2001 Blum PIN PDF

Progress in Neurobiology 65 (2001) 135 172
www.elsevier.com/locate/pneurobio
Molecular pathways involved in the neurotoxicity of 6-OHDA,

dopamine and MPTP: contribution to the apoptotic theory in
Parkinsons disease
David Blum a,b,*, Sakina Torch a, Nathalie Lambeng c, Marie-France Nissou d,
Alim-Louis Benabid e, Remy Sadoul a, Jean-Marc Verna a
a
Unite Mixte INSERM/UJF E0108, Neurodegenerescence et plasticite, CHU Michallon, Pa6illon de Neurologie, BP217,
38043 Grenoble Cedex 9, France
b
Laboratoire de Neurophysiologie, Departement de Neurosciences, ULB-Erasme, 808 route de Lennik, CP601, 1070 Brussels, Belgium
c
CEA-Grenoble, TDC/DBMS, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France
d
Laboratoire dImmunologie, rue de Kimberley, 38130 Echirolles, France
e
INSERM U318, Neurobiologie Preclinique, CHU Michallon, Pa6 B, BP217, 38043 Grenoble Cedex 9, France
Received 5 December 2000; accepted 9 March 2001
Abstract
Parkinsons disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the
substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and
mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process. Recent histological studies performed on brains from PD patients suggest
that nigral cell death could be apoptotic. However, since post-mortem studies do not allow precise determination of the sequence
of events leading to this apoptotic cell death, the molecular pathways involved in this process have been essentially studied on
experimental models reproducing the human disease. These latter are created by using neurotoxic compounds such as
6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or dopamine (DA). Extensive study of
these models have shown that they mimick, in vitro and in vivo, the histological and/or the biochemical characteristics of PD and
thus help to define important cellular actors of cell death presumably critical for the nigral degeneration. This review reports
recent data concerning the biochemical and molecular apoptotic mechanisms underlying the experimental models of PD and
correlates them to the phenomena occurring in human disease. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Parkinsons disease; Apoptosis; 6-OHDA; MPTP; MPP+; Dopamine; Animal models
Abbre6iations: 6-OHDA, 6-hydroxydopamine; 8OHDG, 8-hydroxy-2%deoxy-guanosine; AIF, apoptosis-inducing factor; BIR, baculovirus
IAP-repeat; BRUCE, BIR-repeat-containing ubiquitin conjugating enzyme; CA, catecholamine; CARD, caspase recruitment domain; DA,
dopamine; DED, death effector domain; DISC, death-inducing signal complex; FADD, Fas-associated death domain; GSH, gluthation; IAP,
inhibitory apoptosis protein; ICE, interleukin-1b converting enzyme; iNOS, inducible nitric oxide synthase; JIP, JNK-interacting proteins; JNK,
c-jun N-terminal kinase; JNKK, JNK kinase; aKGDH, a-ketoglutarate deshydrogenase; l-dopa, l-dihydroxyphenylalanine; MAO, monoamine
oxidase; MAO-I, inhibitor of monoamine oxidase; MAP, mitogen-activated protein; MKK, MAP kinase kinase; MPDP+, 1-methyl-phenyl-dihydropyridinium; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NA, noradrenaline; NF-kB, nuclear
factor-kB; NGF, nerve growth factor; NMDA, N-methyl-d-aspartate; NO, nitric oxide; OXPHOS, oxidative phosphorylation system; Par-4,
prostate apoptosis response-4; PARP, poly-ADP-ribose polymerase; PD, Parkinsons disease; PTP, permeability transition pore; ROS, reactive
oxygen species; SAPK, stress-activated protein kinase; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; TNFa tumor necrosis
factor a; TUNEL, (TdT)-mediated dUTP nick-end labeling; VMAT, vesicular monoamine transporters.
* Corresponding author. Tel.: + 32-2-555-41-15; fax: + 32-2-555-41-21.
E-mail address: david.blum@ulb.ac.be (D. Blum).
0301-0082/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S0301-0082(01)00003-X
D. Blum et al. / Progress in Neurobiology 65 (2001) 135172
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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2. Parkinsons disease . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Clinical features. . . . . . . . . . . . . . . . . . . . . . . .
2.2. Nigral degeneration and etiology . . . . . . . . . . . . . .
2.2.1. Nigral degeneration . . . . . . . . . . . . . . . . . .
2.2.2. Etiology . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Vulnerability, oxidative stress and mitochondrial defects
2.3.1. Vulnerability . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Oxidative stress . . . . . . . . . . . . . . . . . . . .
2.3.3. Mitochondrial defects. . . . . . . . . . . . . . . . .
2.4. Involvement of apoptosis in nigral degeneration . . . . .
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3. Contribution of experimental neurotoxins to the study of apoptosis in PD. . . . . .

3.1. Mechanisms of 6-OHDA, dopamine and MPTP neurotoxicity . . . . . . . . . .
3.1.1. Neurotoxicity of 6-OHDA . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.1. General observations . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.2. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.3. Mitochondrial defects . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.4. Specificity of 6-OHDA toxicity . . . . . . . . . . . . . . . . . . .
3.1.2. Neurotoxicity of dopamine . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.2. Oxidative stress and mitochondrial defects. . . . . . . . . . . . .
3.1.2.3. Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3. Neurotoxicity of MPTP . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.2. Mitochondrial defects and oxidative stress . . . . . . . . . . . . .
3.2. Molecular pathways of cell death induced by 6-OHDA, dopamine and MPTP
3.2.1. Overview on the regulation of apoptosis. . . . . . . . . . . . . . . . . . .
3.2.1.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.2. A regulated mode of cell death . . . . . . . . . . . . . . . . . . .
3.2.1.3. Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.4. Bcl-2 family members . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.5. Inhibitor of apoptosis proteins (IAPs) . . . . . . . . . . . . . . .
3.2.2. Apoptosis induced by 6-OHDA. . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. Apoptosis induced by dopamine . . . . . . . . . . . . . . . . . . . . . . .
3.2.4. Apoptosis induced by MPTP . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5. Overview on dopaminergic neurotoxins and apoptosis . . . . . . . . . . .
3.2.5.1. NF-kB transcription factor. . . . . . . . . . . . . . . . . . . . . .
3.2.5.2. The JNK pathway . . . . . . . . . . . . . . . . . . . . . . . . . .
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4. Relevance of experimental data for human nigral degeneration . . . . . . . . . . . . . . . . .
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5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Parkinsons disease (PD) is a widespread neurodegenerative disorder. Even though the neurochemical defects
and the neuropathological characteristics of this disease
are well defined, its etiology is still unknown. Addition-
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ally, given the current absence of neuroprotective therapies, its treatment remains symptomatic. New advances
in molecular neuroscience recently led to the idea that
neuronal degeneration may be stopped and that specific
neuroprotective strategies could be possible. However,
this step will only be attained if the molecular pathways
of neuronal cell death involved in PD are better understood. The determination of molecular mechanisms of
neuronal death in human brain encounters methodological and biological problems. Therefore, in-vivo and
in-vitro models using experimental neurotoxins are essential since they allow the study of degenerating processes as well as new therapeutic approaches.
In this review, we present the recent advances in the
knowledge of the molecular mechanisms underlying PD
and the contribution of experimental models to this
field.
2. Parkinsons disease
Parkinsons disease, first described by James Parkinson in 1817 (Parkinson, 1817), is a neurodegenerative
disorder that can be defined as a syndrome associated
with specific neuropathological lesions.
2.1. Clinical features

Idiopathic PD is clinically defined by both the development of extrapyramidal motor disturbances, such as
bradykinesia, resting tremors, rigidity, and a later loss
of postural reflexes, and a good response to l-dihydroxyphenylalanine (l-dopa) treatment. When possible, a
histo-pathological post-mortem confirmation ensures
the diagnosis (Fahn, 1989; Calne et al., 1992). The
occurrence of this neurodegenerative disorder increases
with age and is considered to affect more than 2% of
the population after 65 years. The mean onset of the
disease is around 60 years, with a mean duration of 13
years (Hughes et al., 1993). Less frequently, PD may
have an onset below 40 years (Golbe, 1991).
2.2. Nigral degeneration and etiology

2.2.1. Nigral degeneration
The main pathological hallmark of idiopathic PD is a
progressive loss of neuromelanin-containing dopaminergic neurons from the substantia nigra pars compacta
(SNpc; about 5% of cell loss per year), a midbrain
structure. Dopaminergic cell loss is associated with the
presence of eosinophilic intraneuronal inclusions, called
Lewy bodies (Tretiakoff, 1919), composed of neurofilaments (Goldman et al., 1983; Pappolla, 1986) and
ubiquitin (Kuzuhara et al., 1988; Lowe et al., 1988;
Mayer et al., 1989). The presence of Lewy bodies is not
restricted to the central nervous system, since they are
also observed in the peripheral nervous system of
parkinsonian patients (Vanderhaeghen et al., 1970) and
in other degenerative disorders, such as Lewy body
disease and amyotrophic lateral sclerosis. In PD, the
loss of nigral neurons follows a specific pattern with a
more susceptible area located laterally in the ventral
137
part of the SNpc (Hirsch et al., 1988; Goto et al., 1989;

Fearnley and Lees, 1991). This results in severe dopamine (DA) depletion in the striatum (Ehringer and
Hornykiewicz, 1960), responsible for the motor symptoms (Lee et al., 1994), especially akinesia. Other lesions, such as degeneration of the dopaminergic ventral
tegmental area (Agid et al., 1990), the noradrenergic
locus cruelus (Greenfield and Bosanquet, 1953) and
the ascending cholinergic pathway from the Meynert
basalis nucleus (Candy et al., 1983), were also observed.
These non-nigral lesions lead to cognitive and psychological impairments, such as dementia, which is estimated to occur in around 30% of PD cases (Aarsland et
al., 1996).
A subclinical phase exists prior to the appearance of
parkinsonism. During this period, striatal compensatory phenomena (Agid et al., 1990; Anglade et al.,
1995), such as increased neuronal activity or sensitization of dopaminergic receptors (Agid et al., 1990),
occur. Therefore, PD is not clinically obvious before at
least 5070% of the dopaminergic neurons are lost. The
first clinical signs, are only observed when the degeneration is already strongly advanced. It follows that the
onset time point of the degeneration cannot be determined. Thus, the etiology of PD remains difficult to
establish exactly and, subsequently, no preventive clinical tools are yet available.
2.2.2. Etiology
Although the etiology of PD remains obscure, environmental or genetic factors might contribute to the
pathogenesis of PD.
The idea of environmental involvement comes from
the discovery of a toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or MPTP (Langston et al., 1983),
which produces selective nigral neuronal death in human and experimental models (see below and also
Gerlach and Riederer, 1996) and induces the motor
symptoms seen in PD (Langston et al., 1983). However,
although MPTP and analogues, such as tetrahydroisoquinolines (Niwa et al., 1987; Naoi et al., 1993) or other
exogenous/endogenous compounds (Gerlach and
Riederer, 1996), such as carbon monoxide (Ringel and
Klawans, 1972; Choi and Cheon, 1999), b-carbolines
(Collins and Neafsey, 1985) or rotenone, a common
pesticide (Betarbet et al., 2000), can produce dopaminergic lesions, none have been clearly shown to be
responsible for the majority of PD cases. It is possible,
however, that many exogenous/endogenous compounds
can induce PD when a susceptible background exists.
Analysis of families with dominant inheritance of the
disease and twin studies support the idea of a genetic
contribution to the etiology of PD. Five to 10% of PD
cases are familial forms. A few cases of autosomal
dominant transmission exist, and certain mutations
were recently identified. In 1997, in ItalianAmerican
138
kindred and Greek families, a gene responsible for

familial parkinsonism was located on the chromosome
4q21q23 (Polymeropoulos et al., 1996). Shortly thereafter, the mutation was identified as an Ala53Thr substitution in the region encoding for a protein known as
a-synuclein (Polymeropoulos et al., 1997). A second
mutation resulting in an Ala30Pro substitution was
later identified (Kruger et al., 1998). Interestingly, it has
been shown that a-synuclein is one of the components
of Lewy bodies (Spillantini et al., 1997). Mice in which
the a-synuclein gene is inactivated present dysfunction
of the nigro-striatal system (Abeliovich et al., 2000).
Expression of human a-synuclein in Drosophila nerve
cells leads to an age-dependent loss of dopaminergic
neurons with the presence of aggregates resembling
Lewy bodies (Feany and Bender, 2000). Similar results
were obtained in flies expressing mutated a-synuclein
genes (A53T or A30P; Feany and Bender, 2000). In
addition, expression of human wild-type a-synuclein in
transgenic mice leads to the appearance of neuronal
inclusions, a deficit in the dopaminergic system and
motor impairments of the animals (Masliah et al.,
2000), and overexpression of wild-type or mutated form
of a-synuclein renders dopaminergic neurons more vulnerable to neurotoxic insults (Zhou et al., 2000). Therefore, this protein and its mutated forms may contribute
to nigral degeneration even if a recent study suggests
that nigral cell death and a-synuclein induction may be
uncoupled (Kholodilov et al., 1999).
A mutation of the parkin gene, located on the locus
6q25.2q27, was also reported in an autosomal case of
juvenile parkinsonism (Kitada et al., 1998), and a mutation was described on the chromosome 2p13 in families
of European origin (Gasser et al., 1998). Mitochondrial
DNA mutations may also contribute to PD triggering
(Schapira, 1998; see also Section 2.3). However, these
mutations have been described in a very small percentage of PD cases characterized by early onset and, at
least for of the a-synuclein mutation, have not been
identified in sporadic parkinsonism (Vaughan et al.,
1998). Furthermore, although positron emission tomography has suggested a possible concordance among
mono- and di-zygotic twins (Burn et al., 1992), genetic
studies performed on monozygotic twins showed that
PD cannot be considered as an inherited disease in
older onset cases (Marttila et al., 1988; Tanner et al.,
1999) The overwhelming majority of PD cases is sporadic and lacks family history. Contribution of genetic
mutations in pathogenesis of PD cannot today explain
sporadic and late-onset cases (Tanner et al., 1999).
2.3. Vulnerability, oxidati6e stress and mitochondrial

