The images are representative fields from 3 independent experiments, all showing the same results. Taken from Reznichenko et al. [47].

ronal differentiation and survival [68–70]. OS seems to be a major stimulus for MAPK cascade, which might lead to cell survival/cell death [for review see 71]. Among the MAPKs the extracellular signal-regulated kinases (ERK1/2) are mainly activated by mitogen and growth factors [72], while p38 and c-jun-N-terminal kinase (JNK) respond to stress stimuli [73]. However, there have been reports where activation of ERK1/2 is thought to mediate neuronal injury such as in focal ischemia [74], in glutamate and oxidized-low-density-lipoprotein-induced toxicity [40, 75] and in cytotoxicity and activation of caspase-3 in the extraneuronal hepatoma HepG2 [76] and HeLa [77] cell lines, respectively. Increasing evidence shows that catechins can protect against neuronal cell death caused by exogenous OS-inducing agents through modulation of ERK activity [40, 44]. In this regard, a number of flavonoids and phenolic antioxidants, at their respective low protective concentrations, were demonstrated to activate the expression of some stress-response genes, such as phase II drug-metabolizing enzymes, glutathione-s-transferase and heme-oxygenase1 [77], likely via activation of the MAPK pathway [78]. More recently, additional signaling pathways, including PI3K/AKT, protein kinase A (PKA) and calcium, have been implicated in the neuroprotective action of catechin flavonoids. These studies have been conducted mainly in extra-neuronal tissue such as skin and heart. For example, topical application of EGCG induces proliferation of human normal epidermal keratinocytes through stimulation of ERK1/2 and AKT [79]. Other investigators reported a rapid activation of endothelial nitric oxide synthase after EGCG treatment by a process that involves PI3K, PKA and AKT in endothelial cells [80] and a decrease in iNOS expression via inactivation of STAT-1alpha in epithelial and colon cell lines [81]. Consistent with this, Townsend et al. [38] have recently reported that in cardiac myocytes EGCG protects against ischemia/reperfusion-induced apoptosis through a mechanism involving reduction of STAT-1 phosphorylation (inactivation) and of his downstream pro-apoptotic target gene, Fas. The discrepancy or divergence in the different signal pathway activation by EGCG may reflect differences in cell tissue (e.g. neuronal vs. peripheral), or in the downstream pathways being under the control of the different kinases, which may diverge into different responses, thereby providing cell function diversity. Anti-Apoptotic Activity EGCG has been reported to exert a biphasic mode of action as a function of concentration. The low micromo-

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lar concentrations are responsible for the anti-apoptotic/ neuroprotective actions of EGCG. This receives further recognition from an experiment employing customized cDNA microarray designed to clarify the molecular mechanisms involved in the cell survival action of EGCG [44]. The results revealed that a low EGCG concentration decreased the expression of pro-apoptotic genes bax, bad, caspases-1 and 6, cyclin-dependent kinase inhibitor p21, cell-cycle inhibitor gadd45, fas-ligand and tumor necrosis factor-related apoptosis-inducing ligand TRAIL, in SHSY5Y neuronal cells. The same group has reported recently that EGCG reduced the expression of several apoptogenic factors when given after long-term serum deprivation of PC12 cells [47]. These findings are supported by an in vivo study showing that two weeks oral consumption of EGCG (2 mg/kg) alone caused a complete disappearance of Bax immunoreactivity, specifically in the dopaminergic neurons of the SNpc [62] and counteracted the robust increase of Bax protein when administered before MPTP intoxication in the same area. Bax alone, or in conjunction with the BH-3-only proteins (e.g. Bad, Bid, Bim, Noxa, Puma), can trigger the opening of the mitochondrial megachannel permeability transition pore (mPTP), or a specific channel in the outer mitochondrial membrane, both of which promote the fall in mitochondrial membrane potential, leading to cytochrome c release and consequent cell death [82, 83]. The decline in Bax expression by EGCG may favor the increase in the ratio of Bcl-2/Bcl-xL to Bax/Bad proteins, thereby contributing to mitochondrial stability and regulation of mPTP [84]. Protection of mitochondrial integrity is of major importance, especially in the case of postmitotic cells such as neurons and heart muscle cells, which are commonly not renewed. Thus, it is not surprising that one of the major neuroprotective strategies in PD, AD and other neurodegenerative diseases, where increased OS, perturbed cellular energy and ion homeostasis have been implicated, includes pharmacological agents directed to specific mitochondrial targets. In this respect, Ginkgo biloba extract EGb 761 or its individual components were shown to protect mitochondria integrity by protecting against uncoupling of oxidative phosphorylation, thereby increasing ATP levels and by increasing the expression of the mitochondrial DNA-encoded COX III subunit of cytochrome oxidase [for review, see 85]. Flavonoids may also affect mitochondrial integrity by increasing GSH levels and preventing the influx of calcium, as previously reported [86, 87].

Multifunctional Catechins for Neuroprotection

Metal-Chelating Activity One cardinal feature of neurodegenerative diseases including AD, PD, Huntington’s disease, ALS, Friedreich’s ataxia, multiple sclerosis, and aceruloplasminemia is the appearance of excessive iron at degenerative neuronal sites [17]. The buildup of an iron gradient in conjunction with ROS (superoxide, hydroxyl radical and nitric oxide) are thought to constitute a major trigger in the demise in all theses diseases. Therefore, the chelation of free cellular ferric and ferrous ions by the different metal chelators make them potential agents to combat iron-induced generation of reactive oxygen radicals (by the Fenton and Haber-Weiss reactions) and aggregation of alpha ()synuclein and A in PD and AD, respectively. Iron Chelation for AD A significant body of evidence point to an ‘amyloid cascade’ event in the pathogenesis of AD, where amyloid precursor protein (APP) is processed to A, by - and -secretases, which spontaneously self-aggregate in the presence of divalent metals (Fe2+, Cu2+) into neurotoxic amyloid fibrils in the neocortex [88]. Iron was shown to promote both deposition of A and OS, which is associated with the plaques [89]. In addition, iron has taken center stage in AD as a consequence of the studies by Rogers and coworkers [90] who described the existence of an iron responsive element (IRE-type II) in the 5UTR region of APP mRNA. APP is post-transcriptionally regulated by iron regulatory proteins (IRPs), which are labile iron pool-sensitive cytosolic RNA proteins binding specifically to the IRE located in the 5 or 3 untranslated regions of iron metabolism-associated mRNAs. Changes in iron status (iron overload or depletion) lead to compensating changes in the IRP/IRE system of translational control of iron homeostasis. For example, the APP 5-UTR conferred translation was selectively down-regulated upon intracellular iron chelation, in a similar manner as the iron-storage protein ferritin, which also possesses an IRE in its 5-UTR mRNA [90]. Iron removal has effects suggestive of inhibition of key enzymes or other metalloproteins, for instance in mimicking hypoxia, whereas many of the effects of iron overload may be the result of signaling associated with OS. Hypoxia and iron chelation have similar effects on genes regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1), a master regulator orchestrating the coordinated induction of an array of hypoxia-sensitive genes [91]. The target genes of HIF are especially related to angiogenesis, cell proliferation/survival and glucose/

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activation of APP mRNA. Neurodegeneration can result from abnormal serum iron transport to the neurons because a disruption in the blood-brain barrier (BBB) or from release from its storage protein ferritin, thereby increasing the free-labile iron pool (ionic iron). Labile iron can increase the production of amyloid precursor protein (APP) by down-regulating the activities of iron regulatory proteins (IRP1 and IRP2, inactivation and proteasomal degradation, respectively), thereby promoting the translation of APP mRNA from its 5-UTR type II. Ionic iron may also cause aggregation of amyloid- peptide (A) to form toxic aggregates, which, in

turn, can initiate OH generation, causing oxidative stress (OS). Increased iron and OS may activate the prolyl hydroxylase enzymes which are key iron and oxygen sensors, leading to proteasomal-mediated degradation of the transcription factor hypoxia-inducible factor 1, a master regulator orchestrating the coordinated induction of a wide array of survival genes. It has been also suggested that IRP2 can be a substrate for prolyl hydroxylase. Neuroprotective agents that can be used to prevent iron-induced neurodegeneration include M30 and HLA20 (bifunctional iron chelator-MAO inhibitors), VK-28, EGCG and curcumin (iron chelators). For a more detailed explanation, read text.

iron metabolism [92]. The mechanism of HIF-1 activation by iron chelation is not well understood. Fe(II)/2oxoglutarate-dependent dioxygenases have been identified that hydroxylate critical proline and asparagine residues in HIF and upon high oxygen levels and iron overload, target HIF for degradation [93]. Thus, these prolyl hydroxylase enzymes act as key iron and oxygen sensors. This may explain the decrease in cell survival genes described in neurodegenerative diseases such as phosphofructokinase and the angiogenic factor VEGF, both regulated by the HIF proteins [94]. Interestingly, the free iron-induced proteasomal-mediated degradation of IRP2 involves also activation a prolyl hydroxylase and is inhibited by iron chelators [95, 96]. Thus, it is possible that IRP2 is a substrate for this enzyme, in a similar way

as HIF, signaling it for protein degradation. This suggests a convergence of both iron and OS to a common pathway triggering the neurotoxic degenerative cascade (for a detailed explanation, see fig. 2). The involvement of metals in the plaques of AD patients and the presence of an IRE in the unstranslated region of APP mRNA, have encouraged the development of iron chelators as a major new therapeutic strategy for the treatment of AD. In fact, intramuscular administration of the prototype iron chelator desferrioxamine (DFO) slowed the clinical progression of AD dementia [97], and some success has also been achieved with another metal-complexing agent, clioquinol [98]. However, clioquinol is highly toxic [99] and DFO has poor penetration across the blood-brain barrier [17].

Fig. 2. Iron-induced neurodegeneration in AD via transcriptional

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Fig. 3. Comparison of the Fe2+-chelating potency of EGCG to other iron chelators. The metal-binding capacity of EGCG was compared to that of DFO and the novel iron chelators VK28, HLA20 and M30, by assessing their ability to compete with ferrozine for the ferrous ions, resulting in decrease in the absorbance at 562 nm. Ferrozine can quantitatively react with Fe2+ to form Fe2+-ferrozine complex with a strong absorbance at 562 nm. In the presence of other chelating agents, the complex formation is disrupted with the result that the absorbance at 562 nm is decreased. 0.1 mM of drug was mixed with 0.1 mM ferrozine in 5% ammonium acetate (pH 7) followed by the addition of 0.02 mM FeSO4. After 2 h incubation, the absorbance (at 562 nm) of resulting solutions was read. Considering that the purpose of this assay was to evaluate the ability of drugs to compete with the iron indicator ferrozine, drugs and ferrozine were used at equal concentrations. Chelating effect of drug on Fe2+ was calculated as follows: Chelating effect (%) = [1–(absorbance of sample at 562 nm)/(absorbance of control at 562 nm)]! 100. The order of chelating potency for complexing Fe2+ in solution is Desferal (DFO) 1 M30 6 VK28 6 HLA20 1 EGCG.

