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-synuclein modulate dopaminergic synaptic content and tone at the synapse?
Department of Pediatrics, Georgetown University, Washington, D.C., USA; and
* Institut de Neurobiologie Alfred FESSARD, C.N.R.S., 91198 Gif-sur-Yvette cedex, France
1 Correspondence: Laboratory of Molecular Neurochemistry, The Research Building, Room W222, 3970 Reservoir Road, NW, Washington, DC 20007, USA. E-mail: sidhua{at}georgetown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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-Synuclein is a key component of the pathological process of neurodegeneration in Parkinson’s disease. Although its contributions to normal physiological conditions remain elusive, converging observations suggest that a primary function of this protein in dopaminergic neurons may be the regulation of dopamine content and synaptic tone at the synapse. We review here cumulative evidence that demonstrates the participation of -synuclein in the life cycle of dopamine from its synthesis, storage, release, and reuptake. The regulatory role of -synuclein on dopamine metabolism is assessed by discussing the experimental evidence supporting each of these observations in the healthy physiological maintenance of dopaminergic neurons, as well as showing how disruption of these events can initiate the observed neurotoxicity of -synuclein and the genesis of the degenerative processes associated with Parkinson’s disease.—Sidhu, A., Wersinger, C., Vernier, P. Does -synuclein modulate dopaminergic synaptic content and tone at the synapse?
Key Words: dopamine transporter • Parkinson’s disease • synucleopathies • MPTP • neurodegeneration • Lewy bodies
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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-SYNUCLEIN, a synaptic protein of unknown function, is a major component of Lewy bodies (LBs), neuronal cytoplasmic inclusions of aggregated proteins characteristic of idiopathic and familial form of Parkinson’s disease (PD) (1
2
3
4
5
6
7
8
9)
. However, -synuclein aggregates are not specific to PD since the protein also accumulates in the neuronal and glial cytoplasmic inclusions of multiple system atrophy (2
, 9
, 10)
or in the LBs of variants of Alzheimer’s disease (7)
, Down syndrome with Alzheimer’s disease (11)
, and other neurodegenerative diseases collectively known as synucleinopathies (2
, 3
, 5
6
7
8
, 12)
. The association of familial PD with alanine30-proline (A30P) and alanine53-threonine (A53T) mis-sense mutations in the gene encoding -synuclein (13
, 14)
, which are not found in idiopathic PD (15
, 16)
or other forms of inherited PD (17)
, has spurred enormous interest in the pathogenesis of -synuclein and its accumulation into LBs.
How -synuclein may be implicated in the pathophysiological process of PD is far from obvious. -Synuclein exhibits different conformations depending on its interacting environment, such as natively unfolded in solution or the formation of -helix when associated with lipid vesicles (18
, 19)
. A prerequisite of -synuclein neuropathy is its oligomerization into soluble protofibrils (20
, 21)
, followed by their coalescence into insoluble fibrils (7
, 21
, 22)
, comprised of ß-sheets and amyloid-like filaments (23
24
25)
, before their aggregation into insoluble fibrillar structures and inclusions, which then accumulate into LBs. Several experimental conditions, including overexpression of the protein, the presence of the A30P or A53T mutations, exposure to neurotoxins and oxidative factors, induce or accelerate -synuclein aggregation both in vivo and in vitro through procedures that remain poorly understood, even though formation of ß-pleated sheets in the structure of -synuclein is a common feature of these pathological conditions (25
26
27
28
29
30
31
32)
. Both the mutant forms of -synuclein and neurotoxins are associated with early-onset or juvenile PD, suggesting a causal link with the neurodegeneration. Once aggregation is initiated, the bioavailability of -synuclein is substantially diminished such that normal physiological functions regulated by this protein may be severely compromised, which in turn could further exacerbate the initial cellular insult. Thus, loss-of-function phenomena need to be taken into account in the pathophysiology of PD, at least as to a gain of toxic function due to protein aggregates.
