Neuroprotection by monoamineoxidase B inhibitors:a therapeutic strategyfor Parkinson’s disease?{

Rinat Tabakman,{ Shimon Lecht, and Philip Lazarovici*

SummaryParkinsonism (PD) is a neurodegenerative disorder of thebrain resulting in dopamine deficiency caused by theprogressive death of dopaminergic neurons. PD is char-acterized by a combination of rigidity, poverty of move-ment, tremor and postural instability. Selegiline is aselective and irreversible propargylamine type B mono-amine oxidase (MAO-B) inhibitor. This drug, which in-hibits dopaminemetabolism, hasbeen effectively used inthe treatment of PD. However, its therapeutic effects arecompromised by its many neurotoxic metabolites. Tocircumvent thisobstacle, anovelMAO-B inhibitor, rasagi-line, was developed. Paradoxically, the neuroprotectivemechanism of propargylamines in different neuronalmodels appears to be independent of MAO-B inhibition.

Recent investigations into the neuroprotective mechan-ism of propargylamines indicate that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MAO-B and/orother unknown proteins may represent pivotal proteinsin the survival of the injured neurons. Delineation of themechanism(s) involved in the neuroprotective effectsexerted by MAO-B inhibitors may provide the key topreventive novel therapeutic modalities. BioEssays26:80–90, 2004. � 2003 Wiley Periodicals, Inc.

Introduction

Parkinson’s disease (PD) is a progressive movement disorder

that affects 1–2%of the adult population over 60 years of age.

The main symptoms are tremor at rest, muscular rigidity and a

decrease in the frequency of voluntary movements (hypoki-

nesia).(1) For many years, it has been known that PD synd-

rome is due to a disorder of the basal ganglia, brain structures

regulating motor activity and innervated by one of the brain’s

major dopaminergic pathways. The neurochemical basis for

the disease was discovered in 1960 by Hornykiewics,(2) who

showed that the dopamine content of the substantia nigra (SN)

and corpus striatum in postmortem PD brains was extremely

low (less than 10% of normal). Pathologically, PD is charac-

terized by progressive degeneration of pigmented brain stem

nuclei, mostly the pars compacta of the SN, along with the

formation of characteristic eosinophilic cytoplasmic inclusions

known as Lewy bodies.(3) The disease progresses slowly for

many years. The clinical symptoms are due to the degenera-

tion (death) of dopaminergic neurons in the SN, resulting in a

dramatic decline in dopamine level. Diagnosis can be made

when at least 60% of the dopaminergic SNc neurons are lost.(4)

Many factors are considered to contribute to the pathogenesis

of PD: genetic,(5) age-related,(6) enviromental toxins,(7) and

oxidative stress.(8,9) In the past decade, novel hypotheses

have been forwarded suggesting important pathological con-

tributions to dopaminergic neuronal cell death: glutamate

neurotoxicity,(10) mitochondrial abnormalities, disturbances in

intracellular calcium homeostasis, altered iron metabolism

and apoptosis.(9,11–14) Although the genes responsible for

a few rare familial cases have been uncovered,(15–17) the

80 BioEssays 26.1 BioEssays 26:80–90, � 2003 Wiley Periodicals, Inc.

Department of Pharmacology, School of Pharmacy, Faculty of

Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.

*Correspondence to: Philip Lazarovici, Department of Pharmacology

and Experimental Therapeutics, School of Pharmacy, Faculty of

Medicine, The Hebrew University, Jerusalem, 91120, Israel.

E-mail: lazph@md.huji.ac.ilyThis review is part of a PhD thesis to be submitted to the Hebrew

University of Jerusalem by TR.zThe scientific views and opinions expressed in this article are solely

those of the authors and are not to be construed as having any

commercial interest.

DOI 10.1002/bies.10378

Published online in Wiley InterScience (www.interscience.wiley.com).

Abbreviations: PD, Parkinson’s disease; UPDRS, unified Parkinson’s

disease rating scale; SN, substantia nigra; DA, dopaminergic; MAO-B,

type B monoamine oxidase; GAPDH, glyceraldehyde-3-phosphate

dehydrogenase; SOD, superoxide dismutase; NADþ, nicotinamide

adenine dinucleotide; FAD, flavin adenine dinucleotide; BDNF, brain-

derived neurotrophic factor; NGF, nerve growth factor; GDNF, glial

derived neurotrophic factor; DMS, desmethylselegiline; MPPþ, 1-

methyl-4-phenyl pyridinium; MPTP, N-methyl-4-phenyl-1,2,3,6-tetra-

hydropyridine; 6-OH-DA, 6-hydroxydopamine; OGD, oxygen–glucose

deprivation; PET, positron emission tomography; fMRI, functional

magnetic resonance imaging; IC50, 50% inhibitory concentration; Ki,

inhibition constant; PC12, pheochromocytoma cells.