A possible novel promising therapeutic approach for treating AD, PD and ALS with non-toxic, brain permeable metal chelators could be the use of the naturally occurring polyphenols, such as EGCG and curcumin, which by being of natural origin may not exert toxic side effects inherent to synthetic drugs. Both compounds have wellcharacterized antioxidant, metal (iron and copper) chelating and anti-inflammatory activities [12, 100] and have been demonstrated to exert neuroprotective activity against a variety of neurotoxic insults, as well as to regulate APP processing and A burden in cell culture and in

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vivo [12, 101]. Our recent studies have shown that prolonged administration of EGCG to mice induced a reduction in holoAPP levels in the hippocampus [46]. Indeed, this effect may be related to the iron-chelating properties of EGCG, leading to a decrease in the free-iron pool. This in turn results in the suppression of APP mRNA translation by targeting the IRE-II sequences in the APP 5-UTR [90], as was recently shown for DFO and the bifunctional amyloid-binding/metal-chelating drug XH1 [102] (fig. 2). The concept of metal chelation as a neuroprotective strategy has led us to the development of non-toxic, lipophilic, brain-permeable iron chelators for progressive neurodegenerative diseases [103]. Compounds such as VK-28 (Varinel) [104] and the multifunctional iron chelators HLA20 and M30 [105, 106], which possess the propargylamine MAO inhibitory and neuroprotective moiety of rasagiline, display good cell permeability and are protective against 6-OHDA toxicity in differentiated P19 cells [107]. Comparative analysis of the Fe2+-chelating potency of EGCG, the prototype DFO and other pharmacological iron chelators, has revealed similar binding potency (fig. 3). Both EGCG and the iron chelator M30 were shown by us to induce a significant downregulation of membrane-associated holoAPP level in the mouse hippocampus (fig. 4), SH-SY5Y and CHO cells expressing the APP ‘Swedish’ mutation (data not shown). This may have a direct influence on A levels and plaque formation, as shown in preliminary studies for XH1 [102]. Indeed, using a nucleation-dependent polymerization model, it has been shown that wine and green tea polyphenols are able to inhibit formation, extension and destabilization of -amyloid fibrils [108]. Other potential beneficial effect of EGCG in AD may be related to our previous studies demonstrating the ability of EGCG to promote the non-amyloidogenic pathway, via a PKC-dependent activation of -secretase, thereby increasing sAPP [46]. sAPP has been demonstrated to posses potent neuroprotective activities against excitotoxic and oxidative insults in various cellular models [109] and it was shown to protect against p53-mediated apoptosis [110]. Moreover, sAPP promotes neurite outgrowth [111], regulates synaptogenesis [112] and exerts trophic effects on cerebral neurons in culture. As sAPP and A are formed by two mutually exclusive mechanisms, stimulation of the secretory processing of sAPP might prevent the formation of the amyloidogenic A. Thus, EGCG may influence A levels, either via translational inhibition of APP or by regulating APP processing. Finally, iron chelation by EGCG may ablate Fe3+-induced aggregation of hyperphosphorylated tau (PHF), the major constituent

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A

B

holoAPP ➤

holoAPP ➤

β-actin ➤

β-actin ➤

120

120

100

100

80 ✽✽

60 40 20

% of control

processing in mice hippocampus. Representative Western blots of levels of holoAPP in the membrane compartment, obtained from hippocampus of mice treated with EGCG (2 mg/kg) (A), or with M30 (5 mg/kg) (B) for 14 days detected with 22C11 antibody (directed to the to APP N-terminus) or with a C-terminus APP antibody, respectively. Densitometric analysis is expressed as percent of the control, untreated animals after normalizing to the levels of -actin. Data are expressed as the mean 8 SEM (n = 6 mice in each group). ** p ! 0.03 vs. control. Figure drawn using information from Levites et al. [46].

% of control

Fig. 4. Effect of EGCG and M30 on APP

80

✽✽

60 40 20

0

0 Control

EGCG (2 mg/kg)

Control

M30 (5 mg/kg)

Fig. 5. Proposed schematic model for EGCG neuroprotective effect via regulation of APP processing and A formation. d Increased levels/activity, f decreased levels/activity. For full explanation, see text.

of neurofibrillary tangles in AD brains [113] (a descriptive explanation is depicted in fig. 5). Iron Chelation for PD Numerous studies have shown that there is a progressive accumulation of iron and ferritin in the SN pars com-

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pacta (pc) of PD patients [17, 27, 114]. Specifically, redox-active iron has been observed in the rim of Lewy body, the morphological hallmark of PD, also composed of lipids, aggregated -synuclein (concentrating in its peripheral halo) and ubiquitinated, hyperphosphorylated neurofilament proteins [115]. -Synuclein associated

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Fig. 6. Possible mechanism of neurotoxin-induced iron uptake, release and interaction with -synuclein resulting in OS initiated neurodegeneration and its prevention by iron-chelating/antioxidants. The mechanism by which 6-OHDA and MPTP induce increase of iron in substantia nigra pars compacta and within the melanin-containing neurons is not known. These neurotoxins may (a) activate the divalent metal transporter 1 (DMT1) which is responsible for iron transport into the brain across the cell membrane; (b) alter the blood-brain barrier (BBB), thereby allowing iron access to the brain; (c) induce release of iron from ferritin which enters the labile (redox-active) pool of iron. It is the labile pool of iron which can initiate the Fenton chemistry in response to the presence of hydrogen peroxide, thus generating the highly reactive hydroxyl radical (OH). The resultant effect is the depletion of cell-reduced

glutathione (GHS), the rate-limiting cofactor of glutathione peroxidase, the main enzymatic pathway in the brain, to eliminate hydrogen peroxide. Labile pool of iron can also cause aggregation of -synuclein to the neurotoxic form, which can also generate OH. The net effect is oxidative-stress-dependent damage to neuron antioxidant mechanism, membrane lipid peroxidation, demise of cell and mitochondrial membrane, protein misfolding and ultimate cell death. Neuroprotective agents that can be used to prevent iron-induced neurodegeneration include M30 and HLA20 (bifunctional iron chelator-MAO inhibitors); desferal, VK-28, R-APO (R-apomorphine) and EGCG (iron chelators); R-APO, EGCG, melatonin and Vit E (vitamin E) (radical scavengers). Sharp arrows indicate positive inputs, whereas blunt arrows are for inhibitory inputs. Reproduced, with minor modifications, from Youdim et al. [106].

with presynaptic membrane is not toxic; however, a number of recent studies [116–118] have shown that it forms toxic aggregates in the presence of iron and this is considered to contribute to the formation of Lewy body via OS, being one of its constituents. Our recent high throughput gene expression study in the SNpc of Parkinsonian brains employing Affymetrix chip technology [29] has revealed a significant increase

on the key iron and oxygen sensor EGLN1 gene coding for an isoform of 2-oxoglutarate-dependent dioxygenase hydroxylase (see previous section). Excessive production of EGLN1 hydroxylase in the SNpc may lead to a fall in IRP2 and subsequent decrease in transferrin receptor (TfR) mRNA and increase in ferritin levels, both subjected to positive and negative transcriptional regulation by IRP2, respectively [119, 120]. Recent studies in knock-

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out mice for IRP2 have revealed accumulation of iron in the striatum with substantial bradykinesia and tremor [121]. Consistent with the pivotal role of iron in neurodegeneration is the finding that the iron chelator desferal prevents cytochrome c-induced--synuclein aggregation and toxicity in vitro [122] and attenuates dopaminergic neurotoxicity in response to neurotoxins MPTP and 6-OHDA in vivo [123, 124]. In line with the iron-chelating feature of EGCG, this polyphenol was shown to prevent -synuclein accumulation and to attenuate IRP2 depletion, in the SNpc of mice intoxicated with MPTP, when given orally for a period of 2 weeks [62]. This may be associated to our previous studies where green tea extract or EGCG prevented DA-containing neuron degeneration and tyrosine hydroxylase activity decrease [30]. In spite of the absence of clinical trials regarding tea polyphenols and PD, epidemiological studies have shown reduced risk of PD associated with consumption of 2 cups/day or more of tea [125] and a much lower prevalence of PD in Chinese population than in white people [126, 127]. Additional studies examining the effect of iron chelation, by either transgenic expression of the iron-binding protein, ferritin, or oral administration of the metal chelator clioquinol, have shown significant attenuation of MPTP-induced neurotoxicity [128]. These findings have now been substantiated with systemic injection of the brain-permeable iron chelator, VK-28, to rats in response to 6-OHDA [104]. In line, nutritional iron deficiency pro-

tects rats against kainate and 6-OHDA [129]. Figure 6 summarizes the mechanism of neurotoxin-induced iron uptake, release and interaction with -synuclein resulting in OS initiated neurodegeneration and its prevention by iron-chelating/antioxidant agents.

Conclusions

It is likely that syndromes such as AD and PD will require multiple- drug therapy to address the varied pathological aspects of the disease. Therefore, the use of compounds with poly-pharmacological activities or cocktail of drugs is a promising therapeutic approach for the treatment of neurodegenerative diseases. Indeed, a wealth of new data indicates that green tea catechins are being recognized as multifunctional compounds for neuroprotection. They act as radical scavengers, iron chelators and modulators of pro-survival genes, and PKC signaling pathway. The use of EGCG as a natural, non-toxic, lipophilic brain permeable neuroprotective drug is advocated for ‘ironing out iron’ from those brain areas where it preferentially accumulates in neurodegenerative diseases [106]. Thus, green tea catechins may have potential disease-modifying action. Future efforts in the understanding of the protective mechanism of action of these polyphenols should concentrate on deciphering the cellular targets affected by these compounds and other neuroprotectants.