A puzzling aspect of -synuclein-mediated cytotoxicity and LBs formation with regard to PD is the preferential and selective neurodegeneration of dopamine producing neurons, such as the substantia nigra. -Synuclein is ubiquitously expressed at high levels in virtually all regions of the brain, where it is believed to account for up to 0.1% of total brain proteins (26
, 27
, 33)
. If high expression levels of -synuclein were the sole cause of protofibril formation and LBs, then most brain regions could be expected to have aggregates of -synuclein. Yet in PD the substantia nigra neurons, which express considerably less -synuclein than other areas of the brain (C. Wersinger, M. Banta, and A. Sidhu, unpublished results), are specifically degenerated whereas other brain regions expressing higher levels of -synuclein are relatively spared. Increasingly then, the evidence suggests the likelihood that the specific vulnerability of these neurons is linked to other factors, the prime candidate being dopamine itself, which necessarily can confer the selectivity for these neurons. A direct link between the presence of dopamine and the induction of -synuclein aggregation may be a key feature of the neurodegeneration of PD. Indeed, in dopamine neurons of the substantia nigra, dopamine is metabolized via enzymatic deamination by monoamine oxidases (MAO), with production of the nontoxic 3,4-dihydroxyphenylacetic acid (DOPAC) and H2O2 (34)
. In turn, H2O2 can be converted to highly toxic hydroxyl radicals in a reaction catalyzed by iron transition metals, which are found at high levels associated with neuromelanin in the substantia nigra pars compacta (35)
. Dopamine can also undergo spontaneous autoxidation, at normal intracellular pH and in the presence of molecular oxygen, into toxic and reactive dopamine-quinones, superoxide free radicals, and hydrogen peroxide (36)
. Moreover, superoxide can be either converted to H2O2 by superoxide dismutases or into labile, but very reactive and cytotoxic, peroxinitrite radicals in the presence of nitric oxide. Since oxidized dopamine has been shown to stabilize the formation of -synuclein protofibrils (20)
, the toxic form of -synuclein (21)
, by inhibiting the conversion of protofibrils into insoluble fibrils (20)
(imbalances in intracellular levels and oxidative stress of dopamine) may constitute the key initial step in a chain of events that ultimately leads to the cell death of these neurons in PD (37
, 38)
.
We will present evidence, both direct and indirect, showing a close relationship between -synuclein and dopamine, whereby this protein probably participates in regulating the biosynthesis, storage into vesicles and release, as well as the reuptake of this neurotransmitter. It appears that -synuclein may be an adaptor protein or a chaperone, which can indeed regulate nearly every step of the biocycle of dopamine in neurons (Fig. 1
). We suggest that a major physiological role is to modulate the function of the dopamine transporter of the plasma membrane, the key molecule dictating dopaminergic content and synaptic tone of dopamine. Conversely, in pathogenic instances that promote imbalances in intracellular dopamine content, and thereby its oxidative state, -synuclein may become a primary target of attack by reactive species generated upon dopamine autoxidation and/or metabolism and may play a central role as a mediator or facilitator of the dopaminergic neurotoxicity that occurs in PD.
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Structural and functional characteristics of -synuclein
Human -synuclein, a highly conserved acidic protein among the vertebrates phylum, is a 140 amino acid protein of 
19 kDa (26
, 39
, 40)
, originally isolated from amyloid plaques of Alzheimer’s disease brains as a protein called the precursor of the non-Aß-amyloid component (or NAC) of plaques (41
, 42)
and from cholinergic vesicle preparations isolated from the electric organ of the ray Torpedo californica (43)
. -Synuclein belongs to a multigene family encoding structurally closely related proteins that are abundantly expressed in various brain regions and include the - and ß-synucleins and synoretin or 
-synuclein (27
, 40
, 44)
. All three members are highly enriched in presynaptic terminals (43
, 45)
, where -synuclein in particular is found in close proximity to, but loosely associated with, synaptic vesicles (45
, 46)
. -Synuclein immunoreactivity appears equally distributed between cytosolic and intracellular membrane fractions (47
48
49)
and is widely expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, thalamus, and cerebellum (45
, 47)
, and also in the amygdala and nucleus accumbens (C. Wersinger, M. Banta, and A. Sidhu, unpublished results). Somata of dopamine neurons of the substantia nigra pars compacta contain only low levels of -synuclein, which in turn may be accumulated inside the terminals of the corresponding projections in the striatum, but this is still a matter of debate (33
, 50
51
52
53
54)
.