Challenges

molecular basis for the more prevalent idiopathic PD remains

unknown. Novel technologies including DNA-microarray(18)

and single nucleotide polymorphisms(19) are expected to

pinpoint the pathogenesis of this disorder. In addition, new

techniques in neuroimaging such as positron emission tomo-

graphy (PET) and functional magnetic resonance imaging

(fMRI) may provide better means of assessing SN function.(20)

In this review, we briefly summarize the emerging knowledge

regarding the complex mechanism of action of type B mono-

amine oxidase (MAO-B) inhibitors, an important family of

drugs used to treat PD. While the search for MAO-B inhibitors

has turned up some promising propargylamine candidates,

their mode of action in ameliorating PD cannot be explained

solely by MAO-B inhibition. Today, the basis for their neuro-

protective mechanism of action is being actively pursued. We

hypothesize that the beneficial role of MAO-B inhibitors in PD

is due to a shift from neurotoxicity to neuroprotection of the

injured dopaminergic neurons.

Apoptosis and Parkinson’s disease

Since 1996, when apoptotic death was discovered in

dopaminergic cells of Parkinsonian patients,(21) many addi-

tional studies have suggested the involvement of an apoptotic

mechanism in this disorder. Apoptosis, a delayed form of cell

death associated with the activation of a ‘‘genetic program’’, is

an important biological process controlling cell number in

various tissues. This mechanism plays an important role in the

development of the nervous system(22) and is also triggered

following pathological events such as stroke and neurode-

generative diseases.(14) The SN of PD patients shows an

increase in caspase activity and in the pro-apoptotic Bax

protein, as well as nuclear translocation of glyceraldehyde-3-

phosphate dehydrogenase (GAPDH), indicating the activation

of apoptotic signals.(23–25) However, it is not clear whether the

degeneration associated with PD involves a fast ‘event’ in

which a significant number of dopaminergic neurons deterio-

rate as a result of unknown factor(s),(26) or a very slow one that

may last several decades.(27) Furthermore, other postmortem

studies, based on nick-end labeling of fragmented DNA,(28)

Bcl-2 and Bax apoptotic protein markers,(27) failed to show a

significant contribution of apoptosis to SN dopaminergic

neuronal cell death. An additional complication adding to the

cell death controversy in PD is the finding that, in postmortem

tissue, apoptotic markers are present in the glia but not in SN

neurons.(29,30) In general, nigrostriatal dopaminergic neurons

are lost with age. This phenomenon is dramatically acceler-

ated in Parkinson’s patients. The grounds for the vulnerability

of the nigrostriatal dopaminergic neurons are unknown and the

precise mechanism of cell death awaits further investigation.

The gene-expression microarray technique is expected to

make a significant contribution to the identification of gene

products involved in cell death and leading to PD.(18)

Therapeutical approaches

to Parkinson’s disease

As the cause of the death of the dopaminergic neurons is not

known, drugs that can arrest and/or reduce and/or delay the

cell death process in dopaminergic neurons are not available.

However, significant symptomatic relief has been obtained by

the use of different dopaminergic drugs that fall into the follow-

ing categories: dopamine precursors (levodopa); compounds

mimicking (agonists) the action of dopamine (bromocriptine);

agents that prevent dopamine degradation (MAO-B inhibitors

such as rasagiline).

Levodopa, a dopamine precursor that crosses the blood–

brain barrier, is initially effective in most patients, but often

loses its efficacy after several years of treatment, the period

varying from one patient to another. Complications associated

with its use are involuntary movements, which occur in many

patients within several years, and an unpredictable ‘‘on–off’’

effect in the course of treatment.(31) Other adverse effects

include queasiness (nausea), a drop in blood pressure

(hypotension), and, occasionally, psychotic symptoms.(31)

Levodopa treatment requires a certain number of live dopami-

nergic neurons to convert this metabolic precursor into the

neurotransmitter dopamine (DA). As a result, the amount of

synaptic DA is increased and the DA deficiency characteristic

of PD is corrected. The disadvantage of this therapeutic

approach is the augmented cell death of large numbers of DA

neurons during PD progression, leading to less DA synthesis

during levodopa treatment and rendering it less effective.