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Review Neurosignals 2005;14:61–70 DOI: 10.1159/000085386

Received: December 6, 2004 Accepted after revision: March 1, 2005

Unique Properties of Polyphenol Stilbenes in the Brain: More than Direct Antioxidant Actions; Gene/Protein Regulatory Activity Sylvain Doré Johns Hopkins University, School of Medicine, Baltimore, Md., USA

Key Words Bilirubin  Biliverdin  Blood flow  Carbon monoxide  Hemin  Iron

Abstract The ‘French Paradox’ has been typically associated with moderate consumption of wine, especially red wine. A polyphenol 3,4’,5-trihydroxy-trans-stilbene (a member of the non-flavonoids family), better known as resveratrol, has been purported to have many health benefits. A number of these valuable properties have been attributed to its intrinsic antioxidant capabilities, although the potential level of resveratrol in the circulation is likely not enough to neutralize free radical scavenging. The brain and the heart are uniquely vulnerable to hypoxic conditions and oxidative stress injuries. Recently, evidence suggests that resveratrol could act as a signaling molecule within tissues and cells to modulate the expression of genes and proteins. Stimulation of such proteins and enzymes could explain some the intracellular antioxidative properties. The modulation of genes could suffice as an explanation of some of resveratrol’s cytoprotective actions, as well as its influence on blood flow, cell death, and inflammatory cascades. Resveratrol stimulation of the expression of heme oxygenase is one example. Increased heme oxygenase activity has led to significant

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protection against models of in vitro and in vivo oxidative stress injury. Resveratrol could provide cellular resistance against insults; although more work is necessary before it is prescribed as a potential prophylactic in models of either acute or chronic conditions, such as stroke, amyotrophic lateral sclerosis, Parkinson, Alzheimer, and a variety of age-related vascular disorders. Copyright © 2005 S. Karger AG, Basel

The ‘French Paradox,’ so named after a population studied in France, is defined by a low incidence of cardiovascular problems despite a diet that is relatively high in saturated fat [1]. Because of the typical levels of wine intake of the French, it has also been postulated that a moderate consumption of red wine could be associated with this paradox. A vast amount of recent literature proposes that it is the polyphenols in red wine that provide the beneficial effect, mainly attributable to their potential to act as antioxidants. These polyphenols are divided into two main categories: flavonoids and non-flavonoids. The flavonoids, found in natural extracts of plants and fruits, are considered to be the most abundant polyphenols, whereas the non-flavonoid stilbenes are considered to be a minor class. The naturally occurring polyphenol stilbene, resveratrol (3,4
,5-trihydroxy-trans-stilbene), has been proposed to possess most of the beneficial health ef-

Sylvain Doré, PhD Johns Hopkins University, School of Medicine ACCM Department, Neuro Research Division, 720 Rutland Ave Ross Research Building 364-365, Baltimore, MD 21205 (USA) Tel. +1 410 614 4859, Fax +1 410 955 7271, E-Mail [email protected]

CEREBRAL ISCHEMIA [11,12] Reduces infarct size (rat) Reduces focal ischemia damage (rat)

AMYOTROPHIC LATERAL SCLEROSIS [13] Directly stimulates BK(Ca) channel activity in vascular endothelial cells (human cells)

AGING [22,23] PARKINSON DISEASE [14]

Mimics beneficial effects of caloric restriction (yeast)

Protects embryonic mesencephalic cells (rat)

SPINAL CORD LESION [8] OH

Protects spinal cord from ischemia-reperfusion injury (rabbit) HO

PAIN [20]

OH

CHEMOPREVENTION [16,17] Inhibits critical cell cycle regulatory proteins (mice) Converts into an anticancer agent by cytochrome P 450 enzymes (human cells)

trans -resveratrol COGNITIVE IMPAIRMENT [21]

Decreases carrageenaninduced hyperalgesia (rat)

Prevents ICV streptozotocin-induced cognitive impairment (rat)

BRAIN TUMORS [15]

BRAIN EDEMA [9]

Reduces paclitaxel-induced apoptosis in neuroblastoma SH-SY5Y cells (human cells)

Inhibits expression of NFkB p65 (rat)

SEIZURE [10,18,19] Reduces generalized tonic-clonic convulsions (rat) Delays FeCl 3-induced epileptiform EEG changes (rat) Reduces kainate-induced lesions in hippocampus (rat)

Fig. 1. Experimental neurologic benefits of resveratrol.

fects of red wine, considering that resveratrol is mainly found in the skin of the grapes, and the skin and seeds are generally not used in processing white wines. Consequently, it has been proposed that resveratrol would potentially be the most active ingredient [2–5]. Given all of this, the antioxidant properties of red wine, then, must be associated with the actions of resveratrol, and we propose that the protective properties are likely due to a unique cascade of intracellular events leading to activation of unique antioxidant pathways. This hypothesis is being proposed, taking into consideration that the modest amount of resveratrol in the blood is not likely to reach a high enough plasmatic level to neutralize free radicals. Consequently, resveratrol is likely to stimulate an intracellular signaling pathway, leading to cytoprotection. As represented here, several genes and proteins have been shown to be potential targets for resveratrol modulation. Heme oxygenase (HO) is a possible candidate. We have shown that resveratrol is a potent inducer of HO protein levels, activity, and cytoprotection [6]. HO’s main function is to cleave heme (Iron-Protoporphyrin-IX), which then liberates iron, generating carbon monoxide and biliverdin, which is rapidly converted to bilirubin.

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By degradation of heme, a prooxidant, into biliverdin/ bilirubin, antioxidants, modulation of HO activity and levels would seem to be cytoprotective against free radical damage. Using in vitro and in vivo models, we have also shown that HO can be neuroprotective [7]. In addition, HO and its metabolites have been associated with antiapoptotic and antiinflammatory actions and are known to have a vasodilatory effect. Several other enzymatic systems have been suggested to be either directly or indirectly modified by different members of the stilbene family. Here, we propose that protective properties of HO are the mechanisms that provide the brain’s resistance to a variety of neurologic stresses. Resveratrol has been shown to have a unique effect on neuronal cell death and inflammatory processes, which are all important therapeutic targets in the development of either acute and/or chronic neurodegenerative diseases [8–10]. These vascular properties become especially important when considering that reduction in cerebral blood flow (CBF), followed by a reperfusion phase, is likely to affect specific neurons and/or the cell types that are especially vulnerable to free radical damage. Consequently, preventing cell death is likely to have a beneficial

Doré

effect on the rate of neuroinflammation and its consequences. Considering all of this together, one can make a valid hypothesis that polyphenol stilbenes can precondition neurons against induced stress damage. Figure 1 contains a list of potential neurologic benefits associated with models of neurologic diseases treated with resveratrol. Of importance is the fact that resveratrol has been shown to be protective in several species. Following publication in Science and Nature of initial reports that suggested a potential biologic role of resveratrol [24, 25], more than 900 original research articles have been published to support the beneficial effect of resveratrol. It is somewhat paradoxical that such a simple, natural component, extracted from plants and fruits, can have such a variety of actions. The flavonoids constitute the majority of phenols in red wine and are mainly divided into three classes: the flavanols, the flavonols, and the anthocyanins. The known flavonoids (hydroxycinnamic acids, benzoic acids, and stilbenes) are less abundant, and the stilbenes are only a minor class of the known flavonoids. Interestingly, during the production of red wine, complex sugars from the grapes are fermented into alcohol, and it is believed that alcohol would be considered a good solvent for extracting polyphenols from the skins and seeds. Considering that resveratrol is mainly found in the skin of the grapes, it has been reported that the amount of phenols within white wine would be much less than in red wine. Although the reported amounts of resveratrol in wine vary, they suggest that approximately 7 mg/l are present in most reds, as compared to 0.5 mg/l in whites [26, 27]. For purposes of comparison, the flavonoid concentrations in red wine have been in the range of 1,300– 1,500 mg/l. Once again, considering all of the beneficial properties of resveratrol, its minimal amount available in wine, if active, is likely to be mediated via activation of intracellular pathways. It is interesting to note, also, that resveratrol exists in two isomers, the cis and the trans, as shown in figure 2. Both of these are found in wine, and, although it appears that only the trans isomer is found in grapes, direct light could cause its isomerization from trans to cis. In our preliminary observations in neurons, we indicated that the active isoform would be mainly the trans resveratrol. The fact that resveratrol, a simple active ingredient, which has been proposed to be responsible for the rationale behind the French Paradox, is present in such small amounts in wine or juice, and the theory concerning its bioactivity by its direct antioxidant capacity deserve reconsideration. Thus, it led us to hypothesize the activa-

Unique Properties of Polyphenol Stilbenes in the Brain

OH HO

HO

light-induced isomerization

OH

OH

OH

trans-resveratrol

cis-resveratrol

Fig. 2. UV-light-induced isomerization of trans- to cis-resvera-

trol.

tion of an antioxidant intracellular pathway. Considering the direct antioxidant properties associated with resveratrol and taking into account the absorption rate and modifications/conjugations, one would have to consume liters of these drinks in order to achieve the necessary plasmatic molar ratio to neutralize the free radicals and achieve the beneficial health effects. To highlight the variability of resveratrol’s effects, figure 3 shows the intracellular consequences of cells treated with the maximal concentration of 25 M, although the list is not exhaustive. We eliminated experiments in which the concentration was higher than 25 M. We believe that it is reasonable to think that a level ^25 M could potentially be achieved under normal conditions and not typically under pharmacologic conditions. Figure 3 shows the expression regulated by resveratrol of different genes and proteins and describes some of the potential functions. Living organisms under aerobic conditions are continuously exposed to potential damage caused by reactive oxygen species (ROS). ROS are produced naturally during normal cellular activity and mitochondrial function. In addition, induced stress is likely to modify these normal functions and simulate the generation of free radical damage. Many of the studies using polyphenols have required pretreatment with resveratrol to exert these beneficial biologic functions. Such pretreatment would require either an increase in the concentration of this polyphenol or stimulate a cascade leading to activation of an endogenous antioxidant system. This activation is critically important to cytoprotection in tissue with a weak, endogenous, antioxidant system. The heart and the brain are two unique examples of tissues with weak defenses, as evidenced by infarct damage following ischemia/reperfusion. Considering the antioxidant properties associated with resveratrol, we have concentrated on the neuroprotective effect of resveratrol and the possibility that this

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Fig. 3. Example of resveratrol-regulated genes/proteins affected in cells (!25 M). HO1 = Heme oxygenase 1; COX-2 = cyclooxygenase 2; eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase; ET-1 = endothelin-1; GADD45 = growth-arrest- and DNA-damage-induced protein 45; TNF = tumor necrosis factor-alpha; ATF3 = activating transcription factor 3; IGFBP = insulin-like growth factor-binding protein.

Fig. 4. Example of potential outcomes from gene/protein regulation (i.e., HO1) and potential enzymatic role in

the brain. Note: This list of potential actions would occur at physiologic levels, while abnormal or pharmacologic levels of these compounds could have deleterious effects.