Structurally, -, ß-, and -synucleins share a common design composed of three modular protein domains, including a highly conserved amino-terminal lipid binding -helix (residues 7-87), a variable internal hydrophobic NAC domain (residues 61-95), and a carboxyl-terminal acidic tail (residues 95-140) composed primarily of glutamate and aspartate residues (27
, 40
, 44)
. Over half of the synuclein molecule (amino acids 7-87) is composed of seven imperfect repeat sequences of 11 amino acids, each with the core consensus sequence motif KTKEGV (26
27
28
, 40
, 46
, 55)
. Although the function of these repeats is unknown, the structure of -synuclein allows the molecule to exist in a poorly structured conformation or as an -helix in the presence of phospholipids (18
, 19)
, suggesting highly dynamic changes depending upon the local cellular milieu. According to recent nuclear magnetic resonance studies, the lipid binding domain is composed of two -helices when bound to phospholipids or synthetic membranes (55
, 56)
. Other observations suggest that there is an O-glycosylated 22 kDa form of -synuclein, a specific substrate for the E3 ubiquitin ligase activity of parkin that accumulates in autosomal recessive PD, leading to juvenile onset of the disease (57)
. - and ß-synuclein are distinguished by the presence of 11 residues in -synuclein that defines the core of the NAC binding domain, the building block of -synuclein aggregates (58
59
60
61)
. Indeed, it has been recently demonstrated that the "GAV motif" (residues 66 to 74 in the NAC domain) may be responsible for aggregation or fibrillization of -synuclein (61)
.
The function(s) of -synuclein is still unknown; it has been implicated in too many cellular processes to draw a unifying view of its physiological contribution. An interesting clue to -synuclein function would be that -synuclein is a chaperone protein closely related to the function of 14-3-3-chaperone molecules (62
, 63)
, with which it shares considerable sequence homology, thus displaying pleiotropic effects in cells. Indeed, -synuclein is involved in many molecular interactions, especially at presynaptic sites implicated in synaptic vesicle formation, axonal transport, and dopamine synthesis and metabolism, which will be now examined in more detail.
-Synuclein effects on synaptic vesicles
-Synuclein is highly enriched in presynaptic terminals. A role of -synuclein in synaptic plasticity has been envisaged, given the strong modulation of -synuclein expression that has been observed in physiological situations of plastic remodeling of neuronal networks, such as song systems in birds or long-term potentiation in the mammalian hippocampus (26
, 27
, 40)
. Indeed, -synuclein may regulate synaptic vesicle mobilization at nerve terminals (Fig. 2
). In a seminal study, Abeliovich et al. (64)
showed that -synuclein knockout mice exhibited significant impairments in synaptic response to tetanic stimulation. Then, Cabin et al. (65)
found that cultured hippocampal neurons, in which -synuclein expression was knocked down with antisense oligonucleotides, had fewer synaptic vesicles than their control littermates, particularly in the reserve pool. The replenishment of docked vesicles by reserve pool vesicles after depletion was slower in the mutant synapses. -Synuclein-depleted cells also exhibited decreased expression levels of synapsin, an essential protein for synaptic vesicle recycling (65
, 66)
. Thus, -synuclein may be required for the genesis and/or maintenance of a subset of a reserve pool of presynaptic vesicles.
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The molecular mechanisms of the action of -synuclein on the formation and maintenance of synaptic vesicles may involve regulation of key components of the metabolic pathways generating phospholipids of the cellular membrane. It should be recalled here that the highly conserved amino-terminal end of -synuclein binds to phospholipids, acquiring an -helical conformation in this lipidic environment (18
, 19
, 56)
. The role of -synuclein as an adaptor or chaperone protein probably extends to phospholipases and lipid binding proteins. Indeed, -synuclein physically interacts with and inhibits the activity of phospholipase D2 (PLD2) and, to a lesser extent, PLD1, through its amino-terminal repeat region (residues 7–87; refs 67
, 68
). PLD2 is a transmembrane enzyme of the plasma and endosomal membranes that hydrolyzes phospholipids such as phosphatidylcholine into lysophosphatidylcholine and phosphatidic acid in response to external stimuli. PLD2-derived phosphatidic acid is able to recruit adaptor molecules, which in turn trigger the building of vesicles from donor membranes (69)
. PLD2 may therefore be important for vesicles formation and indirect evidence shows that PLD2 might regulate the recycling of synaptic vesicles at or near the plasma membrane or endosomal compartments in response to external stimuli (37
, 38)
. Therefore, by modulating PLD2 activity, a putative key function of -synuclein may be to regulate synaptic vesicular recycling (Fig. 2)
.