Drugs that mimic DA (agonists) are less effective than

levodopa.(32) They activate postsynaptic DA receptors in the

SN and improve motor function independently of DA neuron

degeneration.

The first generation of MAO inhibitors, originally synthe-

sized in the 1950s, was used as antidepressants due to their

inhibitory effect on the metabolism of monoamine neurotrans-

mitters. These inhibitors are both irreversible and non-selec-

tive. However, their use as antidepressants was restricted

because of hepatotoxicity(33) and due to an increase in blood

pressure (hypertensive response) following the ingestion of

some foods and drinks containing tyramine,(34) which are nor-

mally metabolized in the gastrointestinal tract by MAO.(34,35)

As a result of MAO inhibition, the ingested tyramine enters the

circulation and is actively taken up by peripheral adrenergic

neurons, displacing stored noradrenaline and giving rise to a

hypertensive response that can be fatal.(36) This side-effect

pharmacological phenomenon(37) was named the ‘‘cheese

effect’’, since many cheeses are rich in tyramine. The next

discovery was made by Johnston,(38) who showed that the

MAO enzyme exists in two forms: A and B. This led to the

synthesis of a new generation of MAO inhibitors exhibiting

greater selectivity toward the individual isoforms. MAO-A

inhibitors are potent antidepressants but, since MAO-A is the

major isoform in the intestine, they induce the ‘‘cheese effect’’.

Challenges

BioEssays 26.1 81

The finding that MAO-B inhibitors have weaker antidepressant

activity, do not induce the ‘‘cheese effect,’’(39) but can amelio-

rate PD symptoms, was the basis for modern Parkinson

therapeutics,(39,40) using the MAO-B inhibitor selegiline and

rasagiline. Selegiline has been approved for use in the USA as

an adjunct to levodopa but not for monotherapy; rasagiline is a

new addition to the MAO-B inhibitor group.

Monoamine oxidases and inhibitors

in Parkinson’s disease

MAO (EC 1.4.3.4) oxidatively deaminates monoamine neuro-

transmitters (norepinephrine, epinephrine, serotonin and

dopamine), as well as exogeneous amines (e.g., tyramine).

The end products of the enzymatic reaction are aldehydes and

hydrogen peroxide, both toxic to DA neurons. The physio-

logical role of MAO is to terminate the action of several

neurotransmitters and to detoxify exogenous monoamines.

Two MAO subtypes, MAO-A and MAO-B, were defined in

1968,(38) based on their differential sensitivity to irreversible

inhibitors. 20 years later, the corresponding cDNAs of these

two subtypes were cloned. Sequence comparison indicated

72.6% amino acid homology.(41,42) The genes encoding the

two subtypes have been localized to human chromosome

Xp11.23-Xp11.4.(43) TheMAO-AandMAO-Bgenesarehomo-

logous at the level of intron–exon organization and in co-factor

FAD-binding site, and both enzymes are located at the outer

mitochondrial membrane.(44) Along with the similarities, MAO-

A and MAO-B display differences in their numbers of amino

acids, 527 and 520, and molecular weights 59.7 kDa and

58.8 kDa, respectively. The in vivo substrate affinity in humans

also differs: MAO-A metabolizes mainly serotonin, norepi-

nephrine and epinephrine, whereas MAO-B metabolizes

dopamine and phenylethylamine.(44) Both MAO-B and MAO-

A are widely distributed in the human body, the ratio between

them varying in different tissues. The highest value (>69) is

found in platelets; lower ratios were detected in the human

brain, the highest of which (5.5) was found in the SN.(45) The

selectivity toward different endogenous substrates, together

with tissue-specific localization, determines the clinical poten-

tial of each isoform. Inhibition of MAO activity in the brain

increases the synaptic level of the neurotransmitters seroto-

nin, noradrenaline and dopamine. This mechanism was

exploited to treat depression, first with nonselective MAO

inhibitors and, more recently, with selective MAO-A inhibitors.

Today however, inhibitors of MAO-A are not the drugs of first

choice in the treatment of depression, mainly because of the

development of novel antidepressants that act by different

pharmacological mechanisms, without eliciting the cheese

effect.