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Doré

Fe 3+-Ferritin

3O2 + NADPH

H2O + NADP +

Cyt P450 Red

O

OH O

OH

+ Ferritin

CO Fe2+

N N

Fe 2+ N

N

HEME OXYGENASE

N HO

HN

NH

O

RESVERATROL

HN

OO

O

Heme OH (Fe2+ -Protoporphyrin IX)

O

OH O

Biliverdin

OH

NADPH + H+

BV Red

other bilirubin metabolites

NADP +

NH

HN

Bilirubin NH

ROS

HN

OO

Fig. 5. Resveratrol mediates heme degrada-

tion by induction of heme oxygenase levels.

pathway could involve the induction of an antioxidant system, such as heme oxygenase. Regulation of HO activity has been shown to be protective in acute and/or chronic neurodegenerative conditions. Figure 4 summarizes some of the potential outcomes of regulation of the HO protein and its activity. As mentioned above, HO catalyzes the degradation of heme, which is mainly a prooxidant, into iron, biliverdin/ bilirubin, and carbon monoxide (fig. 5). Two isoforms of HO have been isolated and characterized [7, 33–35]. A third isoform has been reported, although it does not seem to be translated into a protein [36]. HO1, the first to be isolated [34], is the inducible enzyme and appears to be concentrated mainly in the liver and spleen, an understandable observation considering the high turnover of hemoglobin, which has heme in its core. Under basal conditions, HO1 is barely detectable in the brain, although several reports suggest that, under a variety of stimuli, it can be induced within brain tissue [37–48]. HO2 is an isoform that is constitutively expressed and appears to be concentrated mainly in the brain and testis [48]. Its protein expression level appears to be extremely stable, which is likely to respond to normal cellular homeostasis. Regulation of the HO1 protein and its activity has elicited a great deal of interest. Regulation of HO1 has been suggested to be in response to many cellular and organ

stresses, in order to defend against disruption of any system homeostasis. HO1 has been suggested to have different functions in the brain. It is one of the heat shock proteins, which are stress proteins that are induced in different cells and following different stimuli [37], including hypothermia [38], global ischemia [41], subarachnoid hemorrhage [42], Parkinson disease [47], Alzheimer disease [39, 40], and several other acute and chronic neurologic conditions [43–46]. In a model of transient ischemia, we and others have shown that HO mRNA and proteins are induced [43]. Reports have indicated that induction of HO proteins in neurons would be protective [49], and our preliminary data indicate that resveratrol could induce HO levels within neurons, potentially affording neuroprotection. Consequently, we believe that modulation of HO levels and its activity could be a pathway by which resveratrol present in red wine or other concentrated extracts could potentially protect the nervous system against induced oxidative stress damage. Many heme-containing enzymes are located in the mitochondria, the cytosol, and the endoplasmic reticulum; and they presumably undergo rapid turnover during oxidative stress. HO is the main enzyme, which can degrade free heme, a prooxidant, and maintain levels that would not reach toxicity. Following an ischemic event, tissue injury has been proposed to be mainly due to increased oxidative stress by free radicals

Unique Properties of Polyphenol Stilbenes in the Brain

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generated during the reperfusion phase. We have previously shown that the infarct volume in HO2 knockout (HO2–/–) mice versus wild-type (WT) mice after stroke is approximately double. HO2 is the isoform present under normal basal conditions. Consequently, increasing the activity of heme oxygenase is likely to be protective in stroke. Interestingly, the group led by Maines has demonstrated significant reduction in infarct size after stroke in a transgenic mouse model on which HO1 was over-expressed using a neuron-specific promoter [50]. In addition, we have recently accumulated evidence suggesting that pre-treatment with resveratrol would be a most potent inducer of HO1 in mouse primary neuronal cultures, that it would be sufficient to prevent induced neurotoxicity, and that this neuroprotection was significantly attenuated by the use of a heme oxygenase inhibitor [51]. All together, these results suggest that an increase in HO1 protein levels and its activities within neurons is likely to provide neuroprotection and promote cell survival. Heme Heme metabolism is a crucial metabolic process. It has been postulated that free heme can rapidly be generated from an induced turnover of heme-containing proteins/ enzymes, for example, catalase, glutathione peroxidase, superoxide dismutase, cytochrome, guanylate cyclase, nitric oxide synthase, etc. It has been further suggested that during hypoxia, ischemic injury could trigger significant amounts of heme being released into the intracellular pool. When stimulation of these hemoproteins is degraded, heme becomes free; probably not salvaged, it should be rapidly degraded. HO is the enzyme that can rapidly cleave prooxidant through heme and limit its capacity to enter into generation of a free radical cycle – notably, through its iron molecule. Consequently, HO could be considered an antioxidant enzyme by degradation of the prooxidant heme. Our previous observations indicate that resveratrol could specifically induce HO1 within neurons and potentially protect cells against oxidative stress injury. Such a pathway is an interesting target for resveratrol, considering that it can regulate the redox state of the cell and prevent cells from dying through an oxidative stress-induced cascade. Iron Degradation of heme by HO1 also generates a molecule of iron. Evidence suggests that regulation of HO1 protein levels could modulate the level of intracellular heme. It is postulated that HO can stimulate the efflux of

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iron outside the cell [52]. Homeostasis of iron is a key factor in controlling cell toxicity. As one example, free iron has been considered to be a key ingredient in the Fenton reaction, in which by reacting with H2O2, it generates free radicals. Regulation of cellular homeostasis of iron is a complex and tightly regulated system. It is regulated by many proteins, a number of which are still under extensive characterization. Rapid upregulation of HO1 by resveratrol in neurons could potentially directly affect the intracellular iron level. It has been previously demonstrated that decrease in HO1 activity would be sufficient to change its iron level within the cell. For example, by using HO–/– mice, it has been shown that iron accumulates in several organs [53]. In addition, numerous iron-binding proteins are regulated by intracellular levels of free iron [54]. As an example, ferritin in the cell can sequester numerous molecules of iron, and its intracellular level is tightly regulated to free iron. Therapeutic implications of controlling iron levels within tissues or within cells are numerous. As an example, administration of desferoxamine, a trivalent iron chelator, over a 2-year period slows the clinical progression of symptoms associated with Alzheimer disease [55]. Further study may bear out that the regulation of HO1 levels also regulates the cellular iron homeostasis. Interestingly, very little has been investigated regarding the potential role of resveratrol in regulating iron and determining its potential effect in neurologic disorders. Carbon Monoxide Carbon monoxide (CO) is a gas that is almost exclusively generated in cells by degradation of heme by heme oxygenase [56, 57]. In that it is a gas, carbon monoxide can travel freely through intracellular and extracellular compartments. The CO literature is vast and complex with many controversies that have yet to be resolved [58– 63]. CO is better known to be toxic at high levels. It can also cause death [64]. Interestingly, in the recent literature, low concentrations of CO have been suggested to be protective. Although CO has an affinity slightly lower to its homologue, nitric oxide (NO), which is another gas, it appears that its half-life would be significantly longer. Such an observation could allow carbon monoxide, by binding to heme moiety present on several key enzymes, to modulate their function [65]. Within the cell, physiologic/normal levels of CO generated from degradation of intracellular heme are likely to have multiple biologic functions on several heme-containing proteins. For example, CO has been shown to act as a vasodilator by po-

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tentially binding with soluble guanylate cyclase (sGC) and modulating its vasoactive activities. It can act on calcium-activated potassium channels (KCa channels) and regulate their opening [66, 67]. CO has also been recently reported to have specific antiapoptotic and antiinflammatory actions [68, 69]. Rapid induction of HO1 could be a means by which resveratrol can increase CO levels within physiologic concentrations and allow cells and tissue to benefit from many of CO’s biologic actions, especially in scenarios in which blood flow is reduced and cell survival is compromised.

Antioxidant properties Most neurologic disorders [6]

Inflammation reduction Most neurologic disorders [77,78]

Restoration of normal blood flow Cerebral Ischemia [79] AD (age-related vascular dementia) [7]

Reduction of neuronal cell death In stroke models [43] In induced apoptosis [80] Cholinergic neuron in AD models [81] Dopaminergic neurons in PD models [47]

Bilirubin Bilirubin is known for its toxicity, at high micromolar concentrations, in the central nervous system (CNS), especially in neonates. Although under physiologic concentrations, bilirubin can act as an endogenous antioxidant [70, 71]. In testing a series of antioxidants, bilirubin was shown to have significant superoxide and hydroxyl radical scavenger activity [72]. Using an animal model of hyperbilirubinemia, a protective effect against cerebral ischemia was demonstrated. We have also observed, using primary hippocampal and cortical neuronal cultures, that bilirubin can be protective at low concentrations [73, 74]. Moreover, in previous observations on the potential role of HO in Alzheimer disease pathology [39, 75], levels of bilirubin derivatives in the cerebral spinal fluid were reported to be significantly increased in brains of AD patients as compared to control [76]. An increase in HO1 within AD brains was reported. Increased HO1 levels could potentially increase the bilirubin level within a physiologic range, and such a pathway could explain some of the antioxidant properties associated with resveratrol in relation to neurologic deficits in age-related dementia. Figure 6 briefly summarizes some of the neurologic disorders that can be beneficially affected by the regulation of HO activity. Regulation of genes by resveratrol, such as in the case of HO1, presents a potential mechanism by which its prophylactic use might be considered, under either acute or chronic neurologic disorders. For example, under ischemic stroke conditions, reperfusion frequently occurs after focal ischemia, particularly in the case of cerebral embolism and transient ischemic attack. These can be warning signs of impending stroke. Recurrence is a prevalent phenomenon in patients who have suffered one episode of stroke. Therefore, availability of a prophylactic approach in these patients is an important goal of preventive medicine. Reports that coronary heart disease appears to be reduced in patients who chroni-

Unique Properties of Polyphenol Stilbenes in the Brain

Regulation of iron homeostasis Parkinsonism [82] Ataxia [83] Movement disorders, tremors [84] Restless Leg Syndrome [85] Hallervorden-Spatz Syndrome [86] Aceruloplasminemia [87] Neuroferritinopathy [88]

Others Sleep pattern [89] Circadian rhythm [90,91] Amyotrophic lateral sclerosis [92] Spinal cord lesion [93] Head trauma [94] Brain edema [50] Huntington [95] Kernicterus [94] Brain tumors [97] Chemoprevention [98] Pain [99] Seizure [100] Atherosclerosis [101] Age-related cognitive impairment [102]

Fig. 6. Non-exhaustive list of potential neurologic benefits of res-

veratrol-regulating HO1.

cally consume moderate amounts of red wine support the prospect of a prophylactic role for resveratrol. Consequently, a better understanding of the mechanisms by which polyphenols and red wine can protect against ischemic and toxic insults would be of great interest.

