-Synuclein and Parkinsons disease

ALESSANDRA RECCHIA,1 PATRIZIA DEBETTO,1 ALESSANDRO NEGRO,* DIEGO GUIDOLIN, STEPHEN D. SKAPER, AND PIETRO GIUSTI2 Department of Pharmacology and Anesthesiology, *Centro di Ricerche in Biotecnologie Innovative, Department of Human Anatomy and Physiology, University of Padova, 35131 Padova, Italy; and Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, Harlow, Essex CM19 5AW, U.K.
ABSTRACT

Alpha-synuclein ( -syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in -syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinsons disease (PD). The physiological functions of -syn remain to be fully dened, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of -syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, -syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. -Syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which -syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin-1, chaperone 14-3-3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that -syn-based models represent a paradigm, which is closest to the human pathology. Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D., Giusti, P. -Synuclein and Parkinsons disease. FASEB J. 18, 617 626 (2004) Key Words: -synuclein aggregation dopaminergic neurons animal models of Parkinsons disease

BACKGROUND Parkinsons disease (PD) is the second most common progressive neurodegenerative brain disorder of humans, after Alzheimers disease. PD affects 1% of people beyond 65 years of age (1), with a higher prevalence in men (2); it usually manifests itself in the fth or sixth decade of life. PD is characterized clinically by severe motor symptoms including uncontrollable resting tremor, muscular rigidity, impaired postural reexes, and bradykinesia, which vary between patients (for review, see refs 3, 4). These abnormalities can be accompanied by other symptoms, such as autonomic
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dysfunction, depression, and a general slowing of intellectual processes (5). Pathologically, PD is characterized by the marked degeneration of dopaminergic neurons in the substantia nigra pars compacta, which leads to the depletion of dopamine (DA) in its striatal projections, and of other brainstem neurons, with consequent disruption of the cerebral neuronal systems responsible for motor functions (for review, see refs 3, 4). This neurodegeneration is accompanied by the presence of cytoplasmic (Lewy bodies, LBs) and neuritic (Lewy neurites, LNs) inclusions (6) in the surviving dopaminergic neurons and other affected regions of the central nervous system (CNS), but the mechanism underlying their formation is unclear, as is their pathogenic relevance. PD is an essentially sporadic neurodegenerative disease whose pathogenesis remains largely unknown, despite years of intense research in an attempt to explain the complexity and the relative selectivity of dopaminergic neurodegeneration. Genetic and environmental risk factors (7) have gained more attention of late as possible causes of PD, but their relative contributions in initiating the neurodegenerative process continue to be debated. Based on current knowledge, late-onset idiopathic PD is thought to result from a complex interaction among multiple predisposing genes and environmental factors. In addition to sporadic forms of PD, several rare monogenic familial forms of the disease, characterized by early-onset and an autosomal dominant or recessive pattern of inheritance have been identied. Mutations in four genes have been clearly linked to PD encoding -synuclein ( -syn) (8), parkin (9), ubiquitin carboxy-terminal hydrolase L-1 (10), and DJ-1 (11). Other genes or loci that may cause PD have been mapped in families (for review, see refs 4, 1214). Although familial forms of PD with specic genetic defects represent only a minor part ( 10%) of all cases, they may help to identify key abnormalities in protein pathways that are likely to be involved in the more common, multifactorial sporadic form of the disease.
These authors contributed equally to this work. Correspondence: Department of Pharmacology and Anesthesiology, L.go Meneghetti, 2, 35131 Padova, Italy. E-mail: pietro.giusti@unipd.it doi: 10.1096/fj.03-0338rev
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Mutations in the gene encoding for -syn have received a great deal of attention with the discovery that brillar -syn aggregates are the major components of both LBs and LNs (15, 16), characterizing most familial and sporadic PD. This observation suggests that although -syn is infrequently mutated in PD, other cellular processes that could lead to abnormal metabolism and accumulation of this protein might play an important role in the pathogenesis of sporadic as well as familial disease. This review provides a general overview of newer research on -syn neurobiology potentially relevant to the causes of PD. Results from transgenic and nontransgenic -syn animal models of PD are also reviewed for their contribution to an understanding of normal and pathophysiological roles of -syn in the brain.

LOCALIZATION AND STRUCTURE OF

-SYN

-Syn belongs to the synuclein family that includes -syn and -syn (17, 18), which so far have been described only in vertebrates. -Syn and -syn are predominantly expressed in brain at presynaptic terminals, particularly in the neocortex, hippocampus, striatum, thalamus, and cerebellum (19, 20). -Syn is highly expressed in various areas of the brain, particularly in the substantia nigra, and has been found to be overexpressed in some breast and ovarian tumors (21). The -syn, -syn, and -syn genes have been mapped to human chromosomes 4q21 (22), 5q35 (22), and 10q23 (21), respectively. The sequences of all synucleins are similar (18), although only -syn is implicated in disease. -Syn was originally identied in the electric organ of the Pacic electric eel Torpedo Californica (23). Hundreds of well-conserved -syn protein homologues exist in human, bird, mouse, bovine, and rat, but no homologues have been reported in lower organisms

such as Escherichia coli, yeast, C. elegans, or Drosophila. Human -syn is an abundant presynaptic protein with a perinuclear localization rst identied as the precursor of a peptide, called the non-A component (NAC), present in extracellular amyloid plaques in some forms of Alzheimers disease patients (17, 19). -Syn is an extremely heat-resistant, small acidic protein (14 kDa) composed of 140 amino acid (aa) residues (17). -Syn is a soluble, natively unfolded protein with an extended structure primarily composed of random coils (24), but it may acquire secondary structural elements upon interaction with a number of ligands and proteins (25) that likely alter its native state conformation and lead to -syn adopting partially folded conformations. The sequence of -syn can be subdivided into three distinct domains (Fig. 1). The highly conserved aminoterminal domain of -syn (residues 1-65) includes six copies of an unusual 11 aa imperfect repeat that display variations of a KTKEGV consensus sequence and is unordered in solution, but can shift to an -helical conformation (18, 26) that appears to consist of two distinct -helixes interrupted by a short break (27). The amphipathic -helixes (28) are reminiscent of the lipid binding domains of class A2 apolipoproteins (29). In agreement with these structural features, -syn avidly binds to negatively charged phospholipids and becomes -helical upon binding (24, 25, 29), suggesting that the protein may normally be membrane associated (29). Several recent studies (30 32) have shown that lipidic environments that promote -syn folding also accelerate -syn aggregation, suggesting that the lipidassociated conformation of -syn may be relevant to -syn misfolding in neurodegenerative diseases. The -helix forming domain hosts two independent missense mutations (Fig. 1) at position 53, changing an Ala to Thr (A53T), and at position 30, changing an Ala to Pro (A30P); these have been shown to cause autosomal dominant heritable early-onset PD (33). The A53T

Figure 1. Human -syn sequence and domains. The imperfect KTKEGV repeats are shown by violet columns. Missense mutations at residues 30 (A30P) and 53 (A53T) are shown in red. For further details, refer to text.