Many selective MAO-B inhibitors, potentially applicable to

PD treatment, have been synthesized. They are categorized

according to chemical structure, each including several

derivatives (Table 1). The most-potent and selective group

consists of propargylamine derivatives. These are irreversible

inhibitors, which would ensure continuous dopaminergic

stimulation, considered a major therapeutic goal. Lazabemide,

a 2-aminoethyl carboxamide derivative, which is a reversible,

highly selective antioxidant, was developed as a MAO-B

inhibitor for treatment of PD.(46) Clinical trials with this com-

pound were discontinued in phase III of the clinical study due

to the abnormal liver functions that developed in patients

undergoing this treatment. To date, of the variety of selective

MAO-B inhibitors investigated, only one, selegiline (l-depre-

nyl) (Table 1), has been in clinical use since the mid 1970s.(47)

Rasagiline (N-propargyl-1-R-aminoindan) (Table 1), a novel,

potent, selective, and irreversible inhibitor of MAO-B, absorb-

ed after oral administration,(48,49) is currently being evaluated

in phase III clinical trial.(40) In contrast to selegiline, rasagiline is

not metabolized to potentially toxic amphetaminic metabolites

and its major metabolite, 1-R-aminoindan, has exhibited

beneficial effects in animal models of PD.(50) Because of its

selectivity towards MAO-B, rasagiline does not trigger the

‘‘cheese effect’’,(51) and was found to improve motor and

cognitive function in PD animal models.(50) A recent clinical

trial demonstrated the beneficial influence of rasagiline mono-

therapy in early stages of Parkinson’s disease.(40) To evaluate

the safety and efficacy of rasagiline, a multicenter clinical trial

was carried out in which a total of 404 patients in early stages of

PD were included. The participants were randomly assigned to

groups receiving rasagiline at a dose of 1–2 mg per day or

corresponding placebo. A one-week escalation period was

followed by a 25-week maintenance period. The efficacy of

treatment was evaluated according to the Unified Parkinson’s

Disease Rating Scale (UPDRS); rasagiline monotherapy at

both doses proved effective.(40)

Although inhibition of MAO-B is of prime importance in

controlling PD symptoms, it appears that this inhibition under-

lies many additional effects in the brain. An interesting analogy

can be drawn between PD patients chronically administered

MAO-B inhibitors and knockout mice lacking the gene encod-

ing MAO-B. Chen et al. demonstrated significant upregulation

(�30%) of D2 receptors in the striatum and the functional

supersensitivity of D1 receptors in the mouse nucleus accum-

bens.(52) This upregulation resembles similar regulation of the

dopamine receptors observed in PD patients chronically

treated with MAO-B inhibitors. It has been suggested that

MAO-Bmakes asmaller contribution thanMAO-A todopamine

metabolism in the mouse, as compared with man,(53) making it

difficult to conclude that similar changes occur in MAO-B

inhibitor-treated patients. However, subjects in whom the

MAO-B gene is deleted (atypical Norrie disease) have normal

levels of DA metabolites in their plasma.(54) The parallelism

between MAO-B knockout mice and humans with a deleted

MAO-B gene, suggests a similarity in the lack of change in

DA metabolites levels This additional circumstantial evidence

leads us to postulate that upon treatment with MAO-B

Challenges

82 BioEssays 26.1

inhibitors, DA receptors undergo alterations seen in the MAO-

B knockout mice. However, changes in dopamine receptors

are not the sole effect of the MAO-B inhibitors. Recent studies

show the potential for an additional beneficial activity:

neuroprotection. Cumulative in vivo and in vitro evidence

points to the neuroprotective properties of propargylamine

MAO-B inhibitors, such as selegiline and rasagiline.(55–57)

MAO-B inhibitors and neuroprotection

in different neurotoxic models

Since PD progresses very slowly and over many years, its

diagnosis poses a tremendous challenge. Because the trigger

for dopaminergic cell death is unknown, the therapeutic

strategy in the last 15 years has been based on the DA

precursor levodopa, which raises the DA level. The adverse

effects of the treatment, the fact that this compound does

not arrest dopaminergic cell death and disease progression

and the possible contribution of apoptotic cell death to

Parkinson’s disease, prompted the search for new neuropro-

tective approaches. A neuroprotective therapeutic or disease-

modifying modality may be defined as an intervention that

delays or prevents neuronal cell death and thus affects

disease progression.

Selegiline aroused considerable interest as a potential

neuroprotective drug. As summarized in Tables 2 and 3,

preclinical studies point to the neuroprotective effect of the

Table 1. Classification of MAO-B inhibitors of clinical relevance, according to chemical structure, potency,

mechanism of action and selectivity

Chemical group Chemical structureMAO-B inhibition

(IC50 nM)Mechanismof inhibition

Selectivitya

MAOB/MAOA References

Propargylamine derivatives

Selegiline

6 Irreversible 233 95

Rasagiline 30 Irreversible 100 95

Allylamine derivatives 100 Irreversible 50 95

2-aminoethyl carboxamide

derivatives

Lazabemide

37 Reversible 26568 95

N-allenic indolalkylamine

derivatives

25–25000 Irreversible >1 96

4-substituted

cubylcarbinyl amines

120–260b Irreversible — 97

aRatio between MAO-B and MAO-A (IC50). High value indicates greater selectivity towards MAO-B.bKi.