Acknowledgements This work is supported in part by the NIH grants (AA014911 and AT002113), the Wine Institute, and the ABMR Foundation. The author would like to thank Tzipora Sofare, MA, for her assistance in preparing the manuscript.

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Unique Properties of Polyphenol Stilbenes in the Brain

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69 Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH, Soares MP: Heme oxygenase-1derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cells from tumor necrosis factor-alpha-mediated apoptosis. J Biol Chem 2002;277:17950–17961. 70 Gopinathan V, Miller NJ, Milner AD, RiceEvans CA: Bilirubin and ascorbate antioxidant activity in neonatal plasma. FEBS Lett 1994; 349:197–200. 71 Stocker R, Glazer AN, Ames BN: Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci USA 1987;84:5918–5922. 72 Farrera JA, Jauma A, Ribo JM, Peire MA, Parellada PP, Roques-Choua S, Bienvenue E, Seta P: The antioxidant role of bile pigments evaluated by chemical tests. Bioorg Med Chem 1994;2:181–185. 73 Doré S, Takahashi M, Ferris CD, Zakhary R, Hester LD, Guastella D, Snyder SH: Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad Sci USA 1999; 96: 2445– 2450. 74 Doré S, Snyder SH: Neuroprotective action of bilirubin against oxidative stress in primary hippocampal cultures. Ann NY Acad Sci 1999; 890:167–172. 75 Takahashi M, Doré S, Ferris CD, Tomita T, Sawa A, Wolosker H, Borchelt DR, Iwatsubo T, Kim SH, Thinakaran G, Sisodia SS, Snyder SH: Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer’s disease. Neuron 2000;28:461– 473. 76 Kimpara T, Takeda A, Yamaguchi T, Arai H, Okita N, Takase S, Sasaki H, Itoyama Y: Increased bilirubins and their derivatives in cerebrospinal fluid in Alzheimer’s disease. Neurobiol Aging 2000;21:551–554. 77 Zhuang H, Kim YS, Namiranian K, Doré S: Prostaglandins of J series control heme oxygenase expression: Potential significance in modulating neuroinflammation. Ann N Y Acad Sci 2003;993:208–216. 78 Bishop A, Cashman NR: Induced adaptive resistance to oxidative stress in the CNS: A discussion on possible mechanisms and their therapeutic potential. Curr Drug Metab 2003; 4: 171–184. 79 Goto S, Sampei K, Alkayed NJ, Doré S, Koehler RC: Characterization of a new doublefilament model of focal cerebral ischemia in heme oxygenase-2-deficient mice. Am J Physiol Regul Integr Comp Physiol 2003;285:R222– R230. 80 Doré S, Goto S, Sampei K, Blackshaw S, Hester LD, Ingi T, Sawa A, Traystman RJ, Koehler RC, Snyder SH: Heme oxygenase-2 acts to prevent neuronal cell death in brain cultures and following transient cerebral ischemia. Neuroscience 2000;99:587–592. 81 Calabrese V, Butterfield DA, Stella AM: Nutritional antioxidants and the heme oxygenase pathway of stress tolerance: Novel targets for neuroprotection in Alzheimer’s disease. Ital J Biochem 2003;52:177–181.

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82 Schipper HM, Cisse S, Stopa EG: Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann Neurol 1995; 37: 758–768. 83 Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF: Heme and iron metabolism: Role in cerebral hemorrhage. J Cereb Blood Flow Metab 2003;23:629–652. 84 Thompson K, Menzies S, Muckenthaler M, Torti FM, Wood T, Torti SV, Hentze MW, Beard J, Connor J: Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress. J Neurosci Res 2003;71:46–63. 85 Krieger J, Schroeder C: Iron, brain and restless legs syndrome. Sleep Med Rev 2001; 5: 277– 286. 86 Ponka P: Hereditary causes of disturbed iron homeostasis in the central nervous system. Ann NY Acad Sci 2004;1012:267–281. 87 Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL: Aceruloplasminemia: An inherited neurodegenerative disease with impairment of iron homeostasis. Ann NY Acad Sci 2004;1012:299– 305. 88 Crompton DE, Chinnery PF, Fey C, Curtis AR, Morris CM, Kierstan J, Burt A, Young F, Coulthard A, Curtis A, Ince PG, Bates D, Jackson MJ, Burn J: Neuroferritinopathy: A window on the role of iron in neurodegeneration. Blood Cells Mol Dis 2002;29:522–531.

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97 Hara E, Takahashi K, Tominaga T, Kumabe T, Kayama T, Suzuki H, Fujita H, Yoshimoto T, Shirato K, Shibahara S: Expression of heme oxygenase and inducible nitric oxide synthase mRNA in human brain tumors. Biochem Biophys Res Commun 1996;224:153– 158. 98 Solowiej E, Kasprzycka-Guttman T, Fiedor P, Rowinski W: Chemoprevention of cancerogenesis – the role of sulforaphane. Acta Pol Pharm 2003;60:97–100. 99 Liang DY, Li X, Clark JD: Formalin-induced spinal cord calcium/calmodulin-dependent protein kinase II alpha expression is modulated by heme oxygenase in mice. Neurosci Lett 2004;360:61–64. 100 Carratu P, Pourcyrous M, Fedinec A, Leffler CW, Parfenova H: Endogenous heme oxygenase prevents impairment of cerebral vascular functions caused by seizures. Am J Physiol 2003;285:H1148–H1157. 101 Zhuang H, Littleton-Kearney MT, Doré S: Characterization of heme oxygenase in adult rodent platelets. Curr Neurovasc Res 2004;in press. 102 Law A, Doré S, Blackshaw S, Gauthier S, Quirion R: Alteration of expression levels of neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA in the hippocampi and cortices of young adult and aged cognitively unimpaired and impaired Long-Evans rats. Neuroscience 2000;100:769–775.

Doré

Review Neurosignals 2005;14:71–82 DOI: 10.1159/000085387

Received: September 23, 2004 Accepted after revision: November 8, 2004

Neuroprotective Effects of Huperzine A A Natural Cholinesterase Inhibitor for the Treatment of Alzheimer’s Disease

Rui Wang Xi Can Tang State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Zhangjiang Hi-Tech Park, Shanghai, China

Key Words Huperzine A W Alzheimer’s disease W Acetylcholinesterase W Cholinesterase inhibitor W Cognitive enhancer W Neuroprotection W Oxidative stress W Beta-amyloid W Amyloid precursor protein W Cerebral ischemia W Apoptosis W Apoptotic-related gene W Mitochondria W Glutamate W NMDA receptor W Potassium current

chondria, and interfere with APP metabolism. Antagonizing effects on NMDA receptors and potassium currents may contribute to the neuroprotection as well. It is also possible that the non-catalytic function of AChE is involved in neuroprotective effects of HupA. The therapeutic effects of HupA on AD or VD are probably exerted via a multi-target mechanism.

Abstract Huperzine A (HupA), isolated from Chinese herb Huperzia serrata, is a potent, highly specific and reversible inhibitor of acetylcholinesterase. It has been found to reverse or attenuate cognitive deficits in a broad range of animal models. Clinical trials in China have demonstrated that HupA significantly relieves memory deficits in aged subjects, patients with benign senescent forgetfulness, Alzheimer’s disease (AD) and vascular dementia (VD), with minimal peripheral cholinergic side effects compared with other AChEIs in use. HupA possesses the ability to protect cells against hydrogen peroxide, ß-amyloid protein (or peptide), glutamate, ischemia and staurosporine-induced cytotoxicity and apoptosis. These protective effects are related to its ability to attenuate oxidative stress, regulate the expression of apoptotic proteins Bcl-2, Bax, P53 and caspase-3, protect mito-

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder associated with a global impairment of higher mental function, and presenting an impairment of memory as the cardinal symptom [1]. Histopathological hallmarks of the disease are the extracellular deposition of amyloid ß-peptide (Aß) in senile plaques, the appearance of intracellular neurofibrillary tangles (NFT), a loss of cholinergic neurons, and extensive synaptic changes in the cerebral cortex, hippocampus and other areas of brain essential for cognitive functions. To date, the cause and the mechanism by which neurons die in AD remain unclear, but Aß has been established as a crucial factor in AD pathogenesis. Aß deposition may cause neuronal death via a number of possible mechanisms, including oxidative stress, excitotoxicity, energy depletion, inflammation and apoptosis. Despite this multifactorial etiology, genetics plays a key role in

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Prof. Xi Can Tang, State Key Laboratory of Drug Research Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, 555 Zu Chong Zhi Road Zhangjiang Hi-Tech Park, Shanghai 201203 (China) Tel. +86 21 5080 6710, Fax +86 21 5080 7088, E-Mail [email protected]

disease progression. However, environmental factors (e.g. cytokines, neurotoxins) may be even more important in the development and progression of AD. Several lines of evidence support the involvement of oxidative stress [2, 40]. Oxidative damage, mediated by reactive oxygen species (ROS) generated following cell lysis, oxidative bursts, or an excess of free transition metals, has been hypothesized to play a pivotal role in AD neurodegeneration. On the other hand, postmortem studies provide direct morphological and biochemical evidence that some neurons in the AD brain degenerate via an apoptotic mechanism [30, 51], which may or may not be linked to ROS. The biological mechanism underlying the formation of AD is clearly complex, with many factors contributing to the neuropathology. Thus it is not surprising that a number of different intervention therapies are currently being researched to address distinct aspects of the disease. Huperzine A (HupA) is a novel Lycopodium alkaloid with recognized medicinal properties. In China the folk medicine Huperzia serrata (Qian Ceng Ta) (fig. 1), a source of HupA, has been used for centuries in the treatment of contusions, strains, swelling, schizophrenia, etc. The active principle, HupA, is a potent, reversible, and selective inhibitor of acetylcholinesterase (AChE) [56]. Its potency in AChE inhibition is similar or superior to that of physostigmine, galanthamine, donepezil and tacrine [60, 67]. AChE exists in multiple molecular forms that can be distinguished by their subunit associations and hydrodynamic properties [6, 41]. In mammalian brain, the bulk of AChE occurs as a tetrameric, G4 form (10S) together with much smaller amounts of a monomeric, G1 (4S) form [4, 22]. This phenomenon led us to investigate the possibility of HupA on differential inhibition of AChE forms [93]. We observed that HupA preferentially inhibited tetrameric AChE (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric AChE (G1 form). Donepezil showed pronounced selectivity for G1 AChE in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region [93]. HupA has been found to reverse or attenuate cognitive deficits in a broad range of animal models. Enhancement of learning and memory performance was documented in the passive footshock avoidance paradigm [37, 54, 98, 99], the classic water maze escape task [36, 79], and spatial discrimination in the radial arm maze [72]. Similarly, cognition enhancement was found in a delayed-response task in aged monkeys [78] and in reserpine- or yohimbinetreated monkeys [44]. Beneficial effects were seen not only in intact adult rodents, but also in rodents cognitive-