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mutation has been identied in a large Italian-Greek family (Contursi kindred) (8) that had autosomal dominant PD with LBs. The second mutation, A30P, was later found in a small German family with PD (34). These mutations may make it easier for -syn to be in the random coil state so that aggregation is more likely to occur. It has been suggested that this may be due to acquisition of a -sheet conguration by the protein, which renders it more prone to aggregation and lament formation. It has been shown (35) that the A30P and A53T mutations increase the rate of -syn oligomerization, whereas the rate of mature bril formation is increased and decreased by A53T and A30P mutations, respectively. The central hydrophobic domain of -syn (residues 66-95) is known as the non-A component of plaque (NAC) (17), the second major component of brain amyloid plaques in Alzheimers disease (17, 36). It comprises the highly amyloidogenic part of the molecule that is responsible for the ability of -syn to undergo a conformational change from random coil to -sheet structure (37) and to form A -like protobrils and brils (36, 37). These features distinguish -syn from -syn and -syn, which fail to form copolymers with -syn (36). NAC region carries a phosphorylation site on Ser 87 (38). The acidic carboxyl-terminal domain (residues 96140) of -syn has no recognized structural elements but has a strong negative charge composed primarily of acidic amino acids (18). Different from the amphipathic amino-terminal and the hydrophobic NAC regions, which are highly conserved between species, the carboxyl-terminal region is highly variable in size and in sequence (21). It hosts an acidic domain (residues 125-140) that appears critical for the chaperone-like activity of -syn (39; see section Chaperone-like activity of -syn), as demonstrated by deletion mutants of the carboxyl-terminal region in which the -syn chaperone activity is lost (40, 41). Several phosphorylation sites (Fig. 1) have been detected in the carboxyl-terminal region on Tyr-125, -133, and -136, and on Ser-129 (42). Tyr-125 residues can be phosphorylated by two Src family protein tyrosine kinases, c-Src and Fyn (43, 44). Phosphorylation by Src family kinases does not suppress or enhance the tendency of -syn to polymerize. -Syn has proved to be an outstanding substrate for protein tyrosine kinase p72syk (Syk) in vitro; once it is extensively Tyr-phosphorylated by Syk or tyrosine kinases with similar specicity, it loses the ability to form oligomers, suggesting a putative anti-neurodegenerative role for these tyrosine kinases (42). Considering that the carboxyl-terminal domain of -syn is required for its chaperone-like activity (39), it is conceivable that phosphorylation of these Tyr residues in this region would also affect this property of -syn. However, whether in vivo Tyr-phosphorylation of -syn is mediated by Syk or by another protein tyrosine kinase with overlapping site specicity remains an open question. -Syn can be Ser-phosphorylated by protein kinases
ABNORMAL METABOLISM OF

CKI and CKII (38). The residue Ser-129 is also phosphorylated by G-protein-coupled receptor protein kinases (45). Studies in vitro suggest that phosphorylation at Ser-129 promotes formation of -syn laments as well as oligomers (46) as a consequence of a change in charge distribution and hydrophobicity of -syn carboxyl-terminal region (47). Extensive and selective phosphorylation of -syn at Ser-129 is evident in synucleinopathy lesions, including LBs (46). Other post-translational modications in the carboxyl-terminal, including glycosylation on Ser-129 (47) and nitration on Tyr-125, -133, and -136 (48), may affect aggregation of -syn. A O-glycosylated form of -syn ( Sp22), a specic substrate for ubiquitination by parkin, has been identied by Shimura et al. (9). Although it has not been determined where -syn is glycosylated, a potential target for glycosylation is the carboxyl-terminal Ser-129, hosting O-linked sugars (9). Truncation of the carboxyl-terminal region by proteolysis has been reported to play a role in -syn brillogenesis in various neurodegenerative diseases (35). Full-length as well as partially truncated and insoluble aggregates of -syn have been detected in highly puried LBs (49).

PHYSIOLOGICAL FUNCTIONS OF

-SYN

Several lines of evidence suggest a role of -syn in membrane-associated processes at the presynaptic level. In the Zebra nch, -syn is transiently expressed in a telencephalic area associated with song acquisition during the critical period for song learning (50). In bovine brain, - and -syn are constitutive inhibitors of phospholipase D2 (51), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), thus triggering secretory vesicle production (52). -Syn appears to bind vesicles with high levels of PA (29), and it is possible that this protein may regulate membrane trafcking via vesicle budding or turnover (53). -Syn knockout mice exhibit enhanced DA release at nigrostriatal terminals only in response to paired electrical stimuli, suggesting that -syn is an activity-dependent, negative regulator of dopaminergic neurotransmission (54). -Syn has been shown to bind to fast axonal transport vesicles. Depletion of -syn from cultured primary hippocampal neurons by treatment with antisense oligonucleotides decreased the distal pool of presynaptic vesicles, as visualized by electron microscopy (55). Chaperone-like activity of -syn Chaperones are proteins that prevent irreversible protein aggregation and facilitate the correct folding of non-native proteins through regulated binding and release in vivo (56). -Syn has been suggested to function as a chaperone protein in vivo because, besides lipids (24, 25, 29), it appears capable of interacting with a variety of ligands and cellular proteins (57,
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58), thus modifying their activities. It has recently been reported that the amino-terminal portion of -syn shares 40% aa homology with molecular chaperone 14-3-3 (57), suggesting that the two proteins could subserve the same function. The molecular chaperone 14-3-3 is particularly abundant in brain, where it comprises 1% of total soluble proteins (59). Chaperone 14-3-3 accumulates in LBs, participates in neuronal development and cell growth control (60), and prevents apoptosis by antagonizing BAD, a proapoptotic member of the Bcl-2 family (61; Fig. 2). -Syn binds to many of the same proteins as 14-3-3 (57), including three proteins known to affect cell viability, protein kinase C (PKC), BAD, and extracellular regulated kinase (ERK). -Syn interacts with 14-3-3 (57), and the interaction between the two proteins produces a 54 to 83 kDa protein complex in PD brain (58). This complex is selectively increased in substantia nigra but not in cerebellum or cortex. Thus, -syn may sequester 14-3-3, leading to a reduction in the amount of 14-3-3 protein available to inhibit apoptosis and rendering the cells more susceptible to cellular stresses (58; see also Fig. 2). Both 14-3-3 and -syn bind to tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis (see the section Dopamine and -syn), with divergent consequences: activity is stimulated by 14-3-3 but inhibited by -syn (51). The physical and functional homology between -syn and 14-3-3 suggests that -syn may normally act as a protein chaperone to help the cell deal with the effects of increased stress as part of an initial effort by the cell to protect itself against the accumulation of damaged proteins (57). However, overexpression of wild-type -syn is toxic to dividing cells and overexpression of its mutant forms A53T or A30P exhibits even greater toxicity (57), which may be due to inhibition of PKC and interaction of -syn with BAD, ERK, or other proteins involved in signal transduction (57).