Challenges

BioEssays 26.1 83

propargylamine MAO-B inhibitors selegiline and rasagiline.

This is illustrated by in vitro studies using a variety of

dopaminergic and nondopaminergic neurons taken from

different species and exposed to a range of neurotoxic insults

(excitotoxicity, oxidative stress, depolarization, growth factor

withdrawal and oxygen glucose deprivation). For example,

rasagiline prolongs the survival of cultured primary human and

rat DA neurons under serum-free conditions,(58) reduces

glutamate toxicity in hippocampal neurons,(59) protects nerve

growth factor (NGF)-differentiated pheochromocytoma PC12

cells from oxygen-glucose-deprivation (OGD-ischemia) in-

sult,(57,60) prevents deficits in behavioral parameters following

hypoxia in adult and senescent rats,(61) protects from experi-

mental focal ischemia in the rat,(62) and from closed head injury

in the mouse.(63) The last three are invivomodels. Although the

neuroprotective in vitro effect of selegiline, and even more so

of rasagiline, is clear-cut, the lackof reliable pathophysiological

models for the disease has led to some skepticism among the

Parkinson medical community. The PD models using neuronal

cultures treated with 1-methyl-4-phenyl pyridinium (MPPþ, a

metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetra-

hydropyridine (MPTP)) and 6-hydroxydopamine (6-OH-DA)

Table 2. Neuroprotective in vitro effects of MAO-B inhibitors selegiline and rasagiline

MAO-B Inhibitor Insult ModelNeurotoxicitymechanism

Neuroprotectivemechanism References

Selegiline Excitotoxicity Mesencephalic

dopaminergic, primary

neurons

Depolarization and

calcium overload

Yes

Independent of MAO-B

98

Hypocampal glia and

neurons

Oxidative stress Yes, release of NGF 99

MPPþ Dopaminergic neurons

mesencephalic and

striatal cells

Oxidative stress Yes 100

BSO L-buthionine-(S,R)-

sulfoximine

Mesencephalic neurons Glutathione depletion Yes

Independent of MAO-B

101

Serum and NGF

withdrawal

NGF-partially

differentiated rat

PC12 cells

Oxidative stress and

growth factor

starvation

Yes

Independent of

MAO-B, induction

of transcription

102

Natural cell death

combined with serum

deprivation

Mesencephalic neurons Apoptosis and

oxidative stress

Yes

Serum-enhanced

49

103

104

Serum deprivation and

hypoxia

E1A-NR3 immortalized

retinal neurons

Oxidative stress and

hypoxia

Yesa 105

SIN-1 Neuroblastoma

SH-SY5Y cells

Oxidative stress Yes 77

Rasagiline Natural cell death

combined with serum

deprivation

Mesencephalic neurons Apoptosis and

oxidative stress

Yes

Serum-enhanced

49

103

SIN-1 Neuroblastoma

SH-SY5Y cells

Oxidative stress Yesb 75

76

77

6-OHDA, SIN-1 Neuroblastoma SH-SY5Y

cells

Oxidative stress Yesb

Independent of MAO-B

76

N-methyl(R)salsolinol,

6-OHDA, peroxynitrite

Neuroblastoma

SH-SY5Y cells

Oxidative stress Yesb,c

Independent of MAO-B

106

N-methyl(R)salsolinol SH-SY5Y cells

overexpressing Bcl-2

Oxidative stress Yesd 107

Serum and NGF

withdrawal

Partially differentiated

PC12 cells

Free radical-induced

apoptosis

Yese 48

Oxidative stress Independent of MAO-B

OGD NGF-differentiated

PC12 cells

Oxygen-glucose

deprivation

Yes

Independent of MAO-B

57

60

aRegulation of apoptosis-related gene expression.bStabilization of mitochondrial potential.cSuppresion of caspases and DNA fragmentation.dPrevention of nuclear accumulation of GAPDH.eIncreased gene expression of anti-apoptotic targets (proteins).

Challenges

84 BioEssays 26.1

to trigger cell death, are the most common, and less

disputable. They are the main models used for screening

and developing novel drugs for PD treatment, although the

animals injected with the neurotoxin MPTP do not exhibit Lewy

bodies, a hallmark of PD.