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ly impaired by age [37,79] or by treatment with scopolamine [16, 23, 36, 54, 59, 65, 98], AF64A [73, 9], electroshock [59, 99], cycloheximide [99], NaNO2 [99], or CO2 [37, 98]. In addition, HupA improved cognition in cholinergically lesioned rats [37, 99], and it reduced the spatial working memory deficit induced by lesion of the nucleus basalis magnocellularis [74]. These effects are attributable to increased synaptic ACh. Early on, HupA was found to cause a significant increase in ACh level in rat brain [55, 100]. More recently, a rise in cortical ACh levels has been demonstrated by microdialysis in awake, free-moving rats. In this study HupA was 8- and 2-fold more potent than donepezil and rivastigmine, respectively, and its effect was longer lasting [34]. Clinical trials of HupA in China have demonstrated a meaningful improvement in the memory of aged subjects, individuals with benign senescent forgetfulness and patients with AD. The results indicate minimal peripheral cholinergic side effects compared to other AChEIs in use; even more important, HupA lacks the dose-limiting hepatotoxicity induced by tacrine [75–77, 84, 90]. Adverse effects in the clinical trials were reported at a very low rate and are mainly cholinergic. Examples are dizziness, nausea, gastroenteric symptoms, headaches and depressed heart rate. Several clinical trials have addressed the effects of HupA on vascular dementia (VD). VD is currently considered to be the second most common form of dementia in Europe and the USA. However, in Asia and many developing countries including China, the incidence of VD exceeds that of AD. Multicenter, randomized, double-blind, placebo-controlled clinical trials in China proved that HupA markedly improved the cognitive function of VD [32, 38, 39, 90]. This article will summarize and discuss current research focused on elucidating the neuroprotective effect of HupA.

Protection of HupA against Hydrogen Peroxide and ß-Amyloid Protein-Induced Injury by Attenuating Oxidative Stress

Several neurodegenerative disorders such as AD, cerebral ischemia-reperfusion and head injury are thought to be related to changes in oxidative metabolism. Increased oxidative stress, resulting from free radical damage to cellular function, can be involved in the events leading to AD, and is also connected with lesions called tangles and plaques. Plaques are caused by the deposition of Aß and are observed in the brains of AD patients [7, 40, 45, 48]. Studies show that oxygen radicals initiate amyloid build-

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5 Fig. 1. Chinese herb Huperzia serrata (Qian Ceng Ta). Fig. 5. Anti-apoptotic mechanism of HupA. Black real line arrows = apoptotic-inducing pathway; red real line ar-

rows = affecting sites of HupA; red dash arrows = speculative pathways; (–) = inhibiting; (+) = promoting.

Hup A: A Natural Cholinesterase Inhibitor for Alzheimer’s Disease

Neurosignals 2005;14:71–82

73

Ab 25-35 M

Con

6

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Fig. 2. Reduction of Aß25–35-induced DNA fragmentation by

HupA. Neurons were treated with 20 ÌM of Aß25–35 with or without 1 ÌM of HupA. Fragmented DNA was isolated by NucleoBond DNA and RNA purification kit, electrophoresed with agarose gel, and finally stained with ethidium bromide. M = DNA size marker; Con = control.

up leading to neurodegeneration [19]. HupA has been found to protect against H2O2 and Aß-induced cell lesion, to decrease the level of lipid peroxidation, and to increase antioxidant enzyme activities in rat PC12 and primary cultured cortical neurons [68–70]. Following a 6-hour exposure of the cells to H2O2 (200 ÌM) or a 48-hour exposure of the cells to Aß25–35 (1 ÌM ), a marked reduction in cell survival and the activities of glutathione peroxidase (GSH-Px) and catalase (CAT) was observed, along with an increased production of malondialdehyde (MDA). Pretreatment of the cells with HupA (0.1–10 ÌM) 2 h before H2O2 and Aß exposure caused a significant increase in cell survival. HupA also partly reversed the H2O2- and Aß-induced decrease of GSH-Px and CAT activity, as well as the increase in production of MDA and SOD. All these effects indicate a neuroprotective action. The protective effect of HupA on Aß-induced cell lesion was also observed in NG108–15 cells [86] and primary cultured cortical neurons [71]. In addition to elevating cell survival and GSH-Px and CAT activities while decreasing the level of MDA [70], HupA (0.1–10 ÌM) significantly reduced Aß25–35-induced the formation of reactive oxygen species (ROS) in rat primary cultured cortical neurons [71].

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In rat studies, an intracerebroventricular (i.c.v.) infusion of ß-amyloid 1–40 (800 pmol !3) induced a significant cognitive deficit, morphologic signs of injury and a decrease of cortical choline acetyltransferase activity [64]. Daily i.p. administration of HupA for 12 consecutive days produced partial reversal of the ß-amyloid-induced deficit in learning a water maze task. This treatment ameliorated the loss of choline acetyltransferase activity in cerebral cortex and the neuronal degeneration induced by ß-amyloid 1–40 [64]. HupA also reduced the level of lipid peroxidation and superoxide dismutase in the hippocampus, cerebral cortex and serum of aged rats [49]. In a rat model of chronic cerebral hypo-perfusion, HupA significantly reduced the increases in SOD and lipid peroxide while restoring lactate and glucose to their normal levels [61]. A clinical study also demonstrated a reduction of oxygen free radicals in plasma and erythrocytes from AD patients [77]. These findings indicate that HupA has protective effects against free radical and Aß-induced cell toxicity, which might be beneficial in the treatment of patients with various kinds of dementia. Experience with HupA enantiomers has shown that the neuroprotective properties have no relation to anticholinesterase potency. Thus, preincubation with (+)HupA or (–)-HupA (0.1–10 ÌM ) protected cells with similar potency against Aß toxicity and similar enhancement of survival [86]. This result contrasted with the stereoselectivity of cholinesterase inhibition in vitro and in vivo, in which (–)-HupA is approximately 50-fold more potent than (+)-HupA. In another study, we examined drug effects on the apoptosis induced by incubation with Aß25–35 and on the increase of AChE activity accompanying this reaction. We observed that inhibiting the hydrolyzing activity of AChE without decreasing AChE expression itself did not attenuate the Aß25–35 induced apoptosis [85]. In other words, the ability of HupA to block the catalytic activity of AChE did not parallel its neuroprotective effect. Therefore, the cytoprotective effect of HupA enantiomers may relate to some kind of noncatalytic actions on AChE, or to actions on other cellular targets.

Anti-Apoptotic Effect of HupA

Apoptosis is the process by which neurons die during normal development and is also a feature of chronic and acute neurodegenerative diseases and stroke [82]. The cellular commitment to apoptosis is regulated by the Bcl-2 family of proteins. High levels of Bcl-2 expression will

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Fig. 3. Effects of HupA on H2O2-induced

expression of bcl-2, bax and p53 in PC12 cells by RT-PCR. Cells were exposed to 100 ÌM H2O2, and total RNA was extracted after the indicated recovery period and then subjected to RT-PCR. The PCR products were normalized by ß-actin mRNA. Lane 1: non-treated intact control; lanes 2–5: 0, 2, 6 and 12 h after 30 min H2O2-treatment, respectively. Lanes 6–9 represent the same time points as lanes 2–5 but preincubated with 1 ÌM HupA before H2O2 exposure.

2

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Bcl-2

Bax

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inhibit apoptosis. In contrast, an increased expression of P53 and Bax is associated with the initiation of apoptosis [29]. In accordance with previous reports, studies from our lab demonstrate typical apoptotic changes when neuron-like cells are exposed to stressors such as H2O2, Aß peptide, oxygen-glucose deprivation (OGD), serum deprivation, or the PKC inhibitor, staurosporine. These changes include DNA laddering, cell shrinkage, the generation of nuclear apoptotic bodies, TUNEL positive staining, and other classic hallmarks of apoptosis (fig. 2) [63, 71, 87, 94]. Such abnormalities are markedly relieved by HupA. For example, in rats that received i.c.v. injections of ß-amyloid1–40 (800 pmol ! 3), administration of HupA (0.1, 0.2 mg/kg, i.p.) for 12 consecutive days gave substantial neuroprotection in the brain. This treatment greatly reduced the number of apoptotic-like neurons and partly reversed the down-regulation of Bcl-2 and up-regulation of Bax and P53. Anti-apoptotic effects of HupA were also found in primary cultured neurons. Preincubation with HupA at concentrations higher than 0.01 ÌM led to a large, dose-dependent attenuation of cell toxicity induced by Aß25–35 (20 ÌM). Moreover, HupA (1 ÌM) caused large reductions in the amounts of subdiploid DNA detected in a flow cytometry assay and weakened the ladder pattern on agarose gel electrophoresis, typically seen after exposure to Aß (fig. 2). The anti-apoptotic actions of HupA may involve inhibition of the production or the effects of ROS [71]. We found that preincubation of PC12 cells with HupA before exposure to H2O2 substantially reduced apoptosis, by any of several measures. The same treatment also attenuated H2O2-induced overexpression of bax and p53, while restoring bcl-2 to normal levels (fig. 3) [63]. HupA was also effective in the OGD paradigm. Exposure to OGD for 3 h followed by reoxygenation for 24 h triggers apoptosis

characterized by chromatin condensation, nucleus fragmentation and DNA laddering, accompanied by altered levels of mRNA for c-jun, p53, bcl-2 and bax. In this model, HupA significantly attenuated apoptosis and reduced the up-regulation of c-jun and bax as well as the downregulation of bcl-2 [94]. In the mitochondrial-mediated cell death pathway, a key step is transient opening of the mitochondrial permeability transition (MPT), involving a non-specific increase in the permeability of the inner mitochondrial membrane [25, 28, 53]. In this process, cytochrome c moves from the intermembrane space into the cytoplasm [5] where it binds to another factor (Apaf-1). In the presence of dATP, this complex polymerizes into an oligomer known as the apoptosome. The apoptosome activates the protease, caspase-9, which in turn activates caspase-3. The cascade of proteolytic reactions also activates DNAases, which leads to cell death [83]. When PC12 cells were pre-incubated with HupA at concentrations above 0.01 ÌM, there was a marked neuroprotection against apoptosis induced by ß-amyloid, with a significant reduction in mitochondrial swelling and an improvement in mitochondrial membrane potentials [unpubl. data of this lab]. Pretreatment of rat cortical neurons with HupA (0.01–10 ÌM) significantly elevated cell survival and reduced all signs of apoptosis resulting from exposure to Aß25–35. Further studies focused on caspase activation in primary cultures of rat cortical neurons subjected to a variety of stresses. Measurements of caspase-3-like fluorogenic cleavage demonstrated that HupA (1 ÌM) attenuated an Aß25–35-induced increase in caspase-3 activity at 6, 12, 24, and 48 h [71]. Western blot analyses confirmed these results at the protein level. HupA also inhibited caspase-3 activation in models of apoptosis by serum deprivation