PATHOPHYSIOLOGY OF

-SYN

The rst association between -syn and neurodegenerative diseases was reported by Ueda et al. (17). These authors showed that a short peptide, called NAC, present in puried amyloid plaques of Alzheimers disease patients, is derived from a larger precursor protein (then called non-amyloid component precursor or NACP), which we now know is -syn. Two missense mutations, A53T (8) and A30P (34), in the -syn gene appear to account for rare cases of earlyonset PD in families of European origin. The aggregation and accumulation of these abnormal -syn proteins in dopaminergic neurons have been postulated to be responsible for the subsequent neurodegeneration (34). Very little is known about the structural basis for differences in the brillation between wild-type -syn and its A53T and A30P mutants. Like the wild-type protein, A53T and A30P -syn are natively unfolded under physiological conditions (62), posing a crucial question as to how a single point mutation in the disordered conformation of the protein (i.e., natively unfolded) may affect its aggregation and brillation properties. -Syn aggregation is present not only in autosomal dominant early-onset PD, but also in the classic form of PD and in other CNS disorders, called -synucleinopathies, which include Alzheimers disease, Lewy body dementia, Hallervorden-Spatz syndrome, and multiple system atrophy (Table 1). The assembly of -syn is accompanied by transition from random coil to a -pleated sheet conformation (74). X-ray ber and electron diffraction studies show -syn laments to exhibit a cross- conformation, a characteristic feature of amyloid (75). Common features of surviving dopaminergic neurons in PD are LBs and LNs (76). The latter are found not only in substantia nigra, but also in dorsal motor nucleus of the vagus, nucleus basalis of Meynert, and locus coeruleus (77). LBs are

Figure 2. -Syn aggregation and toxic effects in dopaminergic neurons. A hypothetical scheme depicts various pathways that, leading to aggregation of natively unfolded -syn, oxidative stress, or mitochondrial impairment, cause cell death. For further details, refer to text. DA, dopamine; DOPA, dihydroxyphenylalanine; LBs, Lewy bodies; MAO, monoamine oxidase; ROS, reactive oxygen species; TH, tyrosine hydroxylase; THP, phosphorylated tyrosine hydroxylase; Tyr, tyrosine; UPS, ubiquitin proteasome system.

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TABLE 1. Alpha-synucleinopathies
Disease Reference

Parkinsons disease: Sporadic Familial Dementia with Lewy bodies Pure Lewy bodies dementia Lewy body variant of Alzheimers disease Down syndrome Multiple system atrophy Shy-Drager syndrome Striatonigral degeneration Olivopontocerebellar atrophy Neurodegeneration with brain iron accumulation, type 1 Hallervorden-Spatz syndrome Neuroaxonal dystrophy Other diseases that may have synucleinimmunoreactive lesions Traumatic brain injury Amyotrophic lateral sclerosis Niemann-Pick disease

17 8 63 64 65 66 67 68 69 70 71 71 72 73

abnormal intracytoplasmic inclusions, 525 m in diameter, structurally composed of a dense core and a clearer surrounding halo. Ultrastructurally, LBs contain an eosinophilic core of lamentous and granular material surrounded by radially oriented laments. -Syn is a major component both of LB laments and of dystrophic LNs (62). There are no data implicating - and -syn in neurological disorders. While these proteins are not found in LBs and LNs, it has been reported that patients affected by PD and dementia with LBs display hippocampal neurons containing -, -, and -syn (78). Aggregation mechanisms of -syn Abnormal protein aggregation appears to be a common feature in aging brain and in several neurodegenerative diseases, although a clear role in the disease process remains to be dened. In in vitro models, -syn (or some of its truncated forms) readily assembles into laments resembling those isolated from brain of patients with LB dementia and familiar PD (49). The peptide derived from the central hydrophobic region of -syn (NAC, see Fig. 1) represents a second major intrinsic constituent of Alzheimers plaques (28). Normal -syn and its mutated forms (A53T and A30P) have a random coil conformation and do not form signicant secondary structure in aqueous solution at low concentrations; however, at higher concentrations they are prone to self-aggregate, producing amyloid brils (79). Several differences in the aggregation behavior of the PD-linked mutants and the wildtype protein have been documented. Monomeric -syn aggregates in vitro to form stable brils via a metastable oligomeric (i.e., protobril) state (80). The protobrillization rate of both mutants is higher than that of
ABNORMAL METABOLISM OF