defects
Although the etiology of parkinsonian degeneration
is still ill defined, there is growing interest in the phe-
nomena underlying the degeneration process. In particular, oxidative stress has often been put forward as one
of the major causes of the nigral degeneration (see
Table 1). Its involvement can be established at two
different levels. Indeed, several studies tend to demonstrate that dopaminergic neurons of SNpc are highly
vulnerable to reactive oxygen species, a finding particularly relevant since strong oxidative stress seems to
occur in the degenerating substantia nigra during PD.
2.3.1. Vulnerability
Nigral dopaminergic neurons are particularly vulnerable to degeneration (for reviews, see Hirsch et al.,
1997; Uhl, 1998). For example, basal reactive oxygen
species (ROS) levels are high in dopaminergic neurons.
Indeed, normal enzymatic catabolism of DA induces
the formation of hydrogen peroxide via monoamine
oxidase activity. Moreover, nonenzymatic auto-oxidation of dopamine produces the formation of neuromelanin that potentiates hydroxyl radical formation when
it combines with iron (Fahn and Cohen, 1992; Jellinger
et al., 1992; Jenner et al., 1992). Interestingly, the most
affected nigral neurons in PD were suggested to be
those that contain neuromelanin (Hirsch et al., 1988).
Finally, it was shown that, in the vulnerable part of
SNpc, there is a strong decrease in cells synthesizing
glutathion peroxidase (Damier et al., 1993) leading to a
defect in the basal detoxification capabilities. All these
data underline the predisposition of nigral cells to
oxidative stress and degeneration.
Table 1
Oxidative stress in Parkinsons disease: predisposition and occurrence
Vulnerability factors to oxidati6e Basal dopamine metabolism
stress in normal SNpc
MAO-induced H2O2 generation
Autooxidation-induced ROS
Neuromelanin formation
Oxidati6e stress in SNpc of PD
patients
Iron increase
Increased iron levels
Rise in lactoferrin receptor
expression
Possible decrease in ferritin
Loss in detoxification capacity
Drop in GSH levels
Drop in GSH-peroxidase
expression
Drop in catalase expression
Peroxidation signs
Increase in polyunsaturated
fatty acids
Increase in thiobarbituric acid
Increase in 8-hydroxy-2,
deoxyguanosine
Increase in inducible nitric
oxide synthase activity
139
2.3.2. Oxidati6e stress

This nigral vulnerability fits well with the strong
oxidative stress observed in PD (Table 1). Two main
biochemical phenomena leading to increased ROS generation have been identified within the degenerating
substantia nigra: (1) an increased iron level and (2) a
reduced antioxidant defence level.
In parkinsonian SNpc and at the levels of neuromelanin granules of DA neurons, there is an increase of
iron levels (Dexter et al., 1989a; Hirsch et al., 1991;
Good et al., 1992) possibly related to a (controversial)
decrease in cellular ferritin (Riederer et al., 1989; Dexter et al., 1990; Levay and Bodell, 1993) and a rise in
lactoferrin receptor expression (Leveugle et al., 1996).
Higher iron could lead to an increase in ROS generation due to the presence of neuromelanin. Furthermore,
the ratio between reduced and oxidized glutathione
(GSH/GSSG) is decreased during degeneration thus
enhancing the formation of toxic hydroxyl radicals
(Sofic et al., 1992; Sian et al., 1994). This may represent
one of the earliest biochemical defects in PD. The
impairement of GSH-dependent detoxification may be
due to increased dopamine turnover that could itself
increase basal production of hydrogen peroxide and
then deplete GSH stocks (Riederer et al., 1989). Additionally, relative GSH depletion is accompanied by a
drop in catalase (Ambani et al., 1975) and GSH-peroxidase expression (Kish et al., 1985). All these above
phenomena give increase of the ROS levels and lead to
damage of cellular macromolecules and their subsequent peroxidation. Indeed, PD brains show a decrease
in polyunsaturated fatty acid levels and increased levels
of thiobarbituric acid-reactive compounds (an indicator
or lipid oxidation) and the presence of 8-hydroxy2%deoxy-guanosine (8-OHDG) (Dexter et al., 1989b;
Fahn and Cohen, 1992; Sanchez-Ramos et al., 1994).
Another source of free radicals may arise from activated microglial cells present in degenerating SNpc
(McGeer et al., 1988a,b), which produce nitric oxide
(NO) and cytokines. Indeed, expression of the inducible
nitric oxide synthase (iNOS) is elevated in PD brain
(Hunot et al., 1996). This leads to elevation of NO
levels and to generation of the harmful peroxynitrite
radical. Moreover, glial cell activation could also increase cytokine levels (e.g. TNFa) and ROS or directly
activate the cell death pathway (Boka et al., 1994; Mogi
et al., 1994; Hunot et al., 1997). Finally, high ROS
levels could induce secondary excitotoxicity, raising free
cellular calcium in turn increasing intracellular NO
levels.
cleotide coenzyme Q reductase) of the mitochondrial

respiratory chain. Complex I is located in the inner
mitochondrial membrane and forms a part of the oxidative phosphorylation system (OXPHOS) responsible
for the production of cellular ATP. Decreases in the
activity and immunoreactivity of the reduced form of
the complex I were observed in the SNpc of PD patients (Mizuno et al., 1989; Schapira et al., 1989, 1990;
Hattori et al., 1991). The reasons for this defect are still
unclear, but several studies indicate an alteration in the
mitochondrial genome that encodes several proteins of
the OXPHOS system (Ozawa et al., 1990; Swerdlow et
al., 1996; Schapira, 1998). The mutation rate of mitochondrial DNA (mtDNA) is 10 20 times higher than
for nuclear DNA. This may result from the proximity
of mtDNA with the ROS-generating OXPHOS system,
from oxidative stress (Richter et al., 1988; Ozawa,
1997a) or from the lack of protective histone-like
proteins and the poor DNA repair system in the mitochondria (Clayton et al., 1974; Shadel and Clayton,
1997). A contribution of mtDNA to PD pathogenesis is
also suggested by the recent demonstration of a matrilineal pattern of inheritance in some PD cases (Wooten
et al., 1997; Swerdlow et al., 1998). Therefore, the
mitochondrial complex I defect in PD may involve
mtDNA mutations acting as a susceptibility factor for
the development of PD. Alternatively, the discovery of
MPTP and the potential neurotoxicity of exogenous
rotenone (Betarbet et al., 2000) suggest that environmental factors could also impair mitochondrial function in humans (Langston et al., 1983; Nicklas et al.,
1985; Mizuno et al., 1987a,b). Whatever the nature of
the mitochondrial defect, it may lead finally to decreased ATP levels and impairment of proton pumping
resulting in a decrease in the mitochondrial membrane
potential, which is the first step towards apoptosis
(Desagher and Martinou, 2000).
In conclusion, although PD etiology is still unknown,
this disease could be explained by a genetic susceptibility to environmental or endogenous agents leading to
oxidative damage in a neuronal population that is
already under oxidative stress (Hirsch et al., 1997).
However, despite the many recent advances in the field
of symptomatic treatment of PD, such as deep brain
stimulations (Limousin-Dowsey et al., 1999), no neuroprotective therapy significantly stops or delays the progression of the neurodegeneration. This type of
treatment will only emerge with a better knowledge of
the course and the molecular processes underlying nigral degeneration.
2.3.3. Mitochondrial defects

Impairment of mitochondrial activity also contributes to both ROS generation and nigral cell loss.
The main mitochondrial defect observed in degenerating PD concerns complex I (nicotinamide adenine dinu-
2.4. In6ol6ement of apoptosis in nigral degeneration

Apoptosis was defined as a physiological and regulated mode of cell death. Cells undergoing apoptotic
death exhibit several morphological characteristics,
140
such as chromatin condensation, nuclear fragmentation, blebbing of plasma membrane, cell shrinkage,
cytoplasmic condensation and apoptotic body formation. During this process, organelles, and especially
mitochondria, remain morphologically intact until late
stages. Apoptotic cells are phagocytosed by
macrophages or microglial cells thereby preventing inflammation that occurs during necrosis. This may be a
short process in vivo (Kerr et al., 1972; Clarke, 1990).
Conversely, necrosis is characterised by cytoplasm and
nuclear swelling, loss of plasma membrane integrity and
release of cellular content hastening the immune response (Clarke, 1990).
Pathogenesis of PD involves strong oxidative stress,
reduced antioxidant levels and mitochondrial defects all
known to induce apoptosis in several cellular systems.
In particular, free radicals and GSH depletion have
been shown to trigger active cell death in neurons
(Merad-Boudia et al., 1998). Recently, a decrease in
mitochondrial membrane potential was suggested to be
one of the main phenomena leading to the apoptotic
process (Jacotot et al., 1999; Martinou, 1999).
Since 1996, several studies have pointed out the
potential involvement of apoptosis in Parkinsons disease. The first study was performed by Mochizuki et al.
(1996) using the 3%-end terminal staining of DNA
(TUNEL method) allowing in-situ observation of DNA
fragmentation, characteristic of apoptosis, in the substantia nigra of PD patients (Gavrieli et al., 1992). The
presence of apoptosis was suggested in SNpc of 4/7 late
onset PD, although younger patients did not show any
TUNEL positive staining. Using a similar method,
Kingsbury et al. (1998) observed DNA-end labelling in
10 idiopathic PD cases. The mean number of nigral
apoptotic neurons was estimated to be around 2% with
some observations reaching 12% in seven of 10 postmortem samples. Tatton et al. (1998), using double
staining combining DNA-end labelling and YOYO-1, a
DNA intercalant, clearly observed that DNA-end labelling positive cells also present chromatin condensation. Several groups also observed that nuclear
end-labelling in the degenerating substantia nigra was
accompanied by morphological signs of apoptosis, such
as chromatin clumping, irregular nuclear morphology,
presence of apoptotic bodies and their phagocytosis by
microglia (Tompkins et al., 1997; Kingsbury et al.,
1998; Tatton et al., 1998). Electron microscopy of
degenerating DA neurons was performed by Anglade et
al. (1997). These authors showed that 6% of the observed melanized neurons exhibit ultrastructural hallmarks of apoptosis, such as chromatin condensation,
convolution of nuclear envelope, cell shrinkage and
presence of apoptotic bodies. Some nigral cells dying by
another type of cell death (autophagic type) were also
described. Conversely, few necrotic cells were observed
and corresponded to glia. Interestingly, Anglade et al.
(1997) observed that apoptotic cells never contained

Lewy bodies, possibly extruded from dying cells. The
presence of apoptotic cells in PD brain was also described outside the SNpc, in other regions such as pond
or superior colliculus (Kingsbury et al., 1998) and in
nigral glial cells (Mochizuki et al., 1996; Anglade et al.,
1997; Ko sel et al., 1997; Kingsbury et al., 1998), possibly due to either glial apoptosis or uptake of fragmented DNA. However, other studies questioned the
presence of apoptotic cells in parkinsonian SNpc. Indeed, neither the studies of Dragunow et al. (1995),
Ko sel et al. (1997), Banati et al. (1998), Wu llner et al.
(1999) nor that of Jellinger (2000) were able to demonstrate the presence of apoptosis in degenerating substantia nigra by either DNA end-labelling methods or
morphological observations. In the study of Ko sel et al.
(1997), none of the neurons observed, including those
with Lewy bodies, demonstrated morphological signs of
apoptosis in 22 PD cases. Banati et al. (1998) could not
find any DNA fragmentation in 10 PD cases whatever
the young or old disease onset. Therefore, the presence
of apoptosis in nigral PD remains controversial.
Several parameters could explain these discrepancies.
The assessment of apoptosis using in-situ end-labelling
has to be considered with caution since it was previously suggested that this method can also stain nonapoptotic cells (Charriaut-Marlangue and Ben Ari, 1995;
Lassmann et al., 1995). Therefore, only morphological
studies establish with certainty whether SNpc degeneration in PD is apoptotic or not. Furthermore, several
factors may also influence the occurrence of apoptosis
in post-mortem studies. The time lag between the death
of the patients and the fixation of the tissue could
influence the appearance of apoptotic cells (Banati et
al., 1998) even though some authors suggest that this
parameter is not determinant (Mochizuki et al., 1996;
Tompkins et al., 1997; Kingsbury et al., 1998). Additionally, the use of formalin or paraffin embedded
tissue may be a critical parameter. The appearance of
apoptosis in brain may also depend on agonal conditions of patients. Indeed, it was suggested that antemortem hypoxia may increase, at least, DNA-end
labelling staining (Kingsbury et al., 1998). Here again,
other authors suggest that agonal conditions do not
influence the rate of apoptosis (Mochizuki et al., 1996).
Beyond these parameters, the observed discrepancies
could also be directly related to the degenerating process. Indeed, at present, it is not clear whether the
degenerating process in PD involves an event or a
process (Calne, 1994). In the former hypothesis, as
well as in the one-hit model for inherited degeneration
described by Clarke et al. (2000), it could be assumed
that an environmental or an endogenous factor leads to
an early and great loss of dopaminergic neurons followed by normal ageing. In this case, given that apoptosis is a short process in vivo, it is unlikely that a
significant number of apoptotic neurons can be observed at the time of histopathological observations,
since the pathological event leading to the disappearance of dopaminergic neurons may occur far earlier
than the onset of the disease. In the process hypothesis, the neurodegeneration may last several decades as
an active process throughout the patients life and
could be considered as accelerating ageing. In this case,
the rate of cell death could be so low that, at a given
time, only a small number of cells can be observable.
Following the process hypothesis, Wu llner et al.
(1999) made an attempt to calculate the probability of
encountering apoptotic neurons in brains from PD
patients. If one accepts that the neuronal rate of cell
death in dopaminergic neurons lies between 0.5 and
0.7% per year during normal ageing (McGeer et al.,
1988a,b; Fearnley and Lees, 1991) and that the number
of dopaminergic neurons is around 300000 400000 at
the beginning of degeneration, one expects five to 10
dying neurons every day. In PD brains, the estimated
rate of cell death could be around 5% per year (McGeer
et al., 1988a). Therefore, at best, a maximum of 100
apoptotic neurons could be detected in the SNpc of PD
patients. This estimation is difficult to reconcile with
the high number (5 6%) of apoptotic cells counted by
Anglade et al. (1997) and Tompkins et al. (1997).
Furthermore, in vivo, apoptosis is a fast process occurring within 12 h.
All these methodological and biological problems
may be insufficient to explain such high levels. A high
rate of apoptotic cell death could also occur in the
SNpc in patients with a relatively late and rapid loss of
dopaminergic neurons, taking place after several
decades of normal ageing and induced by an undefined
toxic event. This cannot, however, explain all PD cases.
Other possibilities to explain Anglades and Tompinks
results could be that the clearance rate of phagocytosed
apoptotic cells or the fragmented DNA is very low,
allowing their histological detection a long time after
the onset of cell death, or that the cellular mechanisms
that normally follow the DNA fragmentation are impaired. Therefore, the above data cannot be taken as
proof of the occurrence of apoptosis in the degenerating SNpc. The final proof of active cell death in PD
awaits the demonstration that compounds that inhibit
this phenomenon are capable of delaying or even blocking the course of the disease.
Considering the difficulty of interpreting data obtained from human samples, animal models using neurotoxic compounds that mimic dopaminergic
degeneration seen in PD may be useful to study the
type of cell death occurring in human SNpc. In the
following, we report present knowledge concerning the
toxicity and the molecular mechanisms induced by
three neurotoxic compounds: 6-hydroxydopamine (6OHDA), dopamine (DA) and 1-methyl-4-phenyl-
141
1,2,3,6-tetrahydropyridine (MPTP). These findings are