While a variety of neurological deficits are triggered by the

above-mentioned insults in in vivo animal models, mainly the

MPTP model partially mimics the PD syndrome. In rats fed

selegiline, the lifespan of the animals was increased, in parallel

with enhanced catecholaminergic activity in the brain.(64,65)

This phenomenon was unrelated to MAO-B inhibition and

was a topic of controversy. The neuroprotective effects of the

MAO-B inhibitors selegiline and rasagiline have also been

investigated in mice, gerbils and monkeys (Table 3), in brain-

ischemia, head-trauma, and under MPTP toxicity. As opposed

to the clearly neuroprotective effects of propargylamines in

in vitro cellular systems and in vivo experimental animal

models, clinical studies with selegiline have failed to distin-

guish between the contribution of MAO-B inhibition and

MAO-B induced neuroprotection to the amelioration of clinical

symptoms.(66) Although selegiline delayed the need for levo-

dopa, its effect on disease progression has not been proved.

As for rasagiline, a placebo (control) study revealed that

patients with early PD who were treated with rasagiline for

12 months, showed less decline in neurological symptoms

(UPDRS score) than patients whose rasagiline treatment

was delayed for 6 months. These clinical results cannot be

explained by the purely symptomatic effect of rasagiline,(67)

and they may represent a neuroprotective effect.

Insights into the neuroprotection mechanism:

lack of correlation between neuroprotection

and MAO-B inhibition

The neuroprotective effect of propargylamines, demonstrated

in different insult models, both in vitro and in vivo, suggests that

it is not due to MAO inhibition (Tables 2 and 3). The finding that

propargylamines induce neuroprotection in neuronal cell

cultures lacking MAO-B on the one hand, and the absence

of any neuroprotective effect by clorgyline, a MAO-A inhibitor,

on the other, also support the lack of correlation between

neuroprotection and MAO inhibition.(57) Furthermore, the

propargylamine concentration required for neuroprotection

both in vitro and in vivo is 1 mM, whereas the IC50 or Ki value for

in vitro MAO-B inhibition is in the range of 6–30 nM. As shown

in Tables 2 and 3, the major insults evoking neurotoxicity and,

in many cases, leading to neuronal apoptosis, are depolariza-

tion, calcium overload and oxidative stress. Conceptually,

therefore, the neuroprotective effect of propargylamines might

be grounded in the prevention of cell death or the activation of

pro-survival pathways minimizing cellular stress.

Rasagiline and selegiline exert their antioxidant effect

by upregulating the enzymes involved.(68) Selegiline(69) and

rasagiline,(70) promote free radical scavenging by activating

superoxide dismutase (SOD1 and SOD2) and catalase or by

increasing the SOD protein level,(71) upon chronic adminis-

tration to rats. In vitro experiments in our laboratory using cyclic

voltammetry further substantiated the antioxidant properties of

these compounds. Selegiline displays antioxidant effects

in vivo, which are probably not related to MAO-B inhibition

since selegiline reduced the free radical level at a pM con-

centrations too low to inhibit MAO-B activity.(72) The neuro-

protective effect of selegiline could also be mediated indirectly

via antagonistic modulation of the polyamine’s binding site on

the NMDA receptor, thereby reducing NMDA-receptor-gener-

ated excitotoxicity.(73) Moreover, rasagiline and selegiline act

directly as anti-apoptotic agents, probably by interfering with

the cellular apoptotic cascade (pro-survival) (Table 2). Several

studies have shown that treatment of cells with rasagiline

causes upregulation of putative anti-apoptotic and antioxidant

proteins such as Bcl-2, SOD, glutathione and BCLXL.(74) It has

been suggested that rasagiline protects cells from apoptosis

by stabilizing the mitochondrial membrane potential(75,76)

since such stabilization showed a causal relationship to inhibi-

tion of caspase activity and prevention of DNA fragmentation.