wild-type protein; the brillation rate is lower in A30P and higher in A53T (81, 82). Several mechanisms for -syn aggregation have been proposed (see Fig. 2); those involving the ubiquitin proteasome system (UPS) and oxidative stress have gained the most prominence until now. UPS is the primary biochemical pathway responsible for the degradation of normal and abnormal (mutated, misfolded, or unassembled) intracellular proteins (83). Failure of this system leads to protein accumulation and cell death (84). Degradation via UPS involves two successive steps: initially, the protein substrate is tagged by covalent attachment of multiple ubiquitin molecules through the action of ubiquitin-conjugating enzymes. Subsequently, the tagged protein is degraded by the 26S proteasome, with release of reusable ubiquitin (83). The 26S proteasome belongs to the proteasome family of multicatalytic proteases and is located in the cytoplasm, endoplasmic reticulum, perinuclear region, and nucleus of eukaryotic cells (85). A growing body of evidence suggests that ubiquitin-dependent protein degradation may be impaired in many neurodegenerative diseases, including PD and diffuse LB disease (DLBD). A key pathological feature in PD and DLBD is the formation of ubiquitinated cytoplasmic inclusions (86). In PD, LBs are formed within the dopaminergic neurons of the substantia nigra pars compacta. In DLBD, the LB-ubiquitin is in the form of polyubiquitin chains rather than ubiquitin monomers, as shown by biochemical analyses of isolated cortical LBs from postmortem tissue (87). This observation suggests that polyubiquitinated proteins may accumulate in inclusions as a result of a dysfunction in the proteasome degradation process. Besides ubiquitin, LBs contain -syn, subunits of the 26S proteasome, and other proteins including parkin, 14-3-3 protein (58), and synphilin-1 (15). The last forms a complex with -syn, which is then ubiquitinated by the E3 ubiquitin ligase activity (88). A mutation in parkin leads to autosomal recessive juvenile parkinsonism, which commonly lacks microscopic -syn Lewy-type aggregates (89). Despite the absence of LBs, selective accumulation of the putatively toxic Sp22 has been demonstrated in parkin-linked PD brains (9), suggesting that parkin mutations may predispose to accumulation of -syn in a soluble nonbrillar form. Taken together, these ndings propose that UPS inactivity may contribute to the development of neurodegeneration in PD forms either or not characterized by LB formation. Another mechanism underlying -syn aggregation may involve the action of oxidants, which cause -syn to aggregate and thereby perhaps initiate formation of toxic intermediate oligomers (90, 91), probably due to a kinetic stabilization of the -syn protobril by a dopamine- -syn adduct (30). Other in vitro studies have revealed that overexpression of -syn can induce iron-dependent aggregation (see Fig. 2) (92).
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Dopamine and -syn One school of thought regarding PD pathogenesis is built around a role for reactive oxygen species (ROS), which can cause oxidative stress and mitochondrial impairment, and cell death (93). The metabolism of DA in nigral neurons produces ROS and other highly reactive chemical species, such as DA-quinones and peroxynitrites, either by autoxidation or by MAO Bmediated oxidative deamination (94 96; see also Fig. 2). The production of ROS and quinones contributes to lipid peroxidation, DNA damage, inhibition of mitochondrial respiratory chain activity, and ultimately neuronal cell death (97). Postmortem studies in PD patients provide strong evidence for increased oxidative stress in dopaminergic neurons of the substantia nigra, possibly caused by increased iron content (98), impairment in mitochondrial function, depletion of reduced glutathione, overexpression of Cu, Zn-superoxide dismutase (99), alterations in antioxidant protein levels, lipid peroxidation, protein nitration, and DNA damage (100; for a review, see ref 93; Fig. 2). Overexpression of -syn, especially its mutant forms, may enhance the vulnerability of neurons to DAinduced cell death through an excessive generation of intracellular ROS (101). The degeneration of dopaminergic neurons may also occur because of a decrease in brain capacity to inactivate these reactive compounds (96). Deciencies in the antioxidant capacity in substantia nigra of PD patients have, in fact, been reported (102). Accumulation of -syn, induced by transfection of wild-type human -syn or the A53T or A30P PD-causing mutants triggered apoptosis (Fig. 2) of cultured human fetal dopaminergic neurons, while there is an increase in survival of cultured nondopaminergic human cortical neurons (58). Mutants of -syn induced neurotoxicity at lower -syn concentrations than the wild-type protein in dopaminergic neurons and signicantly reduced neuroprotective activity relative to wild-type protein in cortical neurons (58). The dopaminergic specicity of -syn neurotoxicity was directly related to endogenous DA production and consequent ROS generation, because inhibition of DA synthesis by the highly specic TH inhibitor -methyl-p-tyrosine prevented -syn-induced apoptosis in cultured dopaminergic neurons (58). DA-dependent neurotoxicity was mediated by elevated levels of 54 to 83 kDa soluble protein complexes containing -syn and 14-3-3 protein (Fig. 2), which, by reducing the anti-apoptotic activity of 14-3-3, could increase neuronal vulnerability to ROS generated by endogenous DA metabolism (58). It has been reported that wild-type -syn overexpression may regulate DA biosynthesis, acting on several enzymes that play different roles in this process. The activity of TH, the enzyme catalyzing the rate-limiting step in the biosynthesis of catecholamines (103), may be negatively modulated by -syn either interacting directly with the enzyme (104) or decreasing its gene
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expression (105). In addition, aromatic acid DOPA decarboxylase gene expression is decreased by wildtype -syn overexpression (105). Since, for catalytic activity, TH requires tetrahydrobiopterin (BH4) as a cofactor, any event reducing BH4 availability decreases DA biosynthesis. Wild-type -syn overexpression may also reduce DA synthesis by inhibiting the key enzymes involved in the production of BH4 , including GTP cyclohydrolase and sepiapterin reductase (105). A reduction in free soluble -syn may occur by down-regulation of -syn mRNA or by stimuli that induce brillation. Disinhibition of -syn activity may lead to an increase in DA concentration with subsequent generation of ROS. Collectively, these data point to a key role for -syn in the regulation of DA synthesis. A loss in -syn function consequent to its aggregation or decreased expression, as occurs in PD, may selectively disrupt DA homeostasis and negatively affect dopaminergic neuron survival (104).

ANIMAL MODELS OF PD The classical animal models of PD rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), and, more recently, the agricultural chemicals paraquat and rotenone, to deplete DA (Fig. 3, left panel). These models are able to elicit motor decits in different animal species, e.g., Drosophila, rats, mice, and primates, with the exception of MPTP, which fails to induce a signicant dopaminergic neurodegeneration in rats (106 108). However, neurotoxic models have some limitations regarding, in particular, the acute nature of the dopaminergic neuron loss, which does not allow for the identication of LBs with any of the neurotoxins used except rotenone (106), thus reproducing only the terminal conditions of the human disease. A new neurotoxic PD model has been developed based on impairment of proteasomal function by intranigral injection of the selective proteasome inhibitor lactacystin (109; see also Fig. 3, left panel). Inhibition of 20/26S proteasomal function in the substantia nigra pars compacta induced by lactacystin leads to protein accumulation and inclusion body formation, accompanied by a relatively selective degeneration of dopaminergic neurons and motor dysfunction. This model closely mirrors the neurodegenerative process that occurs in sporadic PD, but not its progressive development. In the attempt to better reproduce the key features of the human PD, particularly the progressive nature of neurodegeneration, a new approach in the development of PD models has been attempted based on the genetic and neuropathological links between -syn and PD. This has produced some transgenic and nontransgenic -syn animal models (106 108; see also Fig. 3, middle and right panels).
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Figure 3. Animal models of Parkinsons disease. The main PD clinical feature of each animal model are reported. For further details, refer to text. LBs, Lewy bodies; MPTP, 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxydopamine; WT -syn, wild-type -synuclein.