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Caspase-3 activity (U/mg protein)

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a

b

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Fig. 4. Effect of HupA on caspase-3 activity (a) and Western blot detection of cytochrome c and COX4 (b) in primary cortical neurons. Neurons under serum deprivation for 24 h. HupA at a concentration of 1 ÌM added to the culture 2 h in advance. a Data expressed as means B SD. Statistical comparison was made using ANOVA followed by Duncan’s test. There was a significant difference between the serum deprivation group and the untreated control group. ## p ! 0.01 compared to control group. * p ! 0.05 compared to serum depri-

vation group. b The mitochondrial and cytosolic fractions were isolated using an ApoAlert cell fractionation kit. They were then processed using the standard Western blot procedure on 12% SDSPAGE and probe with COX4 antibody (F17 kDa) or cytochrome c antibody (F15 kDa). The presence of cytochrome c in the cytosolic fraction after induction indicates that apoptosis involves mitochondrial release of cytochrome c to the cytosol.

and staurosporine treatment. The apoptosis induced by 24 h of serum deprivation was accompanied by enhanced caspase-3 activity and a release of mitochondrial cytochrome c into the cytosol [97]. HupA (0.1–10 ÌM) improved neuronal survival in this model, inhibiting the rise in caspase-3 activity and protein expression [97]. Likewise, cell survival was greatly enhanced when HupA (0.1– 100 ÌM) was introduced 2 h before a 24-hour exposure to 0.5 ÌM staurosporine. Incubation with HupA at dose of 1 ÌM also reduced staurosporine-induced DNA fragmentation, up-regulation of the pro-apoptotic gene, bax, downregulation of the anti-apoptotic gene, bcl-2, and decrease in caspase-3 proenzyme protein level (fig. 4) [87]. A potassium channel with delayed rectifier characteristics may play an important role in Aß-mediated toxicity. The up-regulation of an outward K+ current known as Ik mediates several forms of neuronal apoptosis and could contribute to the pathogenesis of Aß-induced neuronal death. Exposure to a 20-ÌM concentration of Aß25–35 or Aß1–42 is known to enhance the apoptosis-related current, Ik [81]. Interestingly, HupA will reversibly inhibit the fast transient current, IA, and the sustained potassium current, Ik, in CA1 pyramidal neurons acutely dissociated from rat hippocampus [33]. Such effects might contribute to this agent’s anti-apoptotic effect.

In light of these findings and the effects of HupA on apoptosis-related genes, we propose that HupA blocks apoptosis by antagonizing the mitochondrial-dependent caspase pathway, directly or indirectly (fig. 5). The effects of HupA on the intrinsic caspase-3 pathway might be downstream consequences of altered expression of bcl-2 family genes. Functionally, bcl-2 is a potent cell death suppressor, whose over-expression can prevent cell death in response to a variety of stimuli. It is well known that Bcl-2 suppresses apoptosis by inhibiting cytochrome c release from the mitochondria. On the other hand, bax is a death-promoting factor, whose translocation to the mitochondrial membrane leads to a loss of mitochondrial membrane potential and increases mitochondrial permeability. Increased mitochondrial permeability results in the release of cytochrome c followed by activation of caspase-3 [21]. We consider it likely that HupA owes some of its anti-apoptotic effects to an effective antagonism of the up-regulation of bax and the down-regulation of bcl-2, which impairs mitochondria-dependent caspase pathway. At present, however, direct effects of HupA on cytochrome c and caspase-3 and other possible targets are not excluded.

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Effects of HupA on Secretory Amyloid Precursor Protein and Protein Kinase C-·

Aß is a self-aggregating 39–43-amino acid peptide that originates from a larger polypeptide termed Alzheimer’s amyloid precursor protein (APP). Alternate pathways for APP processing have been described: the non-amyloidogenic secretory pathway, which releases a soluble ectodomain (APPs) and prevents Aß formation [15], and the endosomal-lysosomal pathway, which produces amyloidogenic products [24]. The amyloid hypothesis of AD [17, 46] is focused on the potential toxic role of an excessive production of Aß and suggests that the aberrant metabolism of APP is a central pathogenetic mechanism for the disease. Several factors can affect the secretory non-amyloidogenic pathway of APP. For example, the stimulation of phospholipase C (PLC)-coupled receptors, such as muscarinic m1 and m3, has been shown to potentiate the secretion of APP in cell cultures. These effects are probably mediated mainly by protein kinase C (PKC) [43]. It has also been reported that several anticholinesterases affect APP processing in addition to the catalytic function of AChE [18, 42]. Our own studies showed that HupA could alter APP processing in the brains of rats given i.c.v. infusions of Aß1–40, and in otherwise untreated human embryonic kidney 293 (HEK293sw) cells [88]. In the Aß treated rats, levels of APPs and PKC· were significantly decreased by treatment with Aß1–40. These decreases were much reduced by 12 consecutive days of HupA treatment (0.2 mg/ kg, i.p.), but HupA in normal rats caused no change in either APPs or PKC·. In normal HEK293sw cells, on the other hand, the levels of APPs and PKC· rose progressively during an 18-hour exposure to HupA (1 ÌM). However, no significant alternations in the levels of PKC‰ and PKCÂ were found after HupA treatment. Taken together these findings suggest that HupA may affect the processing of APP by up-regulating PKC, especially PKC·. In an attempt to clarify the receptor mechanisms involved in such effects, we treated HEK293wt cells with cholinergic receptor antagonists [unpubl. data]. The nonselective muscarinic antagonist, scopolamine, partly blocked the HupA-induced rise in levels of APPs and PKC·. By contrast the nicotinic antagonist, mecamylamine, had little effect. These results suggest that muscarinic ACh receptors may mediate, at least in part, the effects of HupA on the regulation of APPs and PKC· in HEK293sw cells.

Hup A: A Natural Cholinesterase Inhibitor for Alzheimer’s Disease

Our recent results provide the first demonstration that HupA can reduce the disturbance of PKC and APPs both in rats and in an isolated cell line. The effect of HupA to enhance non-amyloidogenic processing of APP and elevate APPs levels likely depends on the activation of muscarinic receptors and the PLC/PKC cascade. A number of biological activities such as cell proliferation, promotion of cell-substratum adhesion, neurite outgrowth and the prevention of intracellular calcium accumulation and cell death have been attributed to APPs [47]. Since PKC is a key enzyme in signal transduction, and since APPs itself has neuroprotective effects, modulating the levels of these two proteins by HupA may well be beneficial in AD therapy (fig. 6).

Protection of HupA against Hypoxic-Ischemic and Glutamate Induced Brain Injury and Cytotoxicity

Apart from AD, the most common dementia in the elderly is VD. This disorder, like AD, presents a clinical syndrome of intellectual decline produced by ischemia, hypoxia, or hemorrhagic brain lesion. Cerebral ischemia in rats with permanent bilateral ligation of the common carotid arteries (CCA) provides a useful model of VD, in which to investigate the effects of HupA. These animals experience a significant reduction of cerebral blood flow and exhibit learning and memory impairments and neuronal damage resembling those in VD. Daily oral administration of HupA (0.1 mg/kg) to such rats for 14 days produced significant improvement in the learning of a water maze task. Simultaneously there was marked recovery from the decrease in choline acetyltransferase activity in hippocampus and a restoration of SOD, lipid peroxide, lactate and glucose to normal levels [61]. Similar protection was also observed in gerbils given subchronic oral doses of HupA (0.1 mg/kg, twice daily for 14 days) following 5 min of global ischemia [96]. An in vitro model of neuronal ischemia is the rat pheochromocytoma PC12 cell treated with OGD for 30 min. In our hands, this treatment causes death in more than 50% of the cells in culture, along with major changes in morphology and biochemistry, including elevated levels of lipid peroxide, SOD activity and lactate. Cells pretreated for 2 h with HupA (0.1, 1 and 10 ÌM), however, showed increased survival and reduced biochemical and morphologic signs of toxicity. HupA protected PC12 cells against OGD-induced toxicity, most likely by alleviating disturbances of oxidative and energy metabolism [95].

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Fig. 6. Protective effects of HupA through affecting APP metabolism. Black arrows show the process of AD. Blue real line arrows show the proved pathway; blue dash arrow represents speculative pathway; (–) means reducing or inhibiting.

These findings suggested that HupA might be beneficial for VD therapy through its effects on the cholinergic system, the oxygen free radical system and energy metabolism. A protective effect of HupA on hypoxic–ischemic (HI) brain injury has also been found in neonatal rats [62]. A unilateral HI brain injury was produced in 7-day-old rat pups by the ligation of the left CCA followed by 1 h hypoxia with 7.7% oxygen. After 5 weeks, the HI brain injury in these pups caused working memory impairments in

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water maze performance, as shown by an increased escape latency and a reduced time spent in the target quadrant. The combination of CCA ligation and exposure to a hypoxic environment also led to morphologic damage in the ipsilateral striatum, cortex, and also hippocampus, where it produced 12% neuronal loss in the CA1 region. Treatment with HupA at a dose of 0.1 mg/kg conferred significant protection against the behavioral and morphologic consequences of HI injury (fig. 7). The same treatment spared a significant fraction of the CA1 neurons relative

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Fig. 7. Photomicrographs of coronal brain sections stained with cresyl violet at the levels of the striatum (a, b) and the dorsal hippocampus (c, d) for representative saline-treated and huperzine A 0.1 mg/kg-treated rats. Intraperitoneal administration of huperzine A or saline for 5 weeks after hypoxic-ischemic (HI) brain injury in neonatal rats. Note the gross infarction and atrophy in left hemisphere of saline-treated HI rats (a, c) (n = 11) and the subtle reduction in the left hemisphere in huperzine A-treated HI rats (b, d) (n = 12).