This putative physiological function of -synuclein may be tightly regulated by various intracellular seryl/threonyl protein kinases or tyrosyl protein kinases that phosphorylate -synuclein at various sites (70
71
72
73
74)
. For example, phosphorylation of -synuclein by G-protein-coupled receptor kinases lowers the ability of -synuclein to inhibit PLD2 activity and reduces binding of -synuclein to phospholipids (72)
. Thus, through reduction of its tonic inhibition of PLD2, phosphorylated -synuclein might promote vesicle recycling during periods of high neuronal activity and favor synaptic plasticity.
Other data also suggest that -synuclein may modulate vesicles recycling due to its fatty acid binding protein (FABP) properties (49)
. This contention relates to the fact that the vesicle binding, amphipathic amino-terminal region of -synuclein (residues 7-87) bears significant homology to the lipid binding class A apolipoproteins A2 and C1-3, proteins implicated in lipid transport and major components of LBs (1)
. Moreover, short amino acyl stretches in -synuclein amino and carboxyl termini share >55–67% identity with a cytosolic fatty acid binding motif of FABPs (49)
. -Synuclein might therefore transfer fatty acids, which are highly enriched in synaptic vesicles, to sites of synaptic vesicle formation (i.e., early endosomes) and/or regulate, as a lipid chaperone, the turnover or local organization of polyunsaturated fatty acid acyl groups that have been implicated in clathrin-mediated endocytosis (75)
and therefore in vesicles recycling (76)
.
The recent observation that -synuclein and its familial PD-linked mutants bind to calmodulin in a Ca2+-dependent manner and that this binding potentially alters the conformation of -synuclein, with activated calmodulin accelerating the formation of fibrils in vitro suggests that -synuclein may have a role in Ca2+-dependent signaling pathways, including exocytosis, or that the known calcium control of -synuclein function is mediated through an interaction with calmodulin (77)
. Lee et al. (78)
observed a competition of -synuclein between calmodulin and artificial membranes. In these experiments, -synuclein previously bound to liposomes was released upon specific interaction with calmodulin, suggesting that the interaction of -synuclein with calmodulin may regulate the amount of membrane-bound -synuclein. These observations are consistent with the proposal that, in both dopaminergic and nondopaminergic neurons, -synuclein may have a prominent role in neuronal development and plasticity (26
27
28
29
, 79
80
81)
or regenerative sprouting of damaged axons (82
83
84)
and in neuroprotection (80
, 84
85
86
87
88
89
90)
.
Finally, -synuclein has been suggested to interfere with axonal transport of synaptic vesicles (28
, 40)
by interacting with several proteins that either bind to or are part of the cytoskeleton such as tubulin (91
, 92)
, tau (71)
, mitogen-activated protein kinase 1B (MAP1B) (93)
, MAP2 (94)
, synphilin-1 (95
, 96)
, and torsin A (97)
.
Nevertheless, these effects of -synuclein are very general and cannot account for a specific role in the neurodegeneration of dopamine cells in PD. However, emerging studies (37
, 38
, 66
, 98)
suggest that the effects of -synuclein on synaptic vesicles may be responsible for affecting the vesicular content of dopamine, its storage, and release.
-Synuclein regulates the functional activity of tyrosine hydroxylase, modulating the amount of dopamine synthesized
Dopamine is synthesized in selective groups of neurons of the central and peripheral nervous system. In mammals, substantia nigra pars compacta and ventral tegmental area are the most prominent dopamine nuclei in the brain. Dopamine synthesis depends on a rate-limiting step, the conversion of tyrosine into L-DOPA by phosphorylated tyrosine hydroxylase (TH) before being converted into dopamine by the rather ubiquitous aromatic amino acid decarboxylase. Only phosphorylated TH is active, and the phosphorylation and dephosphorylation of TH are primary in the signaling cascade regulating dopamine biosynthesis (99)
. Several cellular signaling events can modulate the functional activities of the kinases and phosphatases that use TH as substrate. Recently, a reciprocal interplay between -synuclein and 14-3-3 proteins for the regulation of TH activity has been suggested (100)
. The chaperone protein 14-3-3 binds to phospho-TH, and is necessary for maximal phosphorylation of the enzyme (101
, 102)
. The 14-3-3 proteins also protect TH from dephosphorylation and increase the half-life of activated TH in neurons (103)
. Conversely, -synuclein has been shown to colocalize with and directly bind to TH, causing an overall net decrease in enzymatic activity and dopamine synthesis (104)
. -Synuclein binds to the dephosphorylated form of TH and tends to maintain TH in an inactive form, in an opposite and symmetric manner to 14-3-3 proteins (104)
.