finally compared to observations performed on postmortem samples of PD patients.
3. Contribution of experimental neurotoxins to the

study of apoptosis in PD
3.1. Mechanisms of 6 -OHDA, dopamine and MPTP

neurotoxicity
3.1.1. Neurotoxicity of 6 -OHDA
3.1.1.1. General obser6ations. 6-Hydroxydopamine is
one of the most common neurotoxins used to experimentally model nigral degeneration in vitro as well as
in vivo. 6-OHDA is a hydroxylated analogue of the
natural dopamine neurotransmitter. It was originally
isolated by Senoh in 1959 (Senoh and Witkop, 1959a,b;
Senoh et al., 1959a,b). Its biological effects were first
demonstrated by Porter et al. (1963, 1965) and Stone et
al. (1963), who showed that 6-OHDA induces noradrenaline depletion in the autonomic nervous system of
the heart. Shortly thereafter, several studies demonstrated its ability to destroy nerve cell endings of sympathetic neurons (Thoenen et al., 1967; Tranzer and
Thoenen, 1967, 1968; Thoenen and Tranzer, 1968).
Such results can be obtained after systemic injection of
the neurotoxin. However, given that 6-OHDA is unable
to cross the bloodbrain barrier, production of central
neuronal lesions can be achieved only after direct intracerebral administration (intracisternally, intraventricularly or directly into the brain parenchyma).
Application of 6-OHDA into lateral ventricles was
previously shown to produce central catecholamine depletion (Ungerstedt, 1968; Bloom et al., 1969; Uretsky
and Iversen, 1970). In experimental models of PD,
6-OHDA is preferentially injected into the striatum, the
substantia nigra or the ascending medial forebrain bundle (especially in rats), destroying nigral dopaminergic
neurons and depleting the striatum of DA neurotransmitter, thus reproducing the physiopathological features responsible for motor impairments in PD.
Unilateral 6-OHDA-induced SNpc degeneration produces an asymetric and quantifiable motor behaviour
after unilateral lesion induced by systemic administration of either DA receptor agonists, l-dopa or dopamine releasing drugs (amphetamine; Hefti et al.,
1980). This allows easy and reliable control of the
extent of the lesion and the potential benefits of therapeutic treatments.
Interestingly, some evidence exists that 6-OHDA can
be considered as a physiological endogenous neurotoxin, as previously reported for the tetrahydroisoquinoline derivate N-methyl(R)salsolinol (Maruyama et
al., 1992; Deng et al., 1995; Naoi et al., 1996; Naoi and
142
Fig. 1. Hypothetical mechanism of 6-OHDA toxicity. 6-OHDA could induce catecholaminergic cell death by three main mechanisms: reactive
oxygen species generated by intra or extracellular auto-oxidation, hydrogen peroxide formation induced by MAO activity or direct inhibition of
the mitochondrial respiratory chain. These events lead to strong oxidative stress amplified by cytoplasmic free calcium and to a decrease in cellular
ATP avaibility, both leading to cell death.
Maruyama, 1999). Indeed, several studies reported the

presence of 6-OHDA in both rat (Senoh and Witkop,
1959a,b; Senoh et al., 1959a,b) and human brain (Curtius et al., 1974) as well as in urine of l-dopa-treated PD
patients (Andrew et al., 1993). As described above
(Section 2.3), nigral dopaminergic neurons contain significant levels of dopamine, hydrogen peroxide and free
iron. A non-enzymatic reaction between these elements
may possibly lead to 6-OHDA formation (Slivka and
Cohen, 1985; Jellinger et al., 1995; Linert et al., 1996).
This dopamine oxidation can occur at concentrations
as low as 50 mM and induces the formation of both
6-OHDA-related quinones and neurotoxic alkaloid 7dihydroxy 1,2,3,4 tetrahydroisoquinoline (Napolitano
et al., 1999).
In-situ 6-OHDA generation can be favored by other
factors. Melanin can increase free iron levels by displacing it from ferritin, thus amplifying the chemical reaction between iron and dopamine. In the presence of
nitrite ions, dopamine can be oxidized by a variety of
hydrogen peroxide-dependent systems to give small
amounts of 6-OHDA and 6-nitrodopamine (Palumbo

et al., 1999). Manganese, an essential trace element, can
also stimulate dopamine auto-oxidation and 6-OHDA
generation (Garner and Nachtman, 1989). The relevance of 6-OHDA to mimic PD models is reinforced by
the fact that ROS generation and mitochondrial defects
are induced by the neurotoxin (Fig. 1).
3.1.1.2. Oxidati6e stress. 6-OHDA was suggested to

induce nigrostriatal dopaminergic lesions via the generation of hydrogen peroxide and derived hydroxyl radicals (Heikkila and Cohen, 1971). Several studies have
confirmed that 6-OHDA produces oxidative stress in
vivo (Permual et al., 1989, 1992; Kumar et al., 1995) as
well as in vitro (Tiffany-Castiglioni et al., 1982; Decker
et al., 1993; Abad et al., 1995; Choi et al., 1999a;
Lotharius et al., 1999). This explains the protective
effects afforded by antioxidants against 6-OHDA toxicity (see Fig. 2; Tiffany-Castiglioni et al., 1982; Davison
et al., 1986; Cadet et al., 1989; Yamada et al., 1997;
Mayo et al., 1998; Blum et al., 2000) and the observa-
143
Fig. 2. 6-OHDA induces oxidative stress in PC12 cells. Effect of catalase (A), superoxide dismutase (SOD; B), glutathion (GSH; C) and
N-acetyl-cystein (NAC; D) on 6-OHDA-induced PC12 cell death. Twenty-four hours after seeding, cells were concomitantly incubated with both
100 mM 6-OHDA and anti-oxidants. Cell viability was determined by Alamar Blue assay 24 h after 6-OHDA treatment. Results are given as a
percentage of the untreated control and represent the mean 9 S.D. of triplicate determinations from a representative experiment. These
experiments show that catalase, GSH and NAC but not SOD (Fig. 1B) were able to significantly block PC12 cell death. (E) Effect of GSH and
NAC on 6-OHDA auto-oxidation rate. Absorbance at 490 nm was determined for 6-OHDA dissolved in culture medium with or without either
GSH or NAC. Each value represents the mean 9 S.D. of six measurements from a representative experiment and is expressed as the percentage
of maximal 6-OHDA auto-oxidation measured after 24 h. We show that, when added to the culture medium, 6-OHDA was completely
auto-oxidized within 3.5 h (half auto-oxidation time of approximatively 10 min). In contrast, in the presence of GSH, half auto-oxidation was
reached within 9.5 h, and complete auto-oxidation was achieved after 24 h. In the presence of NAC, the 6-OHDA degradation was not maximal,
even after a 24 h incubation. This suggest that protective effects of GSH and NAC are related to their ability to inhibit 6-OHDA oxidation. (F)
Toxic effect of high 6-OHDA concentrations on PC12, C6 and NIH3T3 cell survival. Twenty-four hours after seeding, PC12, C6 and NIH3T3
cells were incubated for 24 h together with 250 or 500 mM 6-OHDA. Results show that the non-catecholaminergic cells (C6 glioma and NIH3T3
fibroblastic cells) are less sensitive to 6-OHDA than PC12 cells, suggesting that, at high concentrations, 6-OHDA specificity is not restrained to
catecholaminergic cells. These results are reprinted from Blum et al. (2000). Extracellular toxicity of 6-hydroxydopamine on PC12 cells.
Neuroscience Letters 283, 193 196 with permission from Elsevier Science.
144
tion of decreased cell death in transgenic mice overexpressing superoxide dismutase and glutathion peroxidase (Asanuma et al., 1998; Bensadoun et al., 1998).
The generation of ROS may arise from two distinct
mechanisms, namely deamination by monoamine oxidase or auto-oxidation, and is presumably initiated
and/or amplified by iron via the Fenton reaction1.
6-OHDA, like DA, is a substrate for monoamine oxidase (MAO) (Breese and Traylor, 1971; Karoum et al.,
1993). This enzymatic reaction gives rise to hydrogen
peroxide. An involvement of MAO was suggested following the observation that selegiline, an inhibitor of
MAO (MAO-I), prevents 6-OHDA toxicity (Knoll,
1986; Salonen et al., 1996), even if the protective effects
of MAO-I are subject to caution (Wu et al., 1996).
However, several lines of evidence suggest that ROSmediated 6-OHDA toxicity seems, rather, due to the
generation of hydrogen peroxide and hydroxyl radicals
via a non-enzymatic self-auto-oxidation process. Indeed, under physiological conditions, 6-OHDA undergoes a rapid and nonenzymatic auto-oxidation
(Heikkila and Cohen, 1972; Seitz et al., 2000; SotoOtero et al., 2000) shown to generate several toxic
species including quinones (Saner and Thoenen, 1971),
superoxide radicals, hydrogen peroxide and the highly
reactive hydroxyl radical (Cohen and Heikkila, 1974).
In keeping with this mechanism, the cytotoxicity of
catecholamines, especially 6-OHDA, directly correlates
with their rates of auto-oxidation (see Fig. 2; Graham
et al., 1978; Soto-Otero et al., 2000).
The formation of ROS generated by MAO and autooxidation may be amplified by iron that catalyzes a
Fenton reaction. Indeed, iron levels are increased in
SNpc and striatum after 6-hydroxydopamine injection
(Hall et al., 1992; He et al., 1996; Oestreicher et al.,
1994). The contribution of iron in 6-OHDA toxicity is
also suggested by studies showing that the 6-OHDA-induced deleterious effects are prevented by iron chelating
agents (Ben Shachar et al., 1991; Borisenko et al., 2000)
and that direct injection of iron into the SNpc produces
similar neurotoxic effects (Sengstock et al., 1992, 1994).
In summary, the combined occurrence of monoamine
oxidase activity, auto-oxidation and elevation in iron
levels is responsible for the strong ROS production
following 6-OHDA treatment.
The subsequent oxidative stress reduces cellular antioxidative capabilities (Kumar et al., 1995), impairs
intracellular redox potential regulation (Shiraga et al.,
1993) and causes lipid peroxidation as demonstrated by
the decrease in phospholipid levels and the increase in
malonaldialdehyde content (Kumar et al., 1995). In
addition, 6-OHDA-generated ROS cause: (1) DNA
1
H2O2 + Fe2 + OH + OH + Fe3 + .
strand break, characterized by an elevation in 8-hydroxy-2%-deoxyguanosine levels (8-OHDG), particularly

via an activation of the poly-ADP-ribose polymerase
(PARP; Bruchelt et al., 1991); (2) mutations (Gee et al.,
1992); (3) a disorganization of the cytoskeleton (Davison et al., 1986); and (4) an impairment of glucose and
a-aminoisobutyric acid uptake (Vroegop et al., 1995).
3.1.1.3. Mitochondrial defects. Several lines of evidence

suggest that 6-OHDA impairs mitochondrial function
by various mechanisms. The studies by Glinka and
Youdim (1995) and Glinka et al. (1996, 1998) suggest
that 6-OHDA directly targets the mitochondrial respiratory chain. More precisely, 6-OHDA is able to
interact with and inhibit complex I in isolated brain
mitochondria (Glinka and Youdim, 1995). This effect
appears to be fully reversible and independent of ROS
since neither antioxidants nor iron chelators can alleviate mitochondrial inhibition (Glinka et al., 1996). Nevertheless, this conclusion was not drawn from whole
cell experiments (Wu et al., 1996; Storch et al., 2000),
and 6-OHDA toxicity against SY5Y neuroblastoma
cells was not accompanied by a reduction of ATP
production, ATP/ADP ratio or NAD+ cellular content
(Storch et al., 2000). These results then suggest that, on
entire cells, inhibition of mitochondrial respiration is
not the main mechanism underlying 6-OHDA toxicity.
However, 6-OHDA was shown to induce a ROS-related collapse in mitochondrial membrane potential
(Lotharius et al., 1999) and to be a strong uncoupler of
oxidative phosphorylation, like dinitrophenol (Wagner
and Trendelenburg, 1971). Together, these data implicate mitochondrial impairments in 6-OHDA toxicity,
even if they have to be further elucidated. These alterations could lead to not only alterations of membrane
potential but also, in vivo, to a defect in dopamine
vesiculation, which may increase cytoplasmic levels of
dopamine, both events leading to cell death (see also
Section 3.1.2; see also Uhl, 1998).
3.1.1.4. Specificity of 6 -OHDA toxicity. Numerous
studies have suggested that 6-OHDA specificity towards catecholaminergic cells was due to its uptake by
specific transporters. This assumption is based: (1) on
the chemical analogy between 6-OHDA and dopamine
(DA) and noradrenalin (NA), (2) on the specific accumulation of 6-OHDA into catecholaminergic neurons
(Jonsson and Sachs, 1970, 1971; Ljungdahl et al., 1971),
and (3) on the inhibitory action of catecholamine (CA)
uptake inhibitors on 6-OHDA-induced toxicity (Breese
and Traylor, 1970). Moreover, in agreement with the
above statement, it was also demonstrated that 6OHDA toxicity upon megacaryocyte progenitor cells
directly depends on the activation of catecholamine
transporters (Gordon et al., 1991). This apparently
simple mechanism is questioned by other studies show-
ing that 6-OHDA does not need to enter into the CA