Transgenic -syn models When human wild-type and mutant (A30P and A53T) -syn are overexpressed in Drosophila, the ies develop an adult-onset, remarkably selective loss of DA neurons. This cell loss is associated with progressive motor dysfunction and with the presence of lamentous intraneuronal inclusions that contain -syn (106). It is not clear whether the observed motor decits are due to DA neuron dysfunction, as there is no difference in the toxicity of wild-type and mutant -syn (107). In addition to Drosophila, several transgenic murine models overexpressing human -syn have been developed (110). Although none of these transgenic models faithfully reproduce PD, some do exhibit signicant synucleinopathy-induced neurodegeneration, a key feature of human -syn pathology. These limitations notwithstanding, -syn transgenic mice represent useful tools to investigate the toxicity of -syn in vivo and factors modulating the aggregation of -syn in vivo (106). It remains to be explained why these -syn transgenic mice fail to display any signicant DA neuron abnormalities, particularly in light of the dramatic pathological changes observed in Drosophila DA neurons and the DA-dependent -syn toxicity seen in human neuronal cell cultures (58). One possibility is that rodent DA neurons are especially resistant to -syn toxicity, perhaps due to the relative levels of -syn vs. -syn, -syn, and other intrinsic protective factors. Further studies are needed to fully clarify this issue. Non transgenic -syn models These models are based on delivering -syn directly to the substantia nigra by means of viral vectors. The use of viral vectors to overexpress putative toxic proteins, such as -syn, may offer distinct advantages over transgenic animal models. For example, viral vectors may be
ABNORMAL METABOLISM OF

administered to virtually any location within the brain at any time during the life span of the animal, and are suitable for rats, which have greater utility than mice for behavioral studies (111). Injection of recombinant adeno-associated viral vectors expressing wild-type or the A53T mutated human -syn into rat SN produced a selective damage (30 to 80%) in nigral dopaminergic neurons (112). After 8 wk, striatal DA and TH levels were reduced by 40 50% and were accompanied by signicant motor impairment in those animals where dopaminergic cell loss exceeded 50 60%. At 6 months, signs of neuronal cell body and axonal pathology had subsided, suggesting that surviving neurons had recovered from the initial insult even though -syn expression remained high (112). Similar results have been reported using a HIV-1-derived lentiviral vector associated with wild-type and mutant -syn derived from either rats or humans (111). A progressive loss in nigral and striatal dopaminergic neurons was observed upon intranigral injection, with accumulation of -syn in cytoplasmic structures similar to LBs and LNs (111).

CONCLUSIONS There is now a large body of evidence to support the involvement of both environmental and genetic factors in PD development. It is not inconceivable that PD will be shown to be heterogeneous in its etiology, with underlying genetic factors in some cases and environmental factors predominating in other instances. -Syn is highly expressed in the CNS and is likely to have an important role as a modulator of synaptic plasticity. Recent evidence implicates this protein under certain conditions as a toxin for the dopaminergic system through multiple and complex ways, which remain ill-dened at best. A more complete understanding of
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-syn biology represents a fundamental tool in our comprehension of PD and may lead to the development of novel therapeutic strategies for treating this disease.
We are grateful to Stefano Lovison and David Rota for their excellent technical support. This work was supported by grants from MIUR (Ministero dellIstruzione, dellUniversita ` e della Ricerca) to P.D. and P.G.

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21. 22.

REFERENCES
1. 2. Tanner, C. M. (1992) Occupational and environmental causes of parkinsonism. Occup. Med. 7, 503513 Dluzen, D. E., and McDermott, J. L. (2000) Gender differences in neurotoxicity of the nigrostriatal dopaminergic system: implications for Parkinson's disease. J. Gend. Specif. Med. 3, 36 42 Lotharius, J., and Brundin, P. (2002) Pathogenesis of Parkinson's disease: dopamine, vesicles and alpha-synuclein. Nat. Rev. Neurosci. 3, 932942 Siderowf, A., and Stern, M. (2003) Update on Parkinson disease. Ann. Intern. Med. 138, 651 658 Berrios, G. E., Campbell, C., and Politynska, B. E. (1995) Autonomic failure, depression and anxiety in Parkinson's disease. Br. J. Psychiatry 166, 789 792 Gomez-Tortosa, E., Newell, K., Irizarry, M. C., Albert, M., Growdon, J. H., and Hyman, B. T. (1999) Clinical and quantitative pathologic correlates of dementia with Lewy bodies. Neurology 53, 1284 1291 Warner, T. T., and Schapira, A.H. (2003) Genetic and environmental factors in the cause of Parkinson's disease. Ann. Neurol. 53 (Suppl. 3), S16 S23; discussion S23S25 Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., et al. (1997) Mutation in the alpha-synuclein gene identied in families with Parkinson's disease. Science 276, 20452047 Shimura, H., Schlossmacher, M. G., Hattori, N., Frosch, M. P., Trockenbacher, A., Schneider, R., Mizuno, Y., Kosik, K. S., and Selkoe, D. J. (2001) Ubiquitination of a new form of alphasynuclein by parkin from human brain: implications for Parkinson's disease. Science 293, 263269 Leroy, E., Boyer, R., Auburger, G., Leube, B., Ulm, G., Mezey, E., Harta, G., Brownstein, M. J., Jonnalagada, S., Chernova, T., et al. (1998) The ubiquitin pathway in Parkinson's disease. Nature (London) 395, 451 452 Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., Dekker, M. C., Squitieri, F., Ibanez, P., Joosse, M., et al. (2003) Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299, 256 259 Polymeropoulos, M. H. (2000) Genetics of Parkinson's disease. Ann. N.Y. Acad. Sci. 920, 28 32 Nussbaum, R. L., and Ellis, C. E. (2003) Alzheimer's disease and Parkinson's disease. N. Engl. J. Med. 348, 1356 1364; erratum in. N. Engl. J. Med. 348, 2588 Dawson, T. M., and Dawson, V. L. (2003) Rare genetic mutations shed light on the pathogenesis of Parkinson disease. J. Clin. Invest. 111, 145151 Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature (London) 388, 839 840 Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and Goedert, M. (1998) Alpha-synuclein in lamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA 95, 6469 6473 Ueda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshi moto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saitoh, T. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90, 1128211286 George, J. M. (2002) The synucleins. Genome Biol. 3, 30023006

23. 24. 25.

3. 4. 5. 6.