The A30P and A53T mutant -synucleins have the same inhibitory effects on TH as the wild-type protein (104)
, leading to the proposal that altered regulation of TH by the -synuclein may not play a role in the pathogenesis of PD (37)
. However, in the pathological process, precipitation and aggregation of -synuclein reduce the availability of cytoplasmic -synuclein, leading to an increase of TH activity mediated by 14-3-3 protein. Since the TH activity is highly dependent on the phosphorylation of the enzyme by several kinases, it is probable that the opposite actions of 14-3-3 protein and -synuclein are mediated in part through this phosphorylation process. Indeed, 14-3-3 proteins favor the activity of calcium-calmodulin-dependent kinase (101
, 102)
and of the MAP kinase ERK by acting at different steps of the MAP kinase (105)
and protein kinase C pathways (106)
. In contrast, -synuclein tends to inhibit the activity of ERK through its direct binding to the MAP kinase (107
, 108)
, an effect also seen with the A53T mutant (108)
. This inhibitory effect of -synuclein can be extended to other kinases such calcium-calmodulin-dependent kinases (78)
and PKC (70)
and helps to dampen the TH activity.
The imbalance between the effects of -synuclein and 14-3-3 protein is highly deleterious since overproduction of cytosolic dopamine generates highly reactive species such as quinones and superoxide free radicals. Moreover, the formation of complexes between 14-3-3 and -synuclein, when this latter tends to aggregate, lowers the ability of 14-3-3 to inhibit several steps of the apoptotic process. Isoforms of the 14-3-3 proteins are necessary to maintain in an inactive form proapoptotic proteins in the cytoplasm such as Bad or the kinases Fkhrl1 and Ask1 (100
and references therein). Therefore, trapping of 14-3-3 by -synuclein is certainly one of the factor that may further contribute to rendering the dopamine-synthesizing neuron more sensitive to oxidative stress and to initiate a vicious circle of cell injury and degeneration.
A role for -synuclein on catecholamine storage at nerve terminals?
Is -synuclein able to play a specific role in the vesicular storage and release of dopamine? The observed decrease of paired-pulse depression in striatal terminals of -synuclein knockout or knockdown mice (64
, 65)
suggests that -synuclein may act as a negative regulator of neurotransmitter release, probably via its inhibitory action on PLD2 activity (see above). Nonphosphorylated -synuclein is able to suppress synaptic vesicle formation during periods of low neuronal activity. In PC12 pheochromocytoma cells, the expression of A53T -synuclein mutant, but not the wild-type, resulted in a loss of catecholamine-secreting dense core granules and the capacity for depolarization-induced dopamine release (98)
. These studies were later confirmed when the A53T mutant was found to decrease both potassium-induced dopamine release and vesicular dopamine storage through down-regulation of VMAT2, but with an increase in amphetamine-mediated release of dopamine, indicating a depletion of vesicular dopamine and an accumulation of cytoplasmic dopamine (66)
. In vivo positron emission tomography imaging studies of patients with idiopathic PD show larger reduction in VMAT2 labeling than expected from measurement of neuronal loss marker by 18F-L-DOPA (109)
, suggesting that a selective reduction in dopamine storage vesicles or DA storage ability may be a pathogenic feature of PD.