cells to exert its deleterious effects. Thus, in mesencephalic cultures, 6-OHDA toxicity is not selective for
dopaminergic neurons (Lotharius et al., 1999), and
several cell types devoid of CA transporters (endothelial
cells, C6 glioma cells, NIH3T3, CHO, cortical and
cerebellar neurons) are damaged by 6-OHDA (Fig. 2;
Abad et al., 1995; Offen et al., 1998; Dodel et al., 1999;
Blum et al., 2000; Seitz et al., 2000). Potent blockers of
NA or DA uptake (desipramine, imipamine, cocaine,
GBR12909) did not prevent cytotoxicity (Abad et al.,
1995; Storch et al., 2000). Additionally, 6-OHDA, at
concentrations as high as 600 mM, does not accumulate
in PC12 cells (Decker et al., 1993), and catalase, to
which cells are normally impermeable, strongly protects
against 6-OHDA (Abad et al., 1995; Yamada et al.,
1997).
The above data thus question the specificity of 6OHDA, especially when dealing with in-vivo intracerebral lesions. Several elements could explain such
conflicting data. First, the results against the specificity
were essentially obtained using cell cultures, but the
density of catecholaminergic uptake sites can differ
between normal brain cells and cell lines (Decker et al.,
1993). Secondly, as previously suggested, even if 6OHDA does not actively enter cells, the interactions
between this compound and the catecholamine transporters can contribute to its toxicity (Decker et al.,
1993). Lastly, the observations of a non-specific in-vitro
toxicity of 6-OHDA may be related to the fact that the
doses used for 6-OHDA treatment are usually much
higher in vitro than in vivo (10 100 mg versus less than
10 mg, respectively). The weak concentration used for
intraparenchymal injection in animal models of PD
may ensure relative specificity to catecholaminergic
cells. Further experiments aimed at describing non-specific toxicity of 6-OHDA in vivo towards non-catecholaminergic neurons may help to clarifiy this point.
In conclusion, considering the possible physiopathological involvement of 6-OHDA and the common biochemical processes with those observed in human
disease, this compound remains an interesting tool to
model nigral degeneration.
3.1.2. Neurotoxicity of dopamine

3.1.2.1. General obser6ations. Dopamine (DA) is a natural neurotransmitter of the brain. In dopaminergic neurons, most of this amine (0.1 1 mM) (Jonsson, 1971) is
contained in vesicles that moderate the concentration of
dopamine both in the cytoplasm and in the synaptic
cleft. In certain pathological conditions, such as ischemia or hypoxia, increases in either extracellular or
intracellular levels of dopamine could lead to brain
damage (Slivka et al., 1988). In addition, dopamine
auto-oxidation, which possibly leads to the formation
145
of 6-OHDA and neuromelanin (Slivka and Cohen,

1985; Jellinger et al., 1995), increases with age and may
lead to intracellular toxicity. Finally, in experimental
conditions, DA is toxic in a variety of neuronal and
non-neuronal cells both in vitro (Graham et al., 1978;
Wick, 1978; Michel and Hefti, 1990) and in vivo (Filloux and Townsend, 1993). These observations thus
suggest that dopamine, in some circumstances, may be
potentially deleterious.
3.1.2.2. Oxidati6e stress and mitochondrial defects.

Amine oxidation, deamination by MAO and/or mitochondrial inhibition may be regarded as hypothetical
mechanisms of DA toxicity (Fig. 3). To date, the best
characterized mechanism of DA toxicity consists of its
own oxidation, which produces radicals, toxic quinonic
compounds and melanin (Graham et al., 1978; Hastings
and Zigmond, 1994; Offen et al., 1996). Free radicals
arise primarily from this process but also secondarily
after reactions between melanin and iron (see Sections
2.3 and 3.1.1). Nitric oxide could be a substantial
source of ROS in DA-mediated oxidative stress since:
(1) l-NAME, a NOS inhibitor, inhibits DA-induced cell
death, (2) DA toxicity is reinforced by cyanide, a
compound known to increase intracellular NO levels
(Jones et al., 2000), and (3) NO could react directly
with DA to form toxic nitroso-conjugates (Daveu et al.,
1997). That DA oxidation leads to strong oxidative
stress is also supported by several experiments showing
the protective potency of various antioxidants and the
potentiating effects of GSH depletion (Ziv et al., 1994;
Offen et al., 1995; Gabbay et al., 1996; Hoyt et al.,
1997; Zilkha-Falb et al., 1997; Stokes et al., 2000).
MAO activity could contribute to DA toxicity by
producing both hydrogen peroxide and 3,4-dihydroxyphenylacetaldehyde, which are highly toxic to catecholaminergic cells (Mattammal et al., 1995). However,
considering the controversial results obtained concerning the protection afforded by MAO inhibition against
DA-induced cell death (Cohen and Spina, 1989; Spina
and Cohen, 1989; Jacobsson and Fowler, 1999), a clear
demonstration of MAO involvement is still needed.
Similarly, the potential effects of DA on mitochondrial
respiration are not firmly established. Indeed, DA has
been shown to block the activity of complex I at low
concentrations (Ben Shachar et al., 1995); by contrast,
in two other independent studies using DA at concentrations as high as 10 mM, neither mitochondrial respiration nor the ATP/ADP ratio was modified
(Morikawa et al., 1996; McLaughlin et al., 1998). However, without necessarily affecting ATP levels, DA
could trigger mitochondrial alterations by decreasing
the activity of creatine kinase or adenylate kinase
(Maker et al., 1986). All the potential toxic pathways
involved in DA toxicity finally lead to oxidative stress
and to subsequent cellular damage such as lipid, protein
146
and nucleic alterations (Moldeus et al., 1983; VelezPardo et al., 1997).
3.1.2.3. Specificity. DA toxicity is not strictly specific

to dopaminergic cells and could act by intracellular
and/or extracellular pathways. Some studies show
that nomifensine, a blocker of the dopamine transport system, inhibits DA-induced PC12 cell death
(Jones et al., 2000). Similarly, an oligonucleotide that
decreases the amount of DA transporter in catecholaminergic cells also decreases the neurotoxic
effects of DA (Simantov et al., 1996). Conversely,
some authors report that DA induces cell death of
non-catecholaminergic cells such as thymocytes or
striatal cells (Offen et al., 1995; McLaughlin et al.,
1998) and that compounds such as catalase or superoxide dismutase that remain extracellular protect from
this amine (Masserano et al., 1996; Offen et al.,
1996). In conclusion, the basal mechanisms of DA
toxicity closely ressemble those of 6-OHDA and
mainly involve both extracellular and intracellular
generation of ROS by oxidation of the catechol moiety and, possibly, mitochondrial inhibition.
3.1.3. Neurotoxicity of MPTP

3.1.3.1. General obser6ations. Between 1979 and 1982, a
population of young Californians addicted to a new
synthetic fentanyl derivative developed an irreversible
l-dopa responsive PD (Davis et al., 1979). The analysis
of this synthetic drug showed that it contained around
3% MPTP (Langston et al., 1983). Patients exhibited
symptoms very similar to those of PD, with bradykinesia, rigidity, postural instability and resting tremors.
UDPRS analysis showed that the MPTP-exposed
group shared similar results to the PD patients, except
for the tremors, which were less frequent (Tetrud et al.,
1989). Additionally, post-mortem investigations clearly
confirmed the lesion of the substantia nigra (Davis et
al., 1979). MPTP was thus considered as a powerful
drug to induce nigral degeneration in animals and was
shown to induce PD-like symptoms in several species
including rat, mouse, dog, cat and monkey.
In the monkey, MPTP induces the loss of pigmented
neurons of the SNpc (Hantraye et al., 1993) but rarely
causes the appearance of eosinophilic inclusions resembling Lewy Bodies (Forno et al., 1988). Injections of
Fig. 3. Hypothetical mechanism of dopamine toxicity. Similarly to 6-OHDA, dopamine could induce catecholaminergic cell death by three main
mechanisms: reactive oxygen species generated by intra or extracellular oxidation, hydrogen peroxide formation induced by MAO activity or
direct inhibition of the mitochondrial respiratory chain. These events could lead to strong oxidative stress and to a decrease in cellular ATP
availability, both leading to cell death.
147
Fig. 4. Hypothetical mechanism of MPTP toxicity. MPTP, injected peripherally, crosses the blood brain barrier and is transformed by glial MAO
into the active compound MPP+. The latter crosses the neuronal membrane by a specific uptake mechanism. Once inside the cells, MPP+ leads
to a major inhibition of the respiratory chain but also to oxidative stress, both triggering cell death. The mitochondrial inhibition provokes an
ATP decrease presumably responsible for secondary excitotoxicity inducing a strong and deleterious increase in cytoplasmic calcium levels.
Oxidative stress generated directly by MPP+ or subsequent to mitochondrial inhibition leads to macromolecule peroxidation and cell death.
MPTP into the SN and the medial forebrain bundle as

well as intrastriatal perfusion lead to a massive loss of
DA in the striatum (Chiueh et al., 1992, 1993) and, more
generally, to the depletion of dopaminergic markers in
the nigrostriatal tract (Heikkila et al., 1985). Interestingly, in monkey, the topology of nigral lesion after
MPTP treatment is similar to that observed in human
PD since the pars lateralis of the SNpc appeared more
affected than the medial part (Varastet et al., 1994).
Other structures are also affected by the MPTP in
monkey: locus cruelus (Forno et al., 1986, 1993),
ventral tegmental area or hypothalamus (German et al.,
1988). Similar findings were obtained in mice exhibiting
neuronal loss in both the locus cruleus and hypothalamus (Seniuk et al., 1990) and a decrease in dopamine
and noradrenaline levels in the mesolimbic system and
the cortex, respectively (Heikkila et al., 1989; Date et al.,
1990). The effects of MPTP on animals depend on
several parameters, such as the administration mode,
dosage and animal age (for reviews see Tipton and
Singer, 1993; Gerlach and Riederer, 1996). Interestingly,
studies on mice support the notion that older animals
are more susceptible to MPTP (Jarvis and Wagner,
1985; Ricaurte et al., 1986, 1987) as shown with another

mitochondrial toxin, the 3-nitropropionic acid (Brouillet
et al., 1993).
When administrated to animals, MPTP crosses the
bloodbrain barrier and is converted, mainly in glial
cells, into its effective form, 1-methyl-4-phenylpyridinium (MPP+), by monoamine oxidase B (Fig. 4)
explaining the protective effect of MAO-I B inhibitors
against MPTP neurotoxicity (Chiba et al., 1984). MPP+
then accumulates in dopaminergic cells after selective
uptake by energy-dependent dopamine uptake sites
(Chiba et al., 1985; Pifl et al., 1993). Besides this uptake,
intracytoplasmic accumulation of MPP+ also depends
on two intracellular trapping systems: (1) the neuromelanin that forms a complex with MPP+ and delays its
cytoplasmic release (DAmato et al., 1986, 1987) and (2)
the vesicular monoamine transporters (VMAT) that
confine the neurotoxin to synaptic vesicles (Takahashi et
al., 1997; Staal and Sonsalla, 2000). Free cytosolic
MPP+ finally enters mitochondria by an energy-dependent mechanism (Ramsay and Singer, 1986) inhibiting
the activity of this organelle and leading to a drop in
cellular ATP levels and subsequent cell death.
148
3.1.3.2. Mitochondrial defects and oxidati6e stress. The

best-defined MPTP effect is the potent inhibition of
complex I with not only subsequent inhibition of oxidation of NAD+-linked substrates but also inhibition of
a-ketoglutarate dehydrogenase (Fig. 4; Nicklas et al.,
1985; Mizuno et al., 1988). The mitochondrial inhibition leads to a decrease in cellular ATP levels (Di
Monte et al., 1986), loss of mitochondrial membrane
potential, alterations of calcium homeostasis and radical formation (Fig. 4). Although mitochondrial inhibition can by itself explain MPTP toxicity, several
observations favor a more complex activity mediated
by mitochondria-dependent or independent ROS generation (Johannessen et al., 1985). In support of mitochondrial independence, it was shown that Rho0 cells,
which are devoid of a functional electron transport
chain, and parental cells were equally sensitive to
MPP+ (Przedborski and Jackson-Lewis, 1998).
Several lines of evidence suggest ROS involvement in
MPTP-induced neurotoxicity (Fig. 4). Increases in ROS
have been detected by using, for example, the DCDHFDA ROS-dependent fluorescent probe in vitro (Kitamura et al., 1998) or electron spin resonance in vivo
(Rossetti et al., 1988). In keeping with this free-radical
generation, MPTP was shown to induce a decrease in
GSH and metallothionein content (Desole et al., 1993;
Oishi et al., 1993; Rojas et al., 2000) or an increase in
3-nitrotyrosine formation (Pennathur et al., 1999). Conversely, MPTP toxicity was potentiated by the decrease
of GSH content (Wu llner et al., 1996) and attenuated
by SH-containing antioxidants (Oishi et al., 1993). The
ROS involvement was also confirmed by the cellular
protection provided by increased superoxide dismutase
expression in transgenic mice (Przedborski et al., 1992)
and the potentiation of MPTP effects by either deficiency in superoxide dismutase or glutathion peroxidase
genes (Klivenyi et al., 2000; Zhang et al., 2000).
Several factors contribute to MPTP-induced ROS
generation. As suggested for 6-OHDA, iron may be of
great importance in MPTP toxicity and triggers a Fenton reaction in dopaminergic cells. MPTP increases free
iron levels in SNpc (Mochizuki et al., 1994b; Temlett et
al., 1994), and desferrioxamine, an iron chelator, blocks
its effects (Lan and Jiang, 1997). MPTP also increases
transferrin receptor and lactoferrin transporter expressions (Faucheux et al., 1995; Fillebeen et al., 1999).
Additionally, MPP+, which is a substrate for xanthine
oxidase, may lead to the formation of the MPP radical
(Adams et al., 1993). MPDP+, an intermediate compound in the metabolism of MPTP, can itself induce
superoxide radical formation during its auto-oxidation
(Zang and Misra, 1992).
ROS formation may also be dependent on indirect
excitotoxicity resulting from neuronal impairment of
energy metabolism and the subsequent increase in cytoplasmic calcium (Storey et al., 1992; Chen et al., 1995).
Indeed, NMDA receptor antagonists (Turski et al.,