26.

27. 28. 29.

7. 8.

30.

9.

31.

10.

32.

11.

33. 34.

12. 13. 14. 15. 16.

35.

36.

37. 38.

17.

39.

18.

Iwai, A., Yoshimoto, M., Masliah, E., and Saitoh, T. (1995) The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system. Neuron 14, 467 475 Nakajo, S., Shioda, S., Nakai, Y., and Nakaya, K. (1994) Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA expression in rat brain determined by immunocytochemistry and in situ hybridisation. Brain Res. Mol. Brain Res. 27, 81 86 Lavedan, C. (1998) The synuclein family. Genome Res. 8, 871 880 Spillantini, M. G., Divane, A., and Goedert, M. (1995) Assignment of human alpha-synuclein (SNCA) and beta-synuclein (SNCB) genes to chromosomes 4q21 and 5q35. Genomics 27, 379 381 Maroteaux, L., Campanelli, J. T., and Scheller, R. H. (1988) Synuclein: a neuron-specic protein localized to the nucleus and presynaptic nerve terminal. J. Neurosci. 8, 2804 2815 Eliezer, D., Kutluay, E., Bussell, R., Jr., and Browne, G. (2001) Conformational properties of alpha-synuclein in its free and lipid-associated states. J. Mol. Biol. 307, 10611073 Li, J., Uversky, V. N., and Fink, A. L. (2001) Effect of familial PD point mutations A30P and A53T on the structural properties, aggregation and brillation of human -syn. Biochemistry 40, 11604 11613 Kahle, P. J., Haass, C., Kretzschmar, H. A., and Neumann, M. (2002) Structure/function of alpha-synuclein in health and disease: rational development of animal models for Parkinson's and related diseases. J. Neurochem. 82, 449 457 Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudho, T. C. (2003) A broken -helix in folded -synuclein. J. Biol. Chem. 278, 1531315318 Uversky, V. N., and Fink, A. L. (2002) Amino acid determinants of alpha-synuclein aggregation: putting together pieces of the puzzle. FEBS Lett. 522, 9 13 Davidson, W. S., Jonas, A., Clayton, D. F., and George, J. M. (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 94439449 Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Kinetic stabilization of the alpha-synuclein protobril by a dopamine-alpha-synuclein adduct. Science 294, 6 9 Lee, H. J., Choi, C., and Lee, S. J. (2002) Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form. J. Biol. Chem. 277, 671 678 Lee, H. J., Shin, S. Y., Choi, C., Lee, Y. H., and Lee, S. J. (2002) Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors. J. Biol. Chem. 277, 5411 5417 Clayton, D. F., and George, J. M. (1999) Synucleins in synaptic plasticity and neurodegenerative disorders. J. Neurosci. Res. 58, 120 129 Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Ala30Pro mutation in the gene encoding alphasynuclein in Parkinson's disease. Nat. Genet. 18, 106 108 Rochet, J. C., Conway, K. A., and Lansbury, P. T. (2000) Inhibition of brillization and accumulation of prebrillar oligomers in mixtures of human and mouse -synuclein. Biochemistry 39, 10619 10626 Giasson, B. I., Murray, I. V. J., Trojanowski, J. Q., and Lee, V. M. (2001) A hydrophobic stretch of 12 amino acid residues in the middle of -synuclein is essential for lament assembly. J. Biol. Chem. 276, 2380 2386 El-Agnaf, O. M., and Irvine, G. B. (2002) Aggregation and neurotoxicity of alpha-synuclein and related peptides. Biochem. Soc. Trans. 30, 559 565 Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390 397 Souza, J. M., Giasson, B. I., Chen, Q., Lee, V. M., and Ischiropoulos, H. (2000) Dityrosine cross-linking promotes formation of stable alpha-synuclein polymers. Implication of

624

Vol. 18

April 2004

The FASEB Journal

RECCHIA ET AL.

40. 41.

42.

43.

44.

45. 46.

47.

48.

49. 50.

51.

52.

53.

54.

55.

56. 57.

58.

59.

nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies. J. Biol. Chem. 275, 18344 18349 Kim, T. D., Paik, S. R., and Yang, C. H. (2002) Structural and functional implications of C-terminal regions of alphasynuclein. Biochemistry 41, 1378213790 Park, S. M., Jung, H. Y., Kim, T. D., Park, J. H., Yang, C. H., and Kim, J. (2002) Distinct roles of the N-terminal-binding domain and the C-terminal-solubilizing domain of -synuclein, a molecular chaperone. J. Biol. Chem. 277, 2851228520 Negro, A., Brunati, A. M., Donella-Deana, A., Massimino, M. L., and Pinna, L. A. (2002) Multiple phosphorylation of alphasynuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation. FASEB J. 16, 210 212 Ellis, C. E., Schwartzberg, P. L., Grider, T. L., Fink, D. W., and Nussbaum, R. L. (2001) Alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases. J. Biol. Chem. 276, 3879 3884 Nakamura, T., Yamashita, H., Takahashi, T., and Nakamura, S. (2001) Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125. Biochem. Biophys. Res. Commun. 280, 1085 1092 Pronin, A. N., Morris, A. J., Surguchov, A., and Benovic, J. L. (2000) Synucleins are a novel class of substrates for G proteincoupled receptor kinases. J. Biol. Chem. 275, 2651526522 Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Alpha-synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4, 160 164 McLean, P. J., and Hyman, B. T. (2002) An alternatively spliced form of rodent alpha-synuclein forms intracellular inclusions in vitro: role of the carboxy-terminus in alpha-synuclein aggregation. Neurosci. Lett. 323, 219 223 Takahashi, T., Yamashita, H., Nakamura, T., Nagano, Y., and Nakamura, S. (2002) Tyrosine 125 of alpha-synuclein plays a critical role for dimerization following nitrative stress. Brain Res. 938, 73 80 Crowther, R. A., Jakes, R., Spillantini, M. G., and Goedert, M. (1998) Synthetic laments assembled from C-terminally truncated -synuclein. FEBS Lett. 436, 309 312 George, J. M., Jin, H., Woods, W. S., and Clayton, D. F. (1995) Characterization of a novel protein regulated during the critical period for song learning in the zebra nch. Neuron 15, 361372 Jenco, J. M., Rawlingson, A., Daniels, B., and Morris, A. J. (1998) Regulation of Phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and betasynucleins. Biochemistry 37, 4901 4909 Chen, Y. G., Siddhanta, A., Austin, C. D., Hammond, S. M., Sung, T. C., Frohman, M. A., Morris, A. J., and Shields, D. (1997) Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network. J. Cell Biol. 138, 495504 Rajagopalan, S., and Andersen, J. K. (2001) Alpha-synuclein aggregation: is it the toxic gain of function responsible for neurodegeneration in Parkinson's disease? Mech. Ageing Dev. 122, 1499 1510 Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W. H., Castillo, P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A., et al. (2000) Mice lacking alpha-synuclein display functional decits in the nigrostriatal dopamine system. Neuron 25, 239 252 Murphy, D. D., Rueter, S. M., Trojanowski, J. Q., and Lee, V. M. (2000) Synucleins are developmentally expressed, and alphasynuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons. J. Neurosci. 20, 3214 3220 Slavotinek, A. M., and Biesecker, L. G. (2003) Unfolding the role of chaperones and chaperonins in human disease. Trends Genet. 17, 528 535 Ostrerova, N., Petrucelli, L., Farrer, M., Mehta, N., Choi, P., Hardy, J., and Wolozin, B. (1999) Alpha-synuclein shares physical and functional homology with 14-3-3 proteins. J. Neurosci. 19, 57825791 Xu, J., Kao, S. Y., Lee, F. J., Song, W., Jin, L. W., and Yankner, B. A. (2002) Dopamine-dependent neurotoxicity of alphasynuclein: a mechanism for selective neurodegeneration in Parkinson disease. Nat. Med. 8, 600 606 Boston, P. F., Jackson, P., and Thompson, R. J. (1982) Human 14-3-3 protein, radioimmunoassay, tissue distribution, and ce-