In neurons, synthesized dopamine is immediately taken up and stored into synaptic vesicles through the activity of the vesicular monoamine transporter 2 (VMAT2), preventing its oxidative catabolism by cytoplasmic MAO. VMAT2 is a member of a transporter family of that uses the proton gradient across vesicular membranes as the driving force to sequester monoamines into vesicles (110
111
112)
. The low, acidic pH environment of such vesicles hinders dopamine breakdown by spontaneous autoxidation (112)
. Under normal conditions, efficient dopamine sequestration into storage vesicles is the major mean that protects dopamine neurons from the deleterious effects of dopamine oxidation. It should be stressed that the presence of VMAT2 in presynaptic vesicles attenuates the deleterious effect of MPTP, the most powerful neurotoxin known to induce parkinsonism in human. In VMAT2-heterozygous knockout mice, MPTP is twice toxic as in wild-type mice, probably because VMAT2 is normally responsible for sequestrating MPP+, the toxic metabolite of MPTP (113
; reviewed in ref 114
).
It has been proposed by Lansbury and colleagues that -synuclein protofibrils with an annular shape may form integral pores in membranes, including vesicular membranes, leading to dopamine leakage and increased levels of cytoplasmic dopamine (115
116
117
118
119)
. In that case, both the decrease in cytoplasmic -synuclein and the presence of -synuclein protofibrils will contribute to increase the cellular damage elicited by free, oxidizable dopamine in neurons.
The physiological role of -synuclein in the regulation of synaptic vesicle formation and neuronal plasticity, is probably impaired in dopaminergic neurons during the pathological process of PD. One possibility is that during the pathological process, the formation of protofibrillar aggregates, and later -synuclein precipitates, decrease the levels of available free -synuclein, which normally acts as a neuroprotective component (Fig. 2)
.
-Synuclein modulates the functional activity of the dopamine transporter
One of the main consequences of the presence of -synuclein at the nerve terminals of dopamine-synthesizing neurons is the effect it exerts on the regulation of the dopamine transporter (DAT) of the plasma membrane. The DAT is expressed, as is -synuclein, in presynaptic terminals of substantia nigra neurons where it mediates the reuptake of synaptically released dopamine and accumulates dopamine into dopaminergic nerve terminals (120
, 121
and references therein). DAT is a member of a large gene family of Na+ and Cl–-dependent transporters (122
, 123)
with a common topology of 12 putative transmembrane domains containing numerous consensus sequences for N-linked glycosylation and putative sites for phosphorylation by PKA, PKC, and calmodulin kinase II (124)
. The DAT is a major determinant of dopamine homeostasis and synaptic strength since it is the primary mechanism by which endogenous neurotransmitter is rapidly removed from the synaptic cleft. Increases or decreases in DAT function will concomitantly decrease or increase synaptic dopamine concentrations, respectively, thereby regulating the activity of multiple post- and presynaptic dopamine D1- and D2-like receptors. The DAT is also the sole means by which the parkinsonian syndrome-inducing neurotoxin MPP+ is transported into nigral neurons (125
, 126)
.
The importance of DAT function in the control of synaptic availability of dopamine suggests that its own regulation may be a crucial component in the maintenance of dopaminergic neurotransmission, since enhanced DAT activity would not only decrease extracellular levels of dopamine but also increase intracellular levels of dopamine upon reuptake, resulting in excessive production of reactive oxygen species within the dopaminergic cell (Fig. 2)
. The principal means by which DAT function is regulated is through the rapid shuttling of DAT to and from the plasma membrane. Previous work in cellular expression systems has shown that reduced cellular dopamine uptake mediated by DAT may be directly correlated to the possible phosphorylation of DAT by kinases, causing rapid redistribution and internalization of DAT away from the plasma membrane (127
128
129)
. The participation of kinases such as protein kinase C that are dynamically activated by changes in intracellular [Ca2+] levels, suggests rapid adaptations of the neuron in response to signal transduction-induced changes in calcium flux to attenuate DAT function. Recent data have shown that MAP kinases ERK1 and 2 phosphorylate the DAT upon signal transduction at the plasma membrane, increasing the amount of DAT at the plasma membrane and dopamine reuptake (130)
. Since -synuclein binds to MAPK and tends to inhibit its activity, as shown for the regulation of TH enzyme, it is likely that the presence of -synuclein at the nerve terminals tends to blur MAPK activity and decrease the amount of DAT inserted in the membrane of nerve terminals.