1991) and calcium channel blockers (Kupsch et al.,
1995, 1996) were shown to protect SNpc efficiently
against MPTP. Furthermore, the involvement of the
calcium-dependent nitric oxide synthase activation that
leads to peroxynitrite formation has been suggested by
several studies (Schulz et al., 1995; Przedborski et al.,
1996; Jenner, 1998; Dehmer et al., 2000). Inflammatory
processes can also contribute to MPTP-induced ROS
generated by reactive microglia (Kurkowska-Jastrzebska et al., 1999). Altogether, these data explain the
increases in malondialdehyde levels and lipid peroxidation induced by MPTP (Rios and Tapia, 1987).
However, MPTP toxicity is not specifically restricted
to catecholaminergic cells. In rats, high concentrations
of MPP+ can indeed induce a loss of GABAergic
striatal neurons (Altar et al., 1986). Cell membrane
damage and gliosis induced by iontophoretic diffusion
of MPP+ confirm this non-specific toxicity (Ter Horst
et al., 1992). Moreover, MPP+, which can enter cells by
glutamate transporters (Marini et al., 1989), induces the
death of cerebellar granule cells devoid of any dopamine uptake system (Dipasquale et al., 1991).
In summary, the mechanisms of 6-OHDA, DA and
MPTP imply comparable cellular modifications susceptible to induce apoptosis of dopaminergic cells.
3.2. Molecular pathways of cell death induced by

6 -OHDA, dopamine and MPTP
3.2.1. O6er6iew on the regulation of apoptosis
3.2.1.1. General considerations. Apoptosis (Kerr et al.,
1972) is an active, gene-directed mechanism crucially
involved in the efficient removal of cells, which are
either no longer needed or damaged and thus possibly
dangerous. In metazoans, apoptosis thus plays a pivotal
role in animal development and in maintenance of
tissue homeostasis (Steller, 1995; Jacobson et al., 1997;
Rich et al., 1999; Vaux and Korsmeyer, 1999). As
mentioned above, apoptosis is a morphologically and
biochemically defined mode of cell death characterized
by nuclear and cytoplasmic condensation, loss of mitochondrial membrane potential, DNA fragmentation,
dilatation of the endoplasmic reticulum, alterations in
cell membrane composition and formation of apoptotic
bodies. Because this evolutionarily conserved cell suicide program is tightly controlled, its disregulation
contributes to several diseases such as cancer (Strasser
et al., 1990; Thompson, 1995; Strasser et al., 1997),
autoimmune syndromes (OReilly and Strasser, 1999),
neurodegenerative disorders and ischemic stroke (Barr
and Tomei, 1994).
3.2.1.2. A regulated mode of cell death. The suggestion
of the existence of a common intracellular death pro-
gram was first based on the observations of stereotyped

morphological changes associated with apoptosis in
different organisms and circumstances. It was later
confirmed by genetic studies of cell death in Caenorhabditis elegans demonstrating that the cell death machinery consists of gene products that either promote or
inhibit apoptosis in the nematode (Ellis and Horvitz,
1986; Ellis et al., 1991; Yuan and Horvitz, 1992; Conradt and Horvitz, 1998; Horvitz, 1999). These studies
identified three genes, egl-1, ced-3 and ced-4, absolutely
required for activation and execution of apoptosis in C.
elegans and a critical negative regulator, ced-9. The
Ced-3 caspase, synthesized as a zymogen, requires the
binding of the Ced-4 adapter protein for its auto-activation (Yang et al., 1998a). The apoptosis suppressor
Ced-9 controls Ced-4 activity and can be inhibited
through direct association with Egl-1, a nematode
counterpart of proapoptotic Bcl-2 family members
(Conradt and Horvitz, 1998; del Peso et al., 1998).
The cloning of these genes revealed that they encoded
proteins homologous to mammalian proteins, confirming the conservation of this death pathway through
evolution (Hengartner and Horvitz, 1994a). The protein
encoded by ced-3 is very similar to the human cysteine
protease interleukin-1b converting enzyme (ICE/caspase-1Yuan et al., 1993). Ced-9 is related to the human
Bcl-2 protein (Hengartner and Horvitz, 1994b). The
bcl-2 gene was originally identified as a proto-oncogene
found at the breakpoint of t(14; 18) chromosomal
translocations in low-grade B-cell lymphomas (Tsujimoto et al., 1984), and the resulting elevated constitutive expression promotes cancer by inhibiting cell death.
Other mammalian equivalents of ced-3 and ced-9 were
subsequently discovered. They form the Ced-3/caspase
and Bcl-2 protein families, respectively. Finally, the
only well-characterized mammalian homolog of ced-4 is
Apaf-1, both of which appear to play a critical role in
the conversion of procaspases into active proteases
(Zhou et al., 1997).
During the last several years, a great deal of work
has been dedicated to the identification of key components of the cell death machinery and their mechanims
of action. Although a great variety of intra- and extracellular stimuli are able to trigger apoptosis (induction
phase), their early transducing downstream pathways
all converge into a common execution apoptotic pathway (effector phase) responsible for the dismantling of
the cell (Dragovich et al., 1998; Nunez and del Peso,
1998; Nunez et al., 1998; Raff, 1998). This final stage of
apoptosis occurs through the activation and critical
function of caspases, although recent evidence suggests
the existence of a caspase-independent pathway for
apoptosis (Borner and Monney, 1999), and is regulated
by Bcl-2 family members and a recently identified class
of proteins, the inhibitors of apoptosis proteins (IAPs).
149
3.2.1.3. Caspases. The execution phase of apoptosis is

generally activated by members of a distinct, highly
conserved class of intracellular cysteine proteases,
called caspases, characterized by their almost absolute
specificity for aspartic acid residues in their substrates.
At least 14 members have been identified to date,
including 11 human forms.
These proteases are constitutively expressed in most
cells, residing in the cytosol as zymogens or proenzymes
(about 3050 kDa) composed of three domains: an
NH2-terminal domain, a large subunit ( 20 kDa) and
a small subunit ( 10 kDa) (Nicholson and Thornberry, 1997). This precursor is activated through proteolytic cleavage at the two aspartic acid sites (one site
between the prodomain and the large subunit, and the
other between the large subunit and the small subunit)
that results in the removal of the prodomain and the
release of two fragments ( 20 kDa and 10 kDa)
that assemble to form a heterodimer. Crystal structure
analyses have revealed that active enzymes are actually
heterotetramers, composed of two small and two large
subunits, with two catalytic sites (Walker et al., 1994;
Wilson et al., 1994; Rotonda et al., 1996). Consequently, the processing of proenzymes by cleavage at
internal caspase consensus sites implies that caspases
can activate either their own procaspases or other procaspases in an amplifying proteolytic cascade (Cohen,
1997; Nicholson, 1999).
Based on caspase specificity and prodomain length
and function, caspases can be subdivided into initiators or upstream (caspases-1, -2, -4, -5, -8 -9, -10, -11
and -12) and effectors or downstream caspases (including caspase-3, -6, -7 and 14). Initiator caspases
usually act proximally relative to the initial death stimulus and can activate effector caspases, while effector
caspases that possess short prodomains contribute to
the execution of apoptosis.
In response to proapoptotic signals, specific adaptator proteins bind to the long N-terminal regulatory
prodomains of initiator procaspases. Based on their
sequence homology, two types of prodomain have been
identified: CARD (caspase recruitment domain; caspase-1, -2, -4, -5, -9, -11 and 12) and DED (death
effector domain; caspase-8 and 10). The interactions
between the prodomains of caspases and corresponding
domains in adapter proteins bring the procaspases into
close proximity to one another, thus promoting zymogen aggregation and processing (Muzio et al., 1998;
Srinivasula et al., 1998; Yang et al., 1998a; Li and
Yuan, 1999). According to the induced proximity or
oligomerization model (Muzio et al., 1998), the close
proximity of procaspases in the aggregates may be
sufficient to allow their activation through auto- or
transprocessing.
Initiator caspases activate either directly or indirectly
various effector caspases, which carry out many of the
150
cellular changes leading to the structured dismantling of

apoptotic cells. Effector caspases cleave a number of
structural and regulatory proteins. Most caspase substrates can be divided into two general categories (reviewed in Nunez et al., 1998; Thornberry and Lazebnik,
1998): regulators of apoptosis, which are either activated or inactivated by cleavage (members of the Bcl-2
family: Bid, Bcl-2 and Bcl-x; kinases), and housekeeping or structural proteins whose cleavage is required for
the disassembly of the cells (e.g. DFF45/ICAD, polyADP-ribose polymerase (PARP), nuclear lamins, gelsolin, focal adhesion kinase (FAK), p21-activated
kinase (PAK2), cytokeratins, etc.).
The pathway leading to caspase activation and apoptosis varies according to the apoptotic stimulus. Basically, two main biochemical pathways can be
distinguished (Green, 1998; Budihardjo et al., 1999;
Bratton et al., 2000): one involves the binding of death
receptors to their ligands (Yuan, 1997; Ashkenazi and
Dixit, 1998, 1999); in the other, various forms of cellular stresses (withdrawal of growth factors, DNA damage, toxic drugs, etc.) trigger mitochondrial release of
caspase-activating proteins (Bossy-Wetzel and Green,
1999a; Desagher and Martinou, 2000). However, there
is now some evidence that cross-talk between these two
pathways exists (Li et al., 1998; Luo et al., 1998a;
Bossy-Wetzel and Green, 1999b). The first pathway
involves DED-containing procaspases that can be activated by the binding of extracellular ligands to death
receptors at the cell surface. These death receptors
belong to the TNF receptor superfamily, and possess
both cysteine-rich extracellular domains and an
intracellular cytoplasmic sequence known as the death
domain. For example, CD95 ligand causes CD95/FasApo-1 receptor trimerization and subsequently the
recruitment of the adapter protein FADD (Fas-associated death domain) and initiator procaspase into a
multimeric complex, the DISC (death-inducing signal
complex) where caspase activation takes place. Activated initiator caspases then cleave downstream effector procaspases, such as caspase-3. Caspase-8 is
associated with apoptosis involving the death receptors
TNFR1, DR3 and CD95/Fas-Apo-1, while caspase-10
may play a role in some caspase cascades. In the second
pathway, apoptosis can be triggered by diverse intra
and extracellular stimuli in the absence of death receptor engagement. Their transducing pathways converge
onto the mitochondria, which play a pivotal role in
caspase activation. Indeed, in these situations, apoptotic stimulation results in mitochondrial damage, e.g.
major changes in membrane structure and function
(disruption of the outer membrane, depolarization;
Green and Kroemer, 1998; Loeffler and Kroemer,
2000). One crucial consequence is the release of cytochrome c from the intermembrane space of mitochondria into the cytosol (Green and Reed, 1998; Desagher
and Martinou, 2000). Once released, cytochrome c interacts with the adaptor protein CED-4/Apaf-1 in the
presence of dATP (Zhou et al., 1997; Cecconi, 1999)
allowing its oligomerization and the subsequent recruitment of the initiator procaspase-9, the key initiator
caspase in the mitochondrial pathway, into a complex
termed an apoptosome. It remains, however, to be
clearly determined whether either following activation
caspase-9 is released from the apoptosome to process
effector caspase-3 and -7 or effector procaspases are
recruited and activated within the apoptosome before
their release (Bratton et al., 2000).
During apoptosis, another important mediator released by mitochondria is the mitochondrial intermembrane apoptosis-inducing factor (AIF) (Susin et al.,
1996, 1999; Zamzami et al., 1996). AIF appears to be a
caspase-independent effector responsible for the initial
nuclear disasembly. Indeed, once liberated into the
cytosol, AIF travels to, and concentrates in, the nucleus, where its seems essential for chromatin condensation and large-scale DNA fragmentation (Lorenzo et
al., 1999; Daugas et al., 2000).
3.2.1.4. Bcl-2 family members. Members of the Bcl-2

family are key regulators of apoptosis in general and of
naturally occurring and pathological neuronal death in
particular (Sadoul, 1998). In C. elegans, the activity of
Ced-9 is required to prevent inappropriate cell death
and generally upstream of Ced-4 and Ced-3. Since the
discovery that ced-9 encodes a protein homologous to
the mammalian Bcl-2 protein, a large number of additional Bcl-2-like genes have been identified. Like Bcl-2,
many have anti-apoptotic activities (e.g. Bcl-xL, Bcl-w,
A1/Bfl-1, Mcl-1) while others promote apoptosis (Bax,
Bad, Bak, Bcl-xS, Bid, Bik/Nbk, Bim/Bod, Blk, Bok,
Hrk/Dp5, Diva).
Bcl-2 family members share at least one of four
evolutionarily conserved specific regions of homology,
referred to as Bcl-2 homology domains (BH1BH4),
which mediate protein interactions and are essential for
their function (Reed, 1998; Antonsson and Martinou,
2000). Some members (Bcl-2, Bcl-xL, Bax) contain a
carboxy-terminal transmembrane domain that anchors
them in membranes and in particular in the outer
mitochondrial membrane.
The molecular mechanisms underlying the pro- or
anti-apoptotic functions of Bcl-2 family members, although intensively investigated during the last several
years, still remain unclear, but mitochondria appear to
be one of the main targets for these proteins (Desagher
and Martinou, 2000); indeed, Bcl-2 proteins regulate
apoptosis by controlling cytochrome c release. Each
member may act through different mechanisms at various levels of the apoptotic pathway; moreover, some of
these proteins appear to exhibit qualitatively different
properties, depending on the signaling pathway acti-
vated and/or the cellular context. However, despite