60. 61. 62.

63.

64.

65. 66. 67. 68.

69.

70.

71.

72.

73.

74.

75.

76.

77. 78.

79.

rebrospinal uid levels in patients with neurological disorders. J. Neurochem. 38, 14751482 Fu, H., Subramanian, R. R., and Masters, S. C. (2000) 14-3-3 proteins: structure, function and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617 647 Yuan, J., and Yanker, B. A. (2000) Apoptosis in the nervous system. Nature (London) 407, 802 809 Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Accelerated in vitro bril formation by a mutant alphasynuclein linked to early-onset Parkinson disease. Nat. Med. 11, 1318 1320 McKeith, I. G., Burn, D. J., Ballard, C. G., Collerton, D., Jaros, E., Morris, C. M., McLaren, A., Perry, E. K., Perry, R., Piggott, M. A., et al. (2003) Dementia with Lewy bodies. Semin. Clin. Neuropsychiatry 8, 46 57 Suh, Y. H., and Checler, F. (2002) Amyloid precursor protein, presenilins, and alpha-synuclein: molecular pathogenesis and pharmacological applications in Alzheimer's disease. Pharmacol. Rev. 54, 469 525 Simard, M., and van Reekum, R. (2001) Dementia with Lewy bodies in Down's syndrome. Int. J. Geriatr. Psychiatry 16, 311 320 Burn, D. J., and Jaros, E. (2001) Multiple system atrophy: cellular and molecular pathology. Mol. Pathol. 54, 419 426 Arai, K., Kato, N., Kashiwado, K., and Hattori, T. (2000) Pure autonomic failure in association with human alpha-synucleinopathy. Neurosci. Lett. 296, 171173 Mochizuki, A., Komatsuzaki, Y., and Shoji, S. (2002) Association of Lewy bodies and glial cytoplasmic inclusions in the brain of Parkinson's disease. Acta Neuropathol. (Berlin) 104, 534 537 Dickson, D. W., Liu, W., Hardy, J., Farrer, M., Mehta, N., Uitti, R., Mark, M., Zimmerman, T., Golbe, L., Sage, J., et al. (1999) Widespread alterations of alpha-synuclein in multiple system atrophy. Am. J. Pathol. 155, 12411251 Saito, Y., Kawai, M., Inoue, K., Sasaki, R., Arai, H., Nanba, E., Kuzuhara, S., Ihara, Y., Kanazawa, I., and Murayama, S. (2000) Widespread expression of alpha-synuclein and tau immunoreactivity in Hallervorden-Spatz syndrome with protracted clinical course. J. Neurol. Sci. 177, 48 59 Newell, K. L., Boyer, P., Gomez-Tortosa, E., Hobbs, W., HedleyWhyte, E. T., Vonsattel, J. P., and Hyman, B. T. (1999) Alpha-synuclein immunoreactivity is present in axonal swellings in neuroaxonal dystrophy and acute traumatic brain injury. J. Neuropathol. Exp. Neurol. 58, 12631268 Forman, M. S., Schmidt, M. L., Kasturi, S., Perl, D. P., Lee, V. M., and Trojanowski, J. Q. (2002) Tau and alpha-synuclein pathology in amygdala of Parkinsonism-dementia complex patients of Guam. Am. J. Pathol. 160, 17251731 Spillantini, M. G., Tolnay, M., Love, S., and Goedert, M. (1999) Microtubule-associated protein tau, heparan sulphate and alpha-synuclein in several neurodegenerative diseases with dementia. Acta Neuropathol. (Berlin) 97, 585594 Biere, A. L., Wood, S. J., Wypych, J., Steavenson, S., Jiang, Y., Ana, D., Jacobsen, F. W., Jarosinski, M. A., Wu, G. M., Louis, J. C., et al. (2000) Parkinson's disease-associated alphasynuclein is more brillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs. J. Biol. Chem. 275, 34574 34579 Serpell, L. C., Berriman, J., Jakes, R., Goedert, M., and Crowther, R. A. (2000) Fiber diffraction of synthetic alphasynuclein laments shows amyloid-like cross-beta conformation. Proc. Natl. Acad. Sci. USA 97, 4897 4902 Kahle, P. J., Neumann, M., Ozmen, L., Muller, V., Jacobsen, H., Schindzielorz, A., Okochi, M., Leimer, U., van Der Putten, H., Probst, A., et al. (2000) Subcellular localization of wild-type and Parkinson's disease-associated mutant alpha-synuclein in human and transgenic mouse brain. J. Neurosci. 20, 6365 6373 Forno, L. S. (1996) Neuropathology of Parkinson's disease. Neuropathol. Exp. Neurol. 55, 259 272 Galvin, J. E., Uryu, K., Lee, V. M., and Trojanowski, J. Q. (1999) Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein. Proc. Natl. Acad. Sci. USA 96, 13450 13455 Wood, S. J., Wypych, J., Steavenson, S., Louis, J. C., Citron, M., and Biere, A. L. (1999) -Synuclein brillogenesis is nucle-

ABNORMAL METABOLISM OF

-SYNUCLEIN IN PARKINSONS DISEASE

625

80.