Our laboratories have shown that upon coexpression in Ltk– fibroblasts or in basal conditions in mesencephalic neurons, -synuclein tends to markedly decrease by 30–50% the activity of the DAT by reducing the reuptake of dopamine (131
, 132)
. The reduction in DAT activity was due to decrease in dopamine uptake velocity by the transporter without any change in DAT expression levels. A consequence of the -synuclein-mediated attenuation of DAT activity was that upon exposure of cotransfected cells to dopamine, there was diminished dopamine-induced oxidative stress and cytotoxicity. In the presence of -synuclein, the transporter was dynamically trafficked away from the plasma membrane into the cytoplasm as indexed by reduced DAT presence at the plasma membrane by biotinylation experiments (132)
. From coimmunoprecipitation studies, -synuclein was found to interact directly with the DAT, forming a stable protein:protein heteromeric complex in cotransfected cells, substantia nigra, and mesencephalic neurons. These interactions occurred between the NAC domain (residues 58-107) of -synuclein and the last 22 amino acids of the carboxyl-terminal (CT) tail of DAT (132)
.
Similar to wild-type, the A30P mutant attenuated DAT function, trafficking dopamine transporter away from the plasma membrane and participating in the formation of stable protein:protein complexes, again through the NAC domain (residues 58-107) of A30P and the last 22 amino acids of the CT tail of DAT (133)
. The A53T mutant was unable to modulate DAT function, and subsequent studies showed that this protein interacted only weakly with the transporter (133)
.
In an earlier study -synuclein was found to have an opposite effect on DAT, i.e., to increase the functional activity of the transporter (134)
. This discrepancy is probably due to the fact that that the cells used in this study were batch-transfected and exposed to trypsin after transfection (134)
, suggesting that cell adhesion may play a role in dictating the functional outcome of DAT/-synuclein interactions. Indeed, we subsequently showed that mild trypsinization of cotransfected cells or neurons under conditions that do not cause cell dissociation reversed the attenuation of DAT function by -synuclein, increasing the presence of the transporter at the plasma membrane (132
, 135)
. Most interesting was the finding that the parkinsonian syndrome-inducing agent, MPP+, whose intracellular transport within cells occurs specifically only through the dopamine transporter, also reversed the inhibitory effects of -synuclein on the transporter (132)
. Under all experimental conditions where the dopamine transporter activity was dysregulated, there was increased reuptake of dopamine and dopamine-induced neurotoxicity, suggesting that disruption of the ability of -synuclein to regulate transporter function may be one of the most important determinants in the genesis of dopaminergic neurodegeneration (Fig. 2)
.
Dopamine-linked aberrations convert -synuclein into a toxic molecule
This overview of the physiological and pathological facets of -synuclein points to a tight link between the general roles that -synuclein plays in neurons and the regulation of dopaminergic tone in human mesencephalon neurons while highlighting and reconciling the ubiquitous nature of -synuclein expression with the relative specificity of degeneration of the substantia nigra pars compacta in PD. In a normal situation, the presence of -synuclein in dopamine-synthesizing neurons tends to tone down the amount of cytoplasmic dopamine at nerve terminals, thereby limiting its conversion to highly reactive oxidative molecules. The soluble form of -synuclein is able to 1) decrease the activity of TH (104)
; 2) regulate availability of synaptic vesicles and reuptake by VMAT2 (37
, 38
, 66)
; and 3) reduce the maximal reuptake of dopamine from the extracellular synapse by DAT (131
, 132)
.
However, as soon as -synuclein begins to form protofibrils and precipitates, the bioavailability of the physiological form of soluble -synuclein is decreased and the toxic potential of dopamine at nerves terminals is increased, since the protein can no longer participate in the intricate physiological processes necessary for the maintenance of healthy dopaminergic neurons. These series of events will favor the production of highly reactive derivatives of dopamine in the cytosol of neurons through relief of the inhibitory mechanism exerted by -synuclein on TH activity and/or its phosphorylation-dependent regulation, impaired storage of dopamine into synaptic vesicles, and an influx in the amount of extracellular dopamine taken up by the DAT. Thus, a vicious circle of events is triggered that may significantly contribute to the rather specific degeneration of the dopamine-synthesizing neurons of the mesencephalon in human PD (Fig. 3
).