variations, the current models that are proposed to
explain their action converge on three main events (see
Adams and Cory, 1998; Reed, 1998):
1. The formation of homo- or heterodimers within the
Bcl-2 family. These interactions are regulated by the
BH1, BH2 and BH3 domains. This latter appears to
be crucial for dimerization of proapoptotic proteins
with death-repressor Bcl-2 members and induction
of apoptosis (Li and Yuan, 1999). Thus, the proapoptotic proteins Bax and Bid can antagonize the
activities of the anti-apoptotic members Bcl-2 and
Bcl-xL.
2. The binding to non-homologous proteins. With the
exception of the proapoptotic Bcl-xS, the BH4 domain is exclusively found within the N-terminal
region of anti-apoptotic proteins and is essential for
the anti-apoptotic activity of Bcl-2/Bcl-xL (Huang et
al., 1998). The sequestration of Apaf-1 by antiapoptotic Bcl-2 family members through their BH4
domain has been proposed as a model for caspase
inactivation (Hu et al., 1998; Inohara et al., 1998;
Pan et al., 1998; Song et al., 1999) although recent
evidence questions it (Moriishi et al., 1999). More
recently, it was shown that the BH4 domain of
Bcl-2/Bcl-xL inhibits the activity of the voltage-dependent anion channels, thought to be, along with
Bax, a component of the permeability transition
pore, the mitochondrial megachannel. This prevents
apoptotic mitochondrial changes (cytochrome c release and loss of mitochondrial membrane potential
Dcm) and cell death (Shimizu et al., 2000). In
addition, the BH4 domain can bind to other
proteins regulating apoptosis, including Raf1, rRas,
Bag-1, p53-binding protein-2 and calcineurin (see
Reed, 1998 for a review).
3. The formation of membrane spanning channels.
This model is based on the observation that some
Bcl-2 family members, e.g. Bcl-2, Bcl-xL and Bax,
could form pores or ion channels in synthetic membranes. During apoptosis, the proapoptotic Bax
protein translocates from the cytosol to the mitochondria where it could trigger the opening of either
the mitochondrial megachannel or a specific channel
in the outer mitochondrial membrane both of which
promote cytochrome c release (Antonsson and Martinou, 2000; Desagher and Martinou, 2000). Bcl-2
prevents this latter probably by inhibiting either
insertion of Bax in the mitochondrial membrane or
Bax-channel activity.
The activity of Bcl-2 family members can be regulated through different mechanisms. Cytokines can induce the transcription of several anti-apoptotic genes.
Moreover, in neurons, like many other non-neuronal
cells, DNA damage and excitotoxicity can lead to
apoptosis through a pathway involving the activation
151
of the p53 suppressor gene (White, 1996; Xiang et al.,

1996, 1998; Hughes et al., 1997). P53 is known to
promote cell cycle arrest or apoptosis in proliferating
cells following DNA damage (Levine, 1997; Amundson
et al., 1998) and to be a transcriptional activator of the
Bax gene (Miyashita and Reed, 1995). The mechanism
by which p53 triggers apoptotic response is still not well
understood and may vary according to the cell type
and/or death stimulation (Johnson et al., 1998, 1999;
Cregan et al., 1999); in some forms of neuronal injury,
cell death involves a p53-induced increase in Bax expression followed by a Bax-dependent caspase-3 activation (Xiang et al., 1998). In addition to transcriptional
regulation, several Bcl-2 family members undergo posttranslational modifications or proteolysis, which can
alter their function. For example, the phosphorylation
of the proapoptotic protein Bad in the presence of
growth factors abrogates its ability to promote apoptosis, while its dephosphorylation restores it (Harada et
al., 1999; Wang et al., 1999b); similarly, the phosphorylated form of Bcl-2 is unable to protect from apoptosis
(Srivastava et al., 1999). The cleavage of the death-inducer Bid by caspase-8 induces its translocation to the
mitochondria and the consecutive release of cytochrome c (Li et al., 1998; Luo et al., 1998a). In some
circumstances, Bcl-2 and Bcl-xL are also targets for
caspases, which convert them from prosurvival proteins
to proapoptotic proteins (Cheng et al., 1997; Clem et
al., 1998; Kirsch et al., 1999). Finally, it was shown that
the calpain-mediated cleavage of Bax enhances its cytotoxic activity (Wood and Newcomb, 2000).
3.2.1.5. Inhibitor of apoptosis proteins (IAPs). IAPs were

first discovered in baculovirus following the observation
of their property of inhibiting apoptosis in insect cells
infected by viruses (Clem and Miller, 1994). IAP family
members all possess a RING finger at their C-terminus
and at least one characteristic structural N-terminal
motif of about 70 amino acids, the baculovirus IAP-repeat (BIR), which is involved in proteinprotein interaction and is required for the anti-apoptotic activity of
IAPs (Clem and Duckett, 1997; Uren et al., 1998;
Miller, 1999). To date, six human IAP proteins have
been identified: X-linked IAP (XIAP), HIAP1, HIAP2,
NAIP, BIR-repeat-containing ubiquitin conjugating enzyme (BRUCE) and survivin. Some IAPs appear to
block cell death induced by diverse extracellular stimuli
(UV light, chemotoxic drugs, activation of TNF and
Fas receptors) in various cell types downstream of
mitochondrial events by directly inhibiting specific caspases (Liston et al., 1996; Duckett et al., 1998; LaCasse
et al., 1998; Deveraux et al., 1999).
3.2.2. Apoptosis induced by 6 -OHDA
Proapoptotic effects of 6-OHDA were first shown in
vitro by Walkinshaw and Waters (1994). They observed
152
that low concentrations of 6-OHDA (5 100 mM) induce biochemical and morphological hallmarks of
apoptosis in either cycling or NGF-differentiated catecholaminergic PC12 cells (Fig. 5). Morphologically,
PC12 cells exhibit classical cell shrinkage, chromatin
condensation and membrane blebbing. Biochemically,
this study showed that 6-OHDA induces oligonucleosomal DNA cleavage. These results were later confirmed
in PC12 cells as well as in cerebellar granule cells,
primary mesencephalic dopaminergic cells, the mesencephalic-derived dopaminergic MN9D cell line, neuroblastoma NB41 cells, the murine embryonic carcinoma
P19 cell line, cultured microglial cells and thymocytes
(Oh et al., 1995; Tsao et al., 1996; Blum et al., 1997;
Funa and Ahgren, 1997; Mayo et al., 1998, 1999; Dodel
et al., 1999; Lotharius et al., 1999; Woodgate et al.,
1999).
In vivo, TUNEL positive cells were observed in the

SNpc from 1 to 14 days after injection of low doses of
6-OHDA into the medial forebrain bundle of Sprague
Dawley rats (He et al., 2000; Zuch et al., 2000).
TUNEL positive cells were dopaminergic and shared
typical apoptotic features such as cell shrinkage, nucleus condensation and formation of chromatin clumps.
Intrastriatal injection of 6-OHDA also produces nigral
apoptosis in developing animals (Marti et al., 1997),
although after the second post-natal week, 6-OHDA
induces both apoptosis and necrosis (Burke and
Kholodilov, 1998). Conversely, intranigral injection induces necrosis (Jeon et al., 1995). Therefore, in vivo,
the mode of cell death induced by 6-OHDA may differ
depending on the target site and the animal age. However, when detected, the actual proportion of apoptotic
Fig. 5. Apoptotic pathway induced by 6-OHDA in PC12 cells. (A) Immunoblot analysis of p53, Bax and Bcl-xL proteins in PC12 cells exposed
for various times to 100 mM 6-OHDA. Relative levels of p53 (53 kDa), Bcl-xL (26 kDa) or Bax (21 kDa) proteins were assessed by independent
immunoblottings using equal amounts of proteins from the same lysates of either non-treated PC12 cells or 100 mM 6-OHDA-treated cells for
various times. (B) Analysis of p53, Bax and Bcl-xL relative levels by scanning densitometry from the representative experiment shown in (A). The
mean arbitrary O.D. values were determined and represented as a percentage relative to the non-treated control. These biochemical experiments
show that 6-OHDA induces a time-dependent increase in p53 and Bax expression after treatment, although the Bcl-xL level remains unchanged.
These results support the idea that 6-OHDA-induced cell death is a p53-dependent mechanism leading to the proapoptotic protein Bax activation.
(C) Fluorimetric analysis of caspase-3 like activity in cytoplamsic protein fractions of PC12 cells treated for various times with 100 mM of
6-OHDA. We show that 6-OHDA induces a strong increase in caspase-3 like activity. This activation could be responsible for the nuclear
fragmentation observed in PC12 cells 24 h after treatment as shown in (D). (D) PC12 cell culture treated for 24 h with 100 mM 6-OHDA and
stained with Hoechst 33342. Apoptotic cells showing highly condensed chromatin or nuclear fragmentation (arrows) are observed among viable
cells. The results A, B and D are reprinted from Blum et al. (1997). p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12
cell. Brain Research 751, 139 142 with permission from Elsevier Science. The results presented in (C) are unpublished data obtained by Blum et
al. with the help of Dr. Mathieu, CRSSA, Grenoble, France.
cells is always low both in vivo (He et al., 2000) and in

vitro (Walkinshaw and Waters, 1994; Woodgate et al.,
1999). Thus, in PC12 cells, less than 5% of the entire
population undergoes apoptosis. The reasons for such
low numbers are unclear. In vivo, it may reflect the
short duration of apoptosis. In vitro, this may be
consistent with secondary necrosis occurring after primary apoptosis. Thus, it is difficult to draw a clear
conclusion purely on these morphological and biochemical signs. However, biochemical and molecular studies
demonstrate that cell death induced by 6-OHDA is
actively regulated.
The potential genotoxicity of 6-hydroxydopamine
(Bruchelt et al., 1991 has led to studies of the role of
p53 transcription factor in 6-OHDA-induced cell death.
This protein was reported to be induced in neurons
after various neurotoxic insults (Sakhi et al., 1994;
Grilli and Memo, 1999; Liu et al., 1999) and can
regulate the transcription of genes involved in the apoptotic process (see Section 3.2.1.4). In vitro, we have
described that 6-OHDA induces an early increase of
p53 cellular content in the PC12 cell line (Fig. 5; Blum
et al., 1997). An increase in p53 immunoreactivity has
also been described in the substantia nigra of rats
treated with 6-OHDA (Qin et al., 1997). Additionally,
compounds such as staurosporine and retinoic acid that
modify p53 expression are able to modulate cell death
induced by 6-OHDA in neuroblastoma SY5Y cells
(Tieu et al., 1999). It is known that p53 regulates the
expression of Bcl-2 family proteins, particularly Bax
and Bcl-2 members (Miyashita et al., 1994; Miyashita
and Reed, 1995). This role could be related to the
increase of the proapoptotic Bax protein oberved in
PC12 cells treated with 6-OHDA (Fig. 5;Blum et al.,
1997), although this phenomenon could be p53-independent. Bax activation may be responsible for the
successive disruption of the mitochondrial membrane
potential (Lotharius et al., 1999), release of cytochrome
c into the cytoplasm (Dodel et al., 1999) and activation
of caspases demonstrated after 6-OHDA exposure. Although p53 and Bax involvement in 6-OHDA-induced
cell death is not fully characterized, the implication of
caspases is supported by numerous recent findings.
Activation of caspase 3-like proteases after 6-OHDA
treatment was described in vitro (Fig. 5; Ochu et al.,
1998) as well as in vivo after intrastriatal injection in
adult or young rats (Cutillas et al., 1999; Jeon et al.,
1999). Additionally, caspase implication in 6-OHDA
toxicity was also indirectly suggested by the poly-ADPribose polymerase proteolytic cleavage, one of the
targets of caspase activity, reported in mesencephalic
MN9D and in SK-N-SH neuroblastoma cells after
6-OHDA exposure (Bruchelt et al., 1991; Choi et al.,
1999a).
All these phenomena may explain the protective effects of anti-apoptotic gene expression against 6-
153
OHDA. Indeed, in-vitro Bcl-2 over-expression

efficiently protects PC12 or primary cortical cells
against 6-OHDA toxicity (Offen et al., 1998; Takai et
al., 1998; Blum et al., 2001). In vivo, herpes simplex
virus vector-mediated expression of Bcl-2 prevents 6hydroxydopamine-induced degeneration of neurons
(Yamada et al., 1999). Furthermore, inhibitors of caspase activity were also demonstrated to be efficient
against 6-OHDA (Ochu et al., 1998; Takai et al., 1998;
Dodel et al., 1999; Lotharius et al., 1999; Von Coelln et
al., 2001).
3.2.3. Apoptosis induced by dopamine