Table 3. Neuroprotective in vivo effects of MAO-B inhibitors selegiline and rasagiline

MAO-B Inhibitor Insult ModelNeurotoxicitymechanism

Neuroprotectivemechanism References

Selegiline Permanent or transient

occlusion of cerebral artery

Rat

Mouse

Gerbil

Focal or whole

brain ischemia

Yes 99

108

109

Selegiline Unilateral MPPþ neurotoxicity Sprague-Dawley rat Oxidative stress Yes 110

Independent of MAO-B

Rasagiline, Selegiline MPTP Monkey Oxidative stress Yes 111

Rasagiline Permanent middle cerebral

artery occlusion

Wistar rat Brain ischemia Yes Independent of MAO-B 62

Rasagiline Closed head injury Mouse Mechanical brain

trauma

Yes 48

Challenges

BioEssays 26.1 85

In any event, the propargyl group of rasagiline and selegiline

is essential to MAO-B inhibition and the anti-apoptotic

effect.(77)

Molecular neuroprotective mechanism

of MAO-B inhibitors

The precise target and details of the molecular mechanisms

involved in the neuroprotective effect exerted by propargy-

lamines are not known. A major obstacle in mechanistic

studies on selegiline is that the molecule is largely (/80%)

converted into amphetamines, some of which display neuro-

toxic activity.(78) Cumulative evidence suggests that brain

injury following amphetamine and methamphetamine admin-

istration is due to increased free radical formation and mito-

chondrial damage, leading to a failure in cellular energy

metabolism followed by secondary excitotoxicity-induced

seizures.(78) The neuroprotective activity of selegiline derives

from a minor metabolite, desmethylselegiline (DMS), which

was shown to be neuroprotective in vitro.(79) However, the

amphetamine-like major metabolites may cause cell da-

mage(80) and thus could reverse the potential beneficial

effects of DMS. Indeed, the addition of L-methamphetamine

to oxygen–glucose-deprived PC-12 cells substantially in-

creased cell death.(57) When both L-methamphetamine and

selegiline were added to PC-12 cells exposed to OGD, the

protective effect of the latter was reduced.(57) In contrast to

selegiline, whose neuroprotective activity derives from its

minor metabolite, DMS, that of rasagiline is mediated solely by

the parent drug(57,81) and its major metabolite, 1-R-amino-

indan, is not toxic.(57)

An interesting attempt to identify the target of the propargy-

lamine anti-apoptotic effect was made by Kragten et al. in

1998.(82) They synthesized a selegiline derivative (CGP 3466)

that is neuroprotective but does not inhibit MAO and is not

metabolized to amphetamines. Using affinity binding, labeling

and BIAcore technology (in which CGP 3466 was covalently

bound to the surface of a flow cell with a CM5 sensor chip(82)),

they detected in rat brain lysates four protein bands of

38,43,50 and 299 kDa, all putative endogeneous binding sites

for CGP 3466. One of these proteins (38 kDa) was identified

as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Using GAPDH antisense oligonucleotides, the investigators

established the importance of this enzyme in mediating the

neuroprotective effect of CGP 3466. Although the precise

role of GAPDH in neuronal apoptosis and in the neuroprotec-

tive mechanism of propargylamines awaits further investiga-

tion, GAPDH is the first selective target reported for these

compounds. GAPDH binds nicotinamide adenine dinucleotide

(NADþ)(82) at a site structurally similar to that of the flavin

adenine dinucleotide (FAD)-binding enzymes, to which MAO-

B belongs. Therefore, we propose that propargylamines bind

to GAPDH or other proteins with a tertiary structure similar to

that of the active site of MAO-B, thereby initiating an anti-

apoptotic process in the cells. It is very tempting to suggest

that the active site of these proteins possesses an FAD-

binding domain such as in MAO-B.(83) Recently, the structure

of human MAO-B was determined by crystalization at 3A

resolution. Electron density analysis revealed that pargyline, a

selegiline analog, binds covalently to N5 of the flavine

nucleotide.(83) Therefore, it is conceivable that the insertion

of propargylamines into the FAD pocket(83) of certain proteins

with a structure similar to that of MAO-B will be followed by

covalent binding to FAD or NAD, as in MAO-B, resulting in

neuroprotection.

