Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.     

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have shown that alpha-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed alpha-synuclein stably silenced MN9D/alpha-SYN(-) cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/alpha-SYN(-) and MN9D/CON cells, suggesting that silencing alpha-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (L-DOPA) and DA levels were observed in MN9D/alpha-SYN(-) cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of alpha-synuclein, suggesting that alpha-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional alpha-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.     

Alpha-synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.     

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4-phenylbutyrate (4-PBA) on nigral dopamine (DA) neurons in rotenone-treated mice. 4-PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up-regulation of alpha-synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4-PBA inhibited rotenone-induced neuronal death and decreased the protein level of alpha-synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4-PBA has a neuroprotective effect in the treatment of PD.     

Parkin accumulation in aggresomes due to proteasome impairment

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and by the presence of ubiquitinated cytoplasmic inclusions known as Lewy bodies. Alpha-synuclein and Parkin are two of the proteins associated with inherited forms of PD and are found in Lewy bodies. Whereas numerous reports indicate the tendency of alpha-synuclein to aggregate both in vitro and in vivo, no information is available about similar physical properties for Parkin. Here we show that overexpression of Parkin in the presence of proteasome inhibitors leads to the formation of aggresome-like perinuclear inclusions. These eosinophilic inclusions share many characteristics with Lewy bodies, including a core and halo organization, immunoreactivity to ubiquitin, alpha-synuclein, synphilin-1, Parkin, molecular chaperones, and proteasome subunit as well as staining of some with thioflavin S. We propose that the process of Lewy body formation may be akin to that of aggresome-like structures. The tendency of wild-type Parkin to aggregate and form inclusions may have implications for the pathogenesis of sporadic PD.     

Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain

Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.     

Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments

The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from na?ve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.     

Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.     

Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1–60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H₂O₂ resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H₂O₂ of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.     

Inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alpha-synuclein. Implication for dopaminergic neurodegeneration

Fibrillization of alpha-synuclein (alpha-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit alpha-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of alpha-Syn chain, generating alpha-Syn-quinone adducts. It is likely that the amino groups of alpha-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked alpha-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact alpha-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of alpha-Syn with DA is relevant to the selective dopaminergic loss in PD.     

Differential cytotoxicity of human wild type and mutant alpha-synuclein in human neuroblastoma SH-SY5Y cells in the presence of dopamine

Parkinson's disease (PD) involves loss of dopaminergic neurons in the substantia nigra and is characterized by intracellular inclusions, Lewy bodies, consisting primarily of aggregated alpha-synuclein. Two substitution mutations (A53T and A30P) in alpha-synuclein gene have been identified in familial early-onset PD. To understand the biological changes that incur upon alpha-synuclein-induced cytotoxicity in the presence of dopamine, the current studies were undertaken. Human SH-SY5Y neuroblastoma cells coexpressing the human dopamine transporter [hDAT], and either wild type (wt) or mutant alpha-synucleins, were treated with 50 microM dopamine (DA). In cells expressing wt or A30P alpha-synuclein, DA accelerated production of reactive oxygen species and cell death as compared to cells expressing A53T or hDAT alone. The increased sensitivity of such cells to DA was investigated by measuring changes in cellular ionic gradient, by atomic absorption spectrometry, and cell metabolism, by high-resolution nuclear magnetic resonance spectroscopy. Both wt and A30P alpha-synuclein caused rapid decrease in levels of intracellular potassium, followed by mitochondrial damage and cytochrome c leakage, with decreased cellular metabolism as compared to cells expressing A53T or hDAT alone. Collapse of ionic gradient was significantly faster in A30P (t(1/2) = 3.5 h) than in wt (t(1/2) = 6.5 h) cells, and these changes in ionic gradient preceded cytochrome c leakage and depletion of metabolic energy. Neither wt nor mutant alpha-synuclein resulted in significant changes in ionic gradient or cellular metabolism in the absence of intracellular DA. These findings suggest a specific sequence of events triggered by dopamine and differentially exacerbated by alpha-synuclein and the A30P mutant.     

Degradative organelles containing mislocalized alpha-and beta-synuclein proliferate in presenilin-1 null neurons

Presenilin-1 null mutation (PS1 -/-) in mice is associated with morphological alterations and defects in cleavage of transmembrane proteins. Here, we demonstrate that PS1 deficiency also leads to the formation of degradative vacuoles and to the aberrant translocation of presynaptic alpha- and beta-synuclein proteins to these organelles in the perikarya of primary neurons, concomitant with significant increases in the levels of both synucleins. Stimulation of autophagy in control neurons produced a similar mislocalization of synucleins as genetic ablation of PS1. These effects were not the result of the loss of PS1 gamma-secretase activity; however, dysregulation of calcium channels in PS1 -/- cells may be involved. Finally, colocalization of alpha-synuclein and degradative organelles was observed in brains from patients with the Lewy body variant of AD. Thus, aberrant accumulation of alpha- and beta-synuclein in degradative organelles are novel features of PS1 -/- neurons, and similar events may promote the formation of alpha-synuclein inclusions associated with neurodegenerative diseases.     

Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein.

alpha-Synuclein is a major component of the fibrillary lesion known as Lewy bodies and Lewy neurites that are the pathologic hallmarks of Parkinson's disease (PD). In addition, point mutations in the alpha-synuclein gene imply alpha-synuclein dysfunction in the pathology of inherited forms of PD. alpha-Synuclein is a member of a family of proteins found primarily in the brain and is concentrated within presynaptic terminals. Here, we address the localization and membrane binding