to saline-treated HI group. These results raise the possibility that HupA have potential utility in treating HI encephalopathy in neonates. Glutamate is the main excitatory neurotransmitter in the CNS, with important roles in neurotransmission and functional plasticity. Excitatory amino acid neurotransmitters are also involved in CNS pathology. The deleterious effects of overstimulation with excitatory amino acids have been implicated in a variety of acute and chronic neurodegenerative disorders such as ischemic brain damage, AD and neuronal cell death [11] [for reviews, see 13, 14, 26, 27, 35]. Glutamate-mediated overactivation of receptors induces excessive Ca2+ influx, which results in elevated intracellular Ca2+ concentrations [10, 12] with serious consequences such as necrosis and apoptosis [31]. Blockade of glutamate receptors prevents most of the Ca2+ influx and neuronal cell death induced by glutamate exposure [50, 57]. It has been reported that HupA protects against glutamate-induced toxicity. HupA (100 ÌM) decreased neuronal cell death caused by a toxic level of glutamate (also

100 ÌM). In those experiments, HupA reduced glutamate-induced calcium mobilization but did not affect the increase in intracellular free calcium induced by exposure to high KCl or a calcium activator Bay-K-8644 [58]. HupA dose-dependently inhibited the NMDA-induced toxicity in primary neuronal cells, most likely by blocking NMDA ion channels and the subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites [20]. Wang et al. [66] reported that HupA reversibly inhibited NMDA-induced current in acutely dissociated rat hippocampal pyramidal neurons and blocked specific [3H]MK801 binding in synaptic membranes from rat cerebral cortex. Of all the AChE inhibitors tested, HupA is the most powerful both in protecting mature neurons and in blocking the binding of [3H]MK-801. Studies on the mechanism of receptor inhibition showed that HupA reversibly inhibited NMDA-induced currents. The effect was noncompetitive, and showed neither ‘voltage-dependency’, nor ‘use-dependency’ [89]. Studies of [3H]MK-801 binding in cortex membranes suggest that HupA acts as a noncompetitive antagonist of the NMDA receptors, via a

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competitive interaction with one of the polyamine binding sites [92]. Of interest, natural (–)-HupA and synthetic (+)-HupA reduced the binding of [3H]MK-801 with similar potencies [91] indicating that HupA inhibits NMDA receptors in rat cerebral cortex without stereoselectivity. This result is in dramatic contrast with the stereoselective inhibition of acetylcholinesterase. NMDA-receptor activation also mediates the generation of long-term potentiation (LTP) – a cellular process that underlies learning and memory [3, 52]. There is evidence that the suppressive action of Aß on LTP in both CA1 and dentate gyrus operates via a NMDA receptorindependent pathway that involves cholinergic terminals in the hippocampus. Of some interest, HupA (1.0 ÌM) was found to enhance LTP, while a much lower dose (0.1 ÌM) largely blocked the suppressive effects of Aß on LTP induction [8, 80].

Neuronal cell death caused by overstimulation of glutamate receptors has been proposed as the final common pathway for a variety of neurodegenerative diseases including AD. The ability of HupA to attenuate glutamatemediated neurotoxicity may be one additional reason for considering this agent as a potential therapeutic for dementia and as a means of slowing or halting the pathogenesis of AD at an early stage [20].

Acknowledgements This work was supported in part by the grants from Ministry of Science and Technology of China (G199805110, G1998051115) and National Natural Science Foundation of China (39170860, 39770846, 3001161954, 30123005 and 30271494). The authors are grateful to Professor Stephen W. Brimijoin (Mayo Clinic, USA) for English revision on the manuscript.

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77 Xu SS, Cai ZY, Qu ZW, Yang RM, Cai YL, Wang GQ: Huperzine A in capsules and tablets for treating patients with Alzheimer’s disease. Acta Pharmacol Sin 1999;20:486–490. 78 Ye JW, Cai JX, Wang LM, Tang XC: Improving effects of huperzine A on spatial working memory in aged monkeys and young adult monkeys with experimental cognitive impairment. J Pharmacol Exp Ther 1999;288:814– 819. 79 Ye JW, Shang YZ, Wang ZM, Tang XC: Huperzine A ameliorates the impaired memory of aged rat in the Morris water maze performance. Acta Pharmacol Sin 2000;21:65–69. 80 Ye L, Qiao JT: Suppressive action produced by beta-amyloid peptide fragment 31–35 on longterm potentiation in rat hippocampus is Nmethyl-D-aspartate receptor-independent: it’s offset by (–)huperzine A. Neurosci Lett 1999; 275:187–190. 81 Yu SP, Farhangrazi ZS, Ying HS, Yeh CH, Choi DW: Enhancement of outward potassium current may participate in beta-amyloid peptide-induced cortical neuronal death. Neurobiol Dis 1998;5(2):81–88. 82 Yuan J, Yankner BA: Apoptosis in the nervous system. Nature 2000;407:802–809. 83 Zamzami N, Kroemer G: The mitochondrion in apoptosis: How Pandora’s box opens. Nature Reviews in Molecular and Cellular Biology 2001;21:67–71. 84 Zhang CL, Wang GZ: Effects of huperzine A tablet on memory. New Drugs Clin Remed 1990;9:339–341. 85 Zhang HY, Brimijoin S, Tang XC: Apoptosis induced by ß-amyloid25–35 in acetylcholinesterase-overexpressing neuroblastoma cells. Acta Pharmacol Sin 2003;24:853–858.

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86 Zhang HY, Liang YQ, Tang XC, He XC, Bai DL: Stereoselectivities of enantiomers of huperzine A in protection against amyloid 25–35induced injury in PC12 and NG108–15 cells and cholinesterase inhibition in mice. Neurosci Lett 2002;317:143–146. 87 Zhang HY, Tang XC: Huperzine A attenuates the neurotoxic effect of staurosporine in primary rat cortical neurons. Neurosci Lett 2003; 340:91–94. 88 Zhang HY, Yan H, Tang XC: Huperzine A enhances the level of secretory amyloid precursor protein and protein kinase C-· in intracerebroventricular ß-amyloid-(1–40) infused rats and human embryonic kidney 293 Swedish mutant cells. Neurosci Lett 2004;360:21–24. 89 Zhang JM, Hu GY: Huperzine A, a nootropic alkaloid, inhibits N-methyl-D-aspartate-induced current in rat dissociated hippocampal neurons. Neuroscience 2001;105:663–669. 90 Zhang RW, Tang XC, Han YY, Sang GW, Zhang YD, Ma YX, Zhang CL, Yang RM: Drug evaluation of huperzine A in the treatment of senile memory disorders. Acta Pharmacol Sin 1991;12:250–252. 91 Zhang YH, Chen XQ, Yang HH, Jin GY, Bai DL, Hu GY: Similar potency of the enantiomers of huperzine A in inhibition of [(3)H]dizocilpine (MK-801) binding in rat cerebral cortex. Neurosci Lett 2000;295:116–118.

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92 Zhang YH, Zhao XY, Chen XQ, Wang Y, Yang HH, Hu GY: Spermidine antagonizes the inhibitory effect of huperzine A on [3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex. Neurosci Lett 2002;319:107–110. 93 Zhao Q, Tang XC: Effects of huperzine A on acetylcolinesterase isoforms in vitro: Comparison with tacrine, donepezil, rivastigmine and physostigmine. Eur J Pharmacol 2002; 455:101–107. 94 Zhou J, Fu Y, Tang XC: Huperzine A protects rat pheochromocytoma cells against oxygen-glucose deprivation. NeuroReport 2001; 12:2073–2077. 95 Zhou J, Fu Y, Tang XC: Huperzine A and donepezil protect rat pheochromocytoma cells against oxygen-glucose deprivation. Neurosci Lett 2001;306:53–56. 96 Zhou J, Zhang HY, Tang XC: Huperzine A attenuates cognitive deficits and hippocampal neuronal damage after transient global ischemia in gerbils. Neurosci Lett 2001;313: 137–140. 97 Zhou J, Tang XC: Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons. FEBS Lett 2002;526:21–25. 98 Zhu XD, Tang XC: Facilitatory effects of huperzine A and B on learning and memory of spatial discrimination in mice. Acta Pharmacol Sin 1987;22:812–817. 99 Zhu XD, Tang XC: Improvement of impaired memory in mice by huperzine A and huperzine B. Acta Pharmacol Sin 1988;9: 492–497. 100 Zhu XD, Giacobini E: Second generation cholinesterase inhibitors: Effect of (L)-huperzine A on cortical biogenic amines. J Neurosci Res 1995;41:828–835.

Wang/Tang

Author Index Vol. 14, No. 1–2, 2005

Amit, T. 46 Avramovich-Tirosh, Y. 46 Doré, S. 61 Houghton, P.J. 6 Howes, M.-J. 6 Komatsu, K. 34 Kuboyama, T. 34 Mandel, S.A. 46 Reznichenko, L. 46 Suk, K. 23 Tang, X.C. 71 Tohda, C. 34 Wang, R. 71 Youdim, M.B.H. 46 Zheng, H. 46

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2005 S. Karger AG, Basel

Accessible online at: www.karger.com/nsg

83

Subject Index Vol. 14, No. 1–2, 2005

Acetylcholine 6 Acetylcholinesterase 71 Alzheimer’s disease 6, 34, 71 Amyloid 34, 71 – precursor protein 71 Apoptosis 71 Ashwagandha 34 Astrocytes, neuroinflammation 23 Axon regeneration 34 Bilirubin 61 Biliverdin 61 Blood flow 61 Carbon monoxide 61 Cell signaling 46 Central nervous system, herbal medicine 23 Cerebral ischemia 71 Cholinergic receptors 6 Cholinesterase inhibitors 6, 71 Coffee beans, trigonelline 34 Cognitive enhancer, huperzine A 71

Galantamine 6 Ginseng 34 Glutamate 71 Green tea catechins 46 Hemin 61 Huperzine A 6, 71 Inflammation 23 Iron 61 – chelation 46 Microglia 23 Mitochondria 71 Neurite outgrowth 46 Neuritic atrophy 34 Neurodegeneration 23, 46 Neuroglia 23 Neuroprotection 23, 46, 71 Neurorescue 46 NMDA receptor 71 Oxidative stress 71

Dendrite regeneration 34 L-DOPA 6 Dopamine 6 Dopaminergic receptors 6 (–)-Epigallocatechin-3-gallate 46 Ergot alkaloids 6

Parkinson’s disease 6, 46 Physostigmine 6 Polyphenol stilbenes 61 Potassium current 71 Protein kinase C 46 Synaptic loss 34

Flavonoids 23 Withania somnifera 34

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2005 S. Karger AG, Basel

Accessible online at: www.karger.com/nsg