81.

82.

83. 84. 85. 86. 87.

88.

89.

90.

91. 92.

93. 94.

95. 96.

ation-dependent. Implications for the pathogenesis of Parkinson's disease. J. Biol. Chem. 274, 19509 19512 Volles, M. J., and Lansbury, P. T., Jr. (2002) Vesicle permeabilization by protobrillar alpha-synuclein is sensitive to Parkinson's disease-linked mutations and occurs by a pore-like mechanism. Biochemistry 41, 4595 4602 Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Acceleration of oligomerization, not brillization, is a shared property of both alphasynuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. USA 97, 571576 Conway, K. A., Harper, J. D., and Lansbury, P. T., Jr. (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. Biochemistry 39, 25522563 Ciechanover, A. (2001) Linking ubiquitin, parkin and synphilin-1. Nat. Med. 7, 1108 1109 McNaught, K., Belizaire, R., Isacson, O., Jenner, P., and Olanow, C. W. (2003) Altered proteasomal function in sporadic Parkinson's disease. Exp. Neurol. 179, 38 46 Voges, D., Zwickl, P., and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 10151068 Gibb, W. R., and Lees, A. J. (1988) The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease. J. Neurol. Neurosurg. Psychiatry 51, 745752 Iwatsubo, T., Yamaguchi, H., Fujimuro, M., Yokosawa, H., Ihara, Y., Trojanowski, J. Q., and Lee, V. M. (1996) Purication and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease. Am. J. Pathol. 148, 15171529 Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross, C. A., Dawson V. L., and Dawson, T. M. (2001) Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med. 7, 1144 1150 West, A., Periquet, M., Lincoln, S., Lucking, C. B., Nicholl, D., Bonifati, V., Rawal, N., Gasser, T., Lohmann, E., Deleuze, J. F., et al. (2002) Complex relationship between Parkin mutations and Parkinson disease. Am. J. Med. Genet. 114, 584 591 Hashimoto, M., Hsu, L. J., Xia, Y., Takeda, A., Sisk, A., Sundsmo, M., and Masliah, E. (1999) Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro. NeuroReport 10, 717721 Goldberg, M. S., and Lansbury, P. T., Jr. (2000) Is there a cause-and-effect relationship between alpha-synuclein brillization and Parkinson's disease? Nat. Cell Biol. 2, E115E119 Ostrerova-Golts, N., Petrucelli, L., Hardy, J., Lee, J. M., Farer, M., and Wolozin, B. (2000) The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J. Neurosci. 20, 6048 6054 Jenner, P., and Olanow, C. W. (1998) Understanding cell death in Parkinson's disease. Ann. Neurol. 44, Suppl. 1, S72S84 Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620 1624 Jenner, P. (1998) Oxidative mechanisms in nigral cell death in Parkinson's disease. Mov. Disord. 13, 24 34 Zigmond, M. J., Hastings, T. G., and Perez, R. G. (2002) Increased dopamine turnover after partial loss of dopaminer-

97.

98.

99. 100.

101.

102.

103.

104. 105. 106. 107. 108. 109.

110. 111.

112.

gic neurons: compensation or toxicity. Parkinsonism Relat. Disord. 8, 389 393 Jones, D. C., Gunasekar, P. G., Borowitz, J. L., and Isom, G. E. (2000) Dopamine-induced apoptosis is mediated by oxidative stress and is enhanced by cyanide in differentiated PC12 cells. J. Neurochem. 74, 2296 2304 Berg, D., Gerlach, M., Youdim, M. B., Double, K. L., Zecca, L., Riederer, P., and Becker, G. (2001) Brain iron pathways and their relevance to Parkinson's disease. J. Neurochem. 79, 225 236 Ilic, T., Jovanovic, M., Jovicic, A., and Tomovic, M. (1998) Oxidative stress and Parkinson's disease. Vojnosanit. Pregl. 55, 463 468 Riederer, P., Soc, E., Rausch, W. D., Schmidt, B., Reynolds, G. P., Jellinger, K., and Youdim, M. B. (1989) Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 52, 515520 Junn, E., and Mouradian, M. (2002) Human -synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine. Neurosci. Lett. 320, 146 150 Rabinovic, A. D., Lewis, D. A., and Hastings, T. G. (2000) Role of oxidative changes in the degeneration of dopamine terminals after injection of neurotoxic levels of dopamine. Neuroscience 101, 6776 Zigmond, R, E., Schwarzschild, M. A., and Rittenhouse, A. R. (1989) Acute regulation of tyrosine hydroxylase by nerve activity and by neurotransmitters via phosphorylation. Annu. Rev. Neurosci. 12, 415 461 Perez, R. G., Waymire, J. C., Lin, E., Liu, J. J., Guo, F., and Zigmond, M. J. (2002) A role for alpha-synuclein in the regulation of dopamine biosynthesis. J. Neurosci. 22, 3090 3099 Baptista, M. J., O'Farrell, C., Daya, S., Ahmad, R., Miller, D. W., Hardy, J., Farrer, M. J., and Cookson, M. R. (2003) J. Neurochem. 85, 957968 Dawson, T. M., Mandir, A., and Lee, M. (2002) Animal models of PD: pieces of the same puzzle? Neuron 35, 219 222 Beal, M. F. (2001) Experimental models of Parkinson's disease. Nat. Rev. Neurosci. 2, 325334 Betabert, R., Sherer, T. B., and Greenamyre, J. T. (2002) Animal models of Parkinson's diseases. Bioessays 24, 308 318 McNaught, K. S., Bjorklund, L. M., Belizaire, R., Isacson, O., Jenner, P., and Olanow, C. W. (2002) Proteasome inhibition causes nigral degeneration with inclusion bodies in rats. NeuroReport 13, 14371441 Lee, M. K., and Price, D. L. (2001) Advances in genetic models of Parkinson's disease. Clin. Neurosci. Res. 1, 456 466 Lo Bianco, C., Ridet, J. L., Schneider, B. L., Deglon, N., and Aebischer, P. (2002) Alpha-synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease. Proc. Natl. Acad. Sci. USA 99, 1081310818 Kirik, D., Rosenblad, C., Burger, C., Lundberg, C., Johansen, T. E., Muzyczka, N., Mandel, R. J., and Bjorklund, A. (2002) Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J. Neurosci. 22, 2780 2791 Received for publication August 18, 2003. Accepted for publication December 16, 2003.

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