Only a few studies have described the molecular
mechanisms of DA-induced apoptosis. It was shown
that exposure of neuronal and non-neuronal cells to
DA and also to l-dopa (Walkinshaw and Waters,
1995; Melamed et al., 1998; Corona-Morales et al.,
2000) induces morphological and biochemical hallmarks of apoptosis (Ziv et al., 1994; Simantov et al.,
1996; Coronas et al., 1997; McLaughlin et al., 1998;
Zhang et al., 1998; Zou et al., 1999; Stokes et al., 2000).
Similar findings were obtained after striatal injection of
DA in rats (Hattori et al., 1998).
As we mentioned previously, one of the main effects
of DA-induced oxidative stress involves DNA damage.
In keeping with this mechanism, Daily et al. (1999)
demonstrated an involvement of the p53 transcription
factor in DA-induced cell death. Indeed, although DA
did not increase p53 protein levels, there was an increase of p53 phosphorylation, a sign of its activation,
after treatment by the amine. Moreover, LTR6 cells,
which overexpress p53 at a permissive temperature,
were shown to be more sensitive than cells expressing
inactive p53 (Daily et al., 1999). Finally, this study
showed that DA-induced DNA degradation was only
obtained after p53 activation. DA-induced cell death
was also accompanied by a strong increase in Bax
protein levels (Kang et al., 1998), suggesting that p53
might be able to transactivate the Bax gene after DA
exposure. The effects of Bax activation can be counteracted by Bcl-2 overexpression that blocks DA-induced
apoptosis (Offen et al., 1997; Cadet et al., 2000),
whereas Bcl-2 antisense oligonucleotides increase the
sensitivity of catecholaminergic cells to DA (Masserano
et al., 1996).
In conclusion, although DA pathways are not entirely elucidated, especially concerning caspase activation, cell death pathways induced by DA and 6-OHDA
appear to be similar.
3.2.4. Apoptosis induced by MPTP
Numerous studies suggest that MPP+ is able to
induce apoptosis in vitro in various cell types such as
cerebellar granule cells, GH3 pituitary cells, catecholaminergic cell lines as PC12 SK-NMC, SH-SY5Y
154
and MES 23.5 or in primary mesencephalic dopaminergic cells (Dipasquale et al., 1991; Hartley et al., 1994;
Mochizuki et al., 1994a; Itano and Nomura, 1995;
Desole et al., 1997; Du et al., 1997; Le et al., 1997;
Seaton et al., 1997; Sheehan et al., 1997; Dodel et al.,
1998; Kitamura et al., 1998; Leist et al., 1998;
Chalmers-Redman et al., 1999; Yoshinaga et al., 2000).
In vitro, apoptosis was assessed using light or electron
microscopy (Hartley et al., 1994; Mochizuki et al.,
1994a; Desole et al., 1997; Sheehan et al., 1997; Dodel
et al., 1998; Fall and Bennett, 1999; Yoshinaga et al.,
2000), specific dyes such as annexin V, an early marker
of apoptosis, or YOYO-1 (Kitamura et al., 1998; Leist
et al., 1998; Chalmers-Redman et al., 1999), and, less
specifically, using the TUNEL method (Dipasquale et
al., 1991; Hartley et al., 1994; Mochizuki et al., 1994a;
Itano and Nomura, 1995; Le et al., 1997; Sheehan et
al., 1997; Dodel et al., 1998; Yoshinaga et al., 2000).
Concordant findings have also been established in the
SNpc of mice treated by the MPTP (Tatton and Kish,
1997; Spooren et al., 1998; Fukuda and Tanaka, 2000).
Several recent works suggest that MPTP-induced
apoptosis may be under the control of p53 protein,
Bcl-2 family genes and caspase activity. Two studies
support the involvement of p53 in MPP+ neurotoxicity.
First, Kitamura et al. (1998) demonstrated that MPP+
is able to produce a strong increase in p53 protein
expression in SH-SY5Y neuroblastoma cells. Second, it
was shown interestingly that nigral neurons from p53deficient mice resist chronic administration of MPTP
better than wild-type animals (Trimmer et al., 1996).
Several lines of evidence demonstrated that MPP+induced apoptosis is regulated by members of the Bcl-2
family. Thus, it was described that nigral Bax mRNA
and protein levels were increased in mice after MPTP
exposure (Hasouna et al., 1996; Vila et al., 2001),
whereas animals deficient in this proapoptotic gene
were resistant to the neurotoxin (Vila et al., 2001).
Conversely, Bcl-2 overexpression protects catecholaminergic cells against MPTP/MPP+ toxicity both
in vitro and in vivo (Kitamura et al., 1998; Offen et al.,
1998; Yang et al., 1998b), whereas decreased Bcl-2 and
Bcl-xL levels generated by either pharmacological treatments or genetic modifications enhanced MPP+-induced cell death (Hochman et al., 1998; Kitamura et
al., 1998). These modifications would impair the mitochondrial membrane potential. As assessed by various
potential sensitive fluorescent-dyes, MPP+ was shown
to affect the Dcm in either catecholaminergic or noncatecholaminergic cells (Lambert and Bondy, 1989; Wu
et al., 1990; Camins et al., 1997; Leist et al., 1998;
Seaton et al., 1998; Cassarino et al., 1999; ChalmersRedman et al., 1999). This is in accordance with studies
demonstrating that the blockade of mitochondrial pore
opening, using cyclosporin A or analogues, reduces
MPP+-induced apoptosis (Seaton et al., 1998;
Chalmers-Redman et al., 1999) and that atractosyloside, which keeps the transition pore open, has a
synergistic activity with MPP+ (Cassarino et al., 1999).
The MPTP-induced fall in mitochondrial membrane
potential may also be influenced by Par-4 (prostate
apoptosis response 4), a recently identified 38 kDa
protein, the expression of which is increased in the
substantia nigra of MPTP-treated monkeys and mice
(Duan et al., 1999). Although its exact function is still
unkown, the inhibition of Par-4 activation protects
against dopaminergic cell death induced by both mitochondrial inhibition and iron (Duan et al., 1999). Thus,
this suggests that Par-4, which does not belong to the
Bcl-2 family, may thus be a critical factor for the
regulation of apoptosis in nigral neurons.
MPP+ toxicity was associated with the translocation
of cytochrome c from mitochondria to cytosol (Du et
al., 1997; Dodel et al., 1998; Leist et al., 1998; Cassarino et al., 1999; Yoshinaga et al., 2000). In line with
this, several studies support the involvement of caspases
in the final step of MPP+ toxicity. Indeed, recent data
showed a rise in caspase-3, but also caspase 8 and 1,
activities in the substantia nigra of mice treated with
MPTP (Viswanath et al., 2000). Accordingly, pharmacological or genetic inhibition of caspase activity was
shown to inhibit cell death induced by MPP+ (Le et al.,
1997; Dodel et al., 1998; Leist et al., 1998; Klivenyi et
al., 1999; Bilsland et al., 2000; Viswanath et al., 2000;
Yoshinaga et al., 2000). These findings are indirectly
confirmed by the fact that fodrin, a cytoskeletal
protein, and PARP, which can be cleaved by different
isoenzymes of the caspase family, are proteolyzed under
MPP+ treatment (Kitamura et al., 1998; Leist et al.,
1998).
However, a number of controversial data question
the proapoptotic properties of MPP+. Electron microscopy of the particular dopaminergic cell line
MN9D showed that MPP+ does not induce apoptosis
but rather induces necrosis (Choi et al., 1999b). Additionally, in this cell line, MPP+-induced cell death was
not dependent on p53 or Bax proteins induction and
was not attenuated by either Bcl-2 overexpression or
zVAD-fmk, a broad caspase inhibitor (Choi et al.,
1999b). Soldner et al. (1999) also observed in cycling
PC12 cells that MPP+ does not induce apoptosis or
caspase activation. Interestingly, Lotharius et al. (1999)
showed very little annexin V staining and no loss of the
mitochondrial potential on primary dopaminergic cell
cultures, unlike 6-OHDA. In vivo, Jackson-Lewis et al.
(1995) failed to find apoptotic cells in mice following
short-time spaced injection of MPTP. The reasons for
all these conflicting results are still unclear. Lotharius et
al. (1999) argued that most of the in-vitro studies used
very high concentrations of the neurotoxin and/or did
not identify the affected cell type (or in some cases did
not use dopaminergic cells) or relied solely on TUNEL
Fig. 6. Hypothetical molecular pathways leading to apoptosis triggered by 6-hydroxydopamine and MPP+.
positivity as an index of apoptosis. These authors also

suggested that (1) the complex metabolism of MPTP
itself, (2) the mode and frequency of administration in
vivo and, (3) the cultured neurons could also potentially alter cell susceptibility to death. The reasons for
these differences have to be further resolved.
3.2.5. O6er6iew on dopaminergic neurotoxins and

apoptosis
Despite controversial data, it appears that the toxic
mechanisms induced by 6-OHDA, MPTP and, to a
lesser extent, DA, could be similar and follow a comparable regulated cell-death cascade (Fig. 6). These
compounds induce cellular oxidative stress and mitochondrial inhibition leading to cell death. This inhibition, accompanied by DNA damages, could trigger p53
induction, itself responsible for Bax activation. Altogether, these phenomena induce loss of the mitochondrial membrane potential and caspase activation. These
data are essentially supported by in-vitro studies, although the few data obtained in animals are well
correlated. However, besides the role of p53, Bcl-2
family proteins and caspases, a number of recent studies show that other cellular systems such as NF-kB and
JNK pathways, interconnected with the cell death pathways, may have some role in the modulation of nigral
cell degeneration.
3.2.5.1. NF-sB transcription factor. The transcription
factor NF-kB (nuclear factor kB) is a dimeric complex
of proteins of the Rel-family that can regulate several
cellular functions upon activation by a variety of extracellular stimuli (Piette et al., 1997). NF-kB, frequently
composed of p50 and p65/RelA proteins, is retained in
155
an inactive state in the cytoplasm by interaction with an

inhibitory protein of the IkB family. Upon stimulation,
the IkB protein is phosphorylated and proteolytically
degraded, allowing nuclear translocation of NF-kB, its
binding to cognate DNA sequences and induction of
transcription of target genes (Henkel et al., 1993).
In the nervous system, NF-kB is found in both glia
and neurons (ONeil and Kaltschmidt, 1997) and is
activated in response to neurotoxic stimulations such as
glutamate (Kaltschmidt et al., 1997; Grilli et al., 1996),
b-amyloid peptide (Kaltschmidt et al., 1997), kainate
(Perez-Otano et al., 1996), quinolinic acid (Qin et al.,
1998) or after cerebral vessel occlusion in rat (Clemens
et al., 1997; Seegers et al., 2000). Interestingly, NF-kB
activation is usually considered as a regulator of the
cell-stress response, in particular following oxidative
stress. However, the precise activation pathway(s) used
by oxidants in many cell types are still ill defined (Piette
et al., 1997), although low levels of ROS, especially
H2O2, are thought to act as messengers (Schreck et al.,
1991; Shi et al., 1999). In keeping with this role, NF-kB
is activated by exposure of catecholaminergic cells to
6-OHDA and MPP+ (Blum et al., 2001; Cassarino et
al., 2000; Lee et al., 2001; Panet et al., 2001) but also in
nigral neurons of PD patients (Hunot et al., 1997).
Recently, NF-kB has gained increasing attention for
its possible role as a critical regulator of cell death
(review in Barkett and Gilmore, 1999). However, the
functional significance of these findings is still unclear
and NF-kB activation leads to various effects on neuronal cell death (Lipton, 1997). The inhibition of NFkB activity has indeed been shown to protect cortical
neurons against glutamate or quinolinic acid-induced
neuronal injury both in vitro and in vivo (Grilli et al.,
1996; Qin et al., 1998) and to participate in the neuroprotective effect of melatonin and normelatonin against
hydrogen peroxide insult (Lezoualch et al., 1998a).
Conversely, the anti-apoptotic function of NF-kB is
supported by several studies showing that the activation
of NF-kB protects primary neuronal cells against
b-amyloid (Barger et al., 1995; Mattson et al., 1997),
mediates nerve growth factor (NGF)-promoted survival
of PC12 cells (Taglialatela et al., 1997) and increases the
resistance of neuronal cells against hydrogen peroxide
(Lezoualch et al., 1998b). In that way, NF-kB activation found after 6-OHDA or MPP+ exposure could
correspond to a protective mechanism against deleterious effects of these neurotoxins (i.e. oxidative stress)
(Blum et al., 2001; Cassarino et al., 2000), although the
exact role of this transcription factor in the regulation
of apoptosis is not fully understood. However, a role of
NF-kB-dependent transcription in the regulation of cell
death genes has recently been demonstrated (review in
Barkett and Gilmore, 1999). A few target genes of
NF-kB involved in the regulation of cell death have
been identified. Several reports thus suggest that some
156
bcl-2 family genes, i.e. bcl-xL and bfl-1/A1, contain

NF-kB sites in their promoter (Dixon et al., 1997; Lee
et al., 1999; Zong et al., 1999) and are upregulated
through NF-kB activation (Grumont et al., 1999;
Tamatani et al., 1999; Wang et al., 1999a). From this
point of view, several anti-apoptotic factors besides
Bcl-2 family members are regulated by Rel/NF-kB including inhibitory apoptosis proteins (IAP; Chu et al.,
1997; Wang et al., 1998; Kawakami et al., 1999).
Therefore, NF-kB activation could modulate the occurrence of cell death and would be an interesting
potential target site for neuroprotection.
3.2.5.2. The JNK pathway. The c-jun N-terminal kinase