Hydrogen peroxide (H2O2), which is a by-product of the

enzymatic reaction catalyzed by MAO-B, has been shown to

inhibit GAPDH. This inactivates the glycolytic and mitochon-

drial pathways of ADP phosphorylation, resulting in a drop in

intracellular ATP level and cell death. It was shown that MAO-

B inhibition by propargylamines confers neuroprotection

from H2O2-induced injury due to GAPDH damage.(84) It

was recently demonstrated that the level of GAPDH and its

nuclear accumulation increase in apoptotic models of serum

withdrawal, growth factor deprivation and MPPþ-induced

neurotoxicity, pointing to the enzyme’s essential role in apop-

tosis.(85,86) Furthermore, propargylamines prevented both

these phenomena, suggesting that this activity rather than

MAO-B inhibition, may contribute to the neuroprotective

effect.(85) Another important finding was the subcellular redu-

ction in GAPDH activity in Alzheimer’s disease and Hunting-

ton’s disease.(87) The investigators posited that intracellular

formation of complexes between GAPDH and proteins chara-

cteristic of neurodegenerative disorders, such as a-synuclein

in PD, b-amyloid in Alzheimer’s or huntingtin in Huntington’s,

may represent an emerging cellular phenotype of neurode-

generative disorders The major challenges in this field will be

to further clarify the apoptotic role of GAPDH, the pathophy-

siological meaning of GAPDH complexes, to identify novel

anti-apoptotic and/or pro-survival FAD- or NAD-binding

proteins and to delineate the interaction between these novel

enzymes and propargylamines in neuroprotection.

Neuroprotection strategy perspectives

In the present review, we have focused on the neuroprotection

of MAO-B inhibitors, their chemical characterization and their

cellular and molecular mechanisms of action. It is impossible in

so brief a review to cite all the earlier work recently summarized

by Magyar and Vizi.(88) We have included a few examples to

illustrate the newly emerging apoptotic concept of dopami-

nergic neuronal degeneration and have described selected

studies on the neuroprotective contribution of MAO-B inhi-

bitors such as propargylamines. The majority of in vitro and

in vivo PD studies attribute the neurodegeneration of SN

dopaminergic neurons to a long series of insults such as:

mitochondrial impairment and oxidative stress, Ca2þoverload,

iron metabolism, neurotoxins, excitotoxicity, neurotrophins,

Challenges

86 BioEssays 26.1

oxygen and glucose deprivation, depolarization and inflam-

mation. It is conceivable that the neuroprotective effect

of propargylamines is due to a shift in the neurotoxicity-

neuroprotection balance towards survival (Fig. 1). MAO-B,

GAPDH and other FAD/NAD-binding proteins represent

pivotal targets of the balance, as reflected by the neuropro-

tective effect of selegiline and rasagiline. Therefore, a pharma-

ceutical approach towards neuroprotection would be to

synthesize novel MAO-B/GAPDH inhibitors. Such inhibitors

should help to launch mechanistic studies aimed at elucidating

the molecular interactions of these compounds in anti-apo-

ptotic and/or survival signaling pathways. A crucial need in PD

drug development is the establishment of new in vitro and

in vivo models of the disease. Once these models are avail-

able, it will be necessary to identify neuronal dopaminergic

apoptotic pathways, as well as survival pathways. One of the

most remarkable changes recently observed in the nigro-

striatal region of the PD brain is the decreased level of neuro-

trophins supporting dopaminergic neuron survival, such

as brain-derived neurotrophic factor (BDNF), NGF(89) and

glial-derived neurotrophic factor (GDNF).(90) Therefore, re-

combinant neurotrophins or compounds increasing the local

production of neurotrophins,(91) and/or neurotrophin receptor-

agonists could prove beneficial in the treatment of this

disorder. Development of MAO-B inhibitors with neurotro-

phin-like activity may be a future strategy in PD therapy. This

possibility is supported by the recent finding that rasagiline

increases GDNF production and release by neuroblastoma

cells.(92) Despite the uncertainties and difficulties attendant

upon neuroprotective therapy in PD, clinical trials with innovat-

ive neuroprotective agents must proceed for neuroprotection

to become a reality.

In addition to the use of low molecular weight drugs for PD

treatment, modern therapeutic tactics focus on cell and gene

therapy. Cell therapy includes implantation in the SN of em-

bryonic or neuronal stem cells, which can synthesize and

release dopamine to correct the PD syndrome.(93) Gene

therapy in PD should aim both at supplementing the low SN

dopamine level by introducing the genes encoding dopamine-

synthesizing enzymes into non-dopaminergic cells in the

striatum, and at supporting the survival of dopaminergic

neurons by preventing apoptosis through the introduction of

genes blocking this process.(94)

We anticipate that, in coming years, the cellular pathophy-

siology of Parkinson’s disease will be clarified, allowing the

design of new drugs and novel therapeutic approaches.

Figure 1. Schematic depicting the balance be-

tween neurotoxicity and neuroprotection in Par-

kinson’s disease. Upper part: Normal population of

SN dopaminergic neurons, consisting mainly of

live neurons (blue) and a few dead neurons (red).

Enzymes (green) (targeted by propargylamines)

pivot the balance between cell survival (#) and cell

death ("). Insults shift the balance towards

neurotoxicity; rasagiline and selegiline tip the

balance towards neuroprotection.

Challenges

BioEssays 26.1 87

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Challenges

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