|
This hypothesis certainly does not recapitulate all the possible mechanisms envisaged to exist at the onset of PD, but it has the merit of including other well-demonstrated components of PD etiology, including genetic components and toxins, that may enter, at multiple entry points, into the deleterious cycle of -synuclein protein aggregation and reactive oxygen species production. Starting from the formation of -synuclein protofibrils, it is clear that free radicals such as free iron or iron-centered radicals (35)
and oxidized dopamine (20)
, and catecholamines structurally related to dopamine (20)
, accelerate and stabilize the formation of -synuclein protofibrils by inhibiting the conversion of toxic soluble protofibrils into insoluble fibrils (20)
. The formation of -synuclein protofibrils is clearly facilitated by the A53T mutation in the -synuclein sequence, or by increased concentration of -synuclein, as shown both in vitro (30)
and in transgenic animals (136
, 137)
. Expression of human -synuclein carrying the two missense mutations (A30P and A53T) in nigrostriatal terminals resulted in increased density of the dopamine transporter and enhanced toxicity to the neurotoxin MPTP (137)
. In transgenic Drosophila, the neuronal degeneration resulting from the formation of -synuclein aggregates is preferentially found in neurons that express TH and DAT (138)
. These findings support the view of a close link between -synuclein and components of the dopamine neurons in the pathophysiological process of Parkinsonian neurodegeneration. Thus, it should be stressed again that -synuclein is by no means cytotoxic in itself but that, on the contrary, significant levels of soluble -synuclein tend to protect neurons against several neurotoxic events, including oxidative stress.
The level of protofibrils and aggregated forms of -synuclein is also likely to be modulated through the ubiquitin-proteasome pathway (reviewed in ref 139
). The soluble form of -synuclein cannot be ubiquitinated by the protein parkin, a gene that, when mutated, is linked to early-onset familial PD (140)
. Parkin is a E3 ligase required to transfer ubiquitin onto proteins targeted for proteasome degradation; in transgenic flies, overexpression of parkin can mitigate -synuclein-induced neuritic pathology and suppress its toxicity (141)
. A causal link between a decrease in proteasomal degradation and high amounts of -synuclein aggregates has not yet been demonstrated. However, UCH-L1 (ubiquitin carboxyl-terminal hydrolase L1), another gene linked to a familial form of PD, is also a component of the ubiquitination pathway, rendering even more probable the involvement of this degradation pathway in the pathological process linking -synuclein aggregation to neuronal degeneration (reviewed in ref 142
).
Still another well-demonstrated entry point into the parkinsonian neurodegeneration is exposure to toxins triggering oxidative stress in dopamine neurons. In this respect, studies linking MPP+ and -synuclein effects highlight the central role of the determinants of dopaminergic systems in the induction of the processes leading to -synuclein toxicity (Fig. 3)
. The expression of DAT confers the selectivity of MPP+ toward substantia nigra (143)
. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) readily crosses the blood–brain barrier and is metabolized to MPP+ by astrocytes, and this metabolite is selectively taken up into the dopaminergic neuron by the DAT in an energy-dependent manner. Once in the cell, MPP+ inhibits complex I of the mitochondrial respiratory chain, releasing cytochrome c, which accelerates aggregation of -synuclein (144)
, ultimately affecting the properties of -synuclein. The presence of -synuclein and its A53T mutant enhances the vulnerability of cells to MPP+ exposure (145)
. -Synuclein null mice are essentially resistant to MPTP-induced degeneration of DA neurons and DA release, and this resistance is due to an inability of the toxin to inhibit complex I of the mitochondria (146)
. This further suggests that -synuclein has a facilitatory role in enhancing, either directly or indirectly, the observed toxicity of MPP+.
To conclude, there is ample evidence to propose that overall, -synuclein itself is not a toxic compound, but that it can be converted to a toxic compound in the presence of specific components of the dopaminergic system, thereby conferring the selectivity for degeneration of dopamine-producing neurons seen in PD. Through its peculiar biochemical properties, -synuclein is able to interact differently with numerous molecular systems, contributing in more than one way to the pathophysiology of PD. Considering -synuclein as a central regulator of the dopamine life cycle and as an effector of dopamine-dependent oxidative stress paves the way for a more accurate understanding of both the physiology and the pathology of dopamine neurotransmission.
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ACKNOWLEDGMENTS |
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Received for publication October 21, 2003. Accepted for publication January 5, 2004.
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