(JNK) group of MAP kinases (mitogen-activated
protein), also known as stress-activated protein kinases
(SAPK), represents a group of enzymes that are activated in cells by exposure to cytokines, environmental
stresses and growth factor withdrawal (for review, see
Su and Karin, 1996). Three different genes ( jnk1, jnk2
and jnk3 ) encode JNK proteins, and each of them gives
alternative spliced isoforms (Gupta et al., 1996) whose
in-vitro translation leads to synthesis of two major
proteins of approximatively 46 and 54 kDa. The jnk1
and jnk2 genes are ubiquitously expressed, whereas jnk3
expression is restricted to the brain, heart and testis.
JNKs proteins are activated by dual phosphorylation of
Thr and Tyr residues within a Thr Pro Tyr motif.
This mechanism of activation establishes JNKs as part
of the wider MAP kinase family. The phosphorylation
state of JNKs is regulated by MKK4 and MKK7
(MAP kinase kinases), also called JNK kinases (JNKK)
(Derijard et al., 1995; Tournier et al., 1997), and the
translocation of activated JNKs into the nucleus is
controlled by JIPs (JNK-interacting proteins) and
GSTp (glutathione S-transferase Pi), which retain
JNKs in the cytoplasm as cytoplasmic anchors (Dickens et al., 1997; Adler et al., 1999). Since JNKs are
activated by phosphorylation, they can be inactivated
by Ser/Thr and Tyr protein phosphatases, also called
MAP kinase phosphatases (MKPs) (Hirsch and Stork,
1997). Activated JNK protein kinases phosphorylate
serine residues 63 and 73 of the c-Jun activation domain, leading to increased AP-1 transcription activity
(Derijard et al., 1994; Kyriakis et al., 1994) but also
ATF-2 (Gupta et al., 1995). JNKs can also antagonize
the anti-apoptotic function of Bcl-2 (Park et al., 1997)
possibly by phosphorylation (Maundrell et al., 1997),
and activate the tumor suppressor p53 (Milne et al.,
1995), a proapoptotic transcription factor.
Less is known about the function and activation of
JNKs under physiological conditions. However, although these kinases are activated following nerve-fiber
lesion and could participate in neuronal regeneration
(Herdegen et al., 1998), there is growing evidence to
suggest that JNKs are potent effectors of neuronal
apoptosis both in vivo and in vitro. Thus, previous

studies on the mechanism of neuronal cell death following NGF withdrawal indicate that expression of c-jun is
induced by growth factor removal (Estus et al., 1994;
Ham et al., 1995). Its role in death is confirmed by the
observation that microinjection of antibodies specific
for c-Jun (Estus et al., 1994) or expression of a c-Jun
dominant negative mutant (Ham et al., 1995) protects
sympathetic neurons from cell death induced by NGF
withdrawal. Furthermore, activation of JNKs and the
consequent phosphorylation of c-Jun have been observed in numerous neuronal death models, such as
trophic factor withdrawal in sympathetic neurons
(Virdee et al., 1997; Eilers et al., 1998), cerebellar
granule neurons (Watson et al., 1998; Le-Niculescu et
al., 1999), neuronally differentiated PC12 cells (Xia et
al., 1995; Maroney et al., 1999; Kanamoto et al., 2000;
Lambeng et al., unpublished results) and motoneurons
(Maroney et al., 1998) but also during ceramide-induced apoptosis in PC12 cells (Hartfield et al., 1998) or
HIV-1-induced death of neurons and microglial cells
(Lannuzel et al., 1997).
In an attempt to elucidate the molecular mechanisms
that lead to dopaminergic cell death in Parkinsons
disease, JNKs activation has been analysed during 6OHDA or MPTP treatments. Human SH-SY5Y neuroblastoma cells that are submitted to MPP+ treatment
exhibit JNK activation, dependent on mitochondrial
adenine nucleotide translocator activity, which controls
the mitochondrial membrane potential (Cassarino et
al., 2000). In a murine dopaminergic neuronal cell line
called MN9D, 6-OHDA induces ROS-mediated JNKs
activation and apoptosis (Choi et al., 1999a). Scavenging ROS with antioxidants such as N-acetyl-cysteine or
a cell-permeable superoxide dismutase mimetic inhibits
both JNKs activation and apoptosis induced by 6OHDA (Choi et al., 1999a). Implication of the JNKs
pathway in apoptosis was also observed in 293 cell lines
and primary neonatal rat striatal cell cultures treated by
dopamine (Luo et al., 1998b). In this study, the role of
increased JNK activity, phosphorylation of c-Jun and
subsequent increase in c-Jun protein in apoptosis was
confirmed by the transient expression of a dominant
negative mutant JNKK that prevents both dopamineinduced JNK activation and cell death. In-vivo experiments further implicate the JNK pathway in the
molecular mechanisms of dopaminergic neuronal death.
In fact, prolonged expression of c-Jun is induced in the
substantia nigra of MPTP-treated mice, and the cells
expressing c-Jun are dopaminergic neurons (Nishi,
1997). CEP-1347, an inhibitor of JNK activation, attenuates MPTP-mediated nigrostriatal dopaminergic loss,
indicating that the JNK signaling pathway may be
activated by MPTP administration (Saporito et al.,
1999). Furthermore, MPTP increases levels of phosphorylated JNK and JNK kinase MKK4 in the nigrostri-
atal system. These phosphorylations were inhibited by

CEP-1347 at a dose that attenuates MPTP-induced
dopaminergic loss (Saporito et al., 2000). These data
implicate the JNK signaling pathway in MPTP-mediated nigrostriatal dopaminergic death and suggest that
it may be activated in the degenerative process in
Parkinsons disease.
Finally, intrastriatal dopamine injection produces a
strong activation of AP-1 composed by c-fos, c-jun and
phosphorylated c-Jun protein (Luo et al., 1999). Interestingly, activity of the transcription factor NF-kB is
also stimulated by dopamine injections. Both JNK and
NF-kB can be activated in response to NMDA neurotoxicity in cortical cell cultures (Ko et al., 1998) or after
treament with MPP+ in neuroblastoma cells (Cassarino
et al., 2000) and may be substrates of a common kinase
named MEKK1 (Lee et al., 1998), but this result needs
further confirmation (Karin and Delhase, 1998). These
last data strongly suggest that the apoptotic cascade,
besides the proteins directly involved, is controlled by
connex transcriptional systems such as JNK and NFkB pathways, themselves interconnected. All these interconnections and their regulatory checkpoints need to
be studied further in experimental models to grasp the
complete scheme of events occurring in catecholaminergic cell degeneration.
4. Relevance of experimental data for human nigral

degeneration
All the data presented here strongly suggest that
experimental in-vitro and in-vivo models of PD may
help to determine which factors could be critical in the
molecular pathways leading to degeneration of nigral
cells. However, these results are only indicative and
need post-mortem confirmation to estimate the real
occurrence of such processes. Only a small amount of
data are available to date, probably due to the complexity of obtaining and interpreting results obtained in
human samples (see Section 2.4). Involvement of p53 in
nigral cell death is still ill defined. One study showed an
increase in neuronal and glial p53 immunoreactivity in
the SNpc of PD patients (De la Monte et al., 1998).
This modification was located in both degenerating
(atrophic and depigmented neurons) and intact neurons, whereas Lewy bodies containing neurons were
p53-negative (De la Monte et al., 1998). Nevertheless,
these findings were not confirmed by Jellinger (2000).
Interestingly, only the former study suggested nigral
apoptosis. It is therefore possible that, if apoptosis
occurs in parkinsonian SNpc, it involves a p53-dependent mechanism. This remains to be further seen.
Other authors aimed to demonstrate an involvement
of Bcl-2 related proteins. It was recently suggested that
Bax immunoreactivity is increased in melanized neu-
157
rons of parkinsonian SNpc, supporting the occurrence

of apoptosis in PD (Tatton, 2000). Some authors also
described that Bcl-2 protein levels were increased in the
SNpc but also in caudate nucleus, putamen and globus
pallidus of parkinsonian patients by either Western
blotting (Marshall et al., 1997) or enzyme-linked immunosorbent assay (Mogi et al., 1996). These results
were interpretated as a compensatory increase of Bcl-2
in the basal ganglia, indicating an adaptative response
to stress. However, no alteration of Bax or Bcl-2 expression, assessed by either immunohistochemistry or
hybridization, was detected in three other studies (Vyas
et al., 1997; Wu llner et al., 1999; Jellinger, 2000). Moreover, in contradition with the results of Tatton (2000),
no relationship has been established between the presence of Lewy bodies and the level of Bax immnoreactivity, suggesting that Bax increase could not be restricted
to damaged nigral neurons (Tortosa et al., 1997). It is
possible, however, that Lewy body-containing neurons
only expressed Bcl-2-related proteins for a short time
and that the peak of expression occurs in depigmented
neurons irreversibly engaged in the death process.
The loss of mitochondrial membrane potential, presumably a direct consequence of Bax activation, is not
well documented, probably due to the difficulty in
performing functional studies on post-mortem samples.
However, mtDNA deletion and oxidative stress could
lead to a decrease in the mitochondrial potential, as
suggested in degenerating human SNpc (Ozawa et al.,
1997b). This could also be expected considering the
probable involvement of caspases in nigral degeneration. Indeed, unlike the results of Jellinger (2000), two
recent studies demonstrated, by either immunohistochemical or enzymatic methods, that caspase-3, but
also caspase-1, activities were increased in the SNpc of
PD patients (Mogi et al., 2000; Tatton, 2000). Additionally, a positive correlation between the number of
caspase-3 positive neurons in the SNpc, ventral tegmental area and central gray matter, and the percentage of
cell death in PD was established, suggesting that the
presence of this protease may be a vulnerability factor
to degeneration in PD (Hartmann et al., 2000). Finally,
the Ala53Thr mutation of the a-synuclein gene abolishes the negative control that the normal form of this
protein exerts on caspase activation, thus suggesting
that caspase activation critically contributes to autosomal dominant forms of PD (Da Costa et al., 2000).
The involvement of other regulators of apoptosis in
nigral degeneration is unclear. NF-kB was shown to be
activated in nigral cells of PD patients (Hunot et al.,
1997), although its contribution to the cell death process was not determined. Concerning the JNK pathway, although only one study mentioned that c-jun is
not activated in degenerating SNpc (Jellinger, 2000),
there is no available information on the role of JNKs
or MKK in PD brains.
158
All these data are not sufficient to clearly ascertain

the exact molecular pathways involved in parkinsonian
SNpc but suggest that common mechanisms could exist
in dopaminergic cell death induced in human or after
exposure to neurotoxic compounds.
5. Conclusion
Throughout this review, we have presented evidence
suggesting that experimental models reproduce the
main cellular modifications occurring in PD. 6-OHDA,
DA and MPTP are able to induce two of the main
biochemical defects in PD, namely oxidative stress and
mitochondrial inhibitions as well as histological lesions.
Other aspects of the human disease are, however, not
reproduced (Hantraye, 1998): the precise anatomical
lesion, the time course of the disease (decades vs. weeks
at best), the occurrence of long-term compensatory
mechanisms which could influence the degeneration
rate and the associated molecular mechanisms etc.
Nevertheless, these models helped to bring new insights
and new hypotheses to the molecular events occurring
in PD. Although not fully understood, convergent data
do exist between the results obtained in human disease
and experimental models. It is also important to consider that the study of various models can avoid taking
epiphenomena for general facts, and, conversely, they
underline the common mechanisms presumably important for the human disease. Therefore, the continuous
interaction between models and human neuropathology
will undoubtely help to determine precisely the crucial
actors of nigral cell death and to elaborate new specific
and neuroprotective strategies.
Acknowledgements
This work was supported by INSERM, Universite
Joseph Fourier and MESR. D.B. holds financial support from Fondation pour la Recherche Me dicale
(France), Fondation Simone et Cino Del Duca (France)
et Fonds National pour la Recherche Scientifique (Belgium). We thank Prof. J.P. Brion, Dr M.C. Galas, Dr
D. Gall, Dr P. Laduron, Dr V. Gaveau, Dr R. Hourez
and Prof. S.N. Schiffmann for critical reading of the
manuscript and Dr F.J. Hemming for English revision.
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Progress in Neurobiology 65 (2001) 135 172

Molecular pathways involved in the neurotoxicity of 6-OHDA,

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

3. Contribution of experimental neurotoxins to the study of apoptosis in PD. . . . . .

4. Relevance of experimental data for human nigral degeneration . . . . . . . . . . . . . . . . .

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

2.1. Clinical features

2.2. Nigral degeneration and etiology

part of the SNpc (Hirsch et al., 1988; Goto et al., 1989;

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

kindred and Greek families, a gene responsible for

2.3. Vulnerability, oxidati6e stress and mitochondrial

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

2.3.2. Oxidati6e stress

cleotide coenzyme Q reductase) of the mitochondrial

2.3.3. Mitochondrial defects

2.4. In6ol6ement of apoptosis in nigral degeneration

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

(1997) observed that apoptotic cells never contained

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

1,2,3,6-tetrahydropyridine (MPTP). These findings are

3. Contribution of experimental neurotoxins to the

3.1. Mechanisms of 6 -OHDA, dopamine and MPTP

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

Maruyama, 1999). Indeed, several studies reported the

amounts of 6-OHDA and 6-nitrodopamine (Palumbo

3.1.1.2. Oxidati6e stress. 6-OHDA was suggested to

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

H2O2 + Fe2 + OH + OH + Fe3 + .

strand break, characterized by an elevation in 8-hydroxy-2%-deoxyguanosine levels (8-OHDG), particularly

3.1.1.3. Mitochondrial defects. Several lines of evidence

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

ing that 6-OHDA does not need to enter into the CA

3.1.2. Neurotoxicity of dopamine

of 6-OHDA and neuromelanin (Slivka and Cohen,

3.1.2.2. Oxidati6e stress and mitochondrial defects.

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

and nucleic alterations (Moldeus et al., 1983; VelezPardo et al., 1997).

3.1.2.3. Specificity. DA toxicity is not strictly specific

3.1.3. Neurotoxicity of MPTP

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

MPTP into the SN and the medial forebrain bundle as

1985; Ricaurte et al., 1986, 1987) as shown with another

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

3.1.3.2. Mitochondrial defects and oxidati6e stress. The

Indeed, NMDA receptor antagonists (Turski et al.,

3.2. Molecular pathways of cell death induced by

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

gram was first based on the observations of stereotyped

3.2.1.3. Caspases. The execution phase of apoptosis is

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

cellular changes leading to the structured dismantling of

3.2.1.4. Bcl-2 family members. Members of the Bcl-2

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

vated and/or the cellular context. However, despite

of the p53 suppressor gene (White, 1996; Xiang et al.,

3.2.1.5. Inhibitor of apoptosis proteins (IAPs). IAPs were

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

In vivo, TUNEL positive cells were observed in the

D. Blum et al. / Progress in Neurobiology 65 (2001) 135172

cells is always low both in vivo (He et al., 2000) and in

OHDA. Indeed, in-vitro Bcl-2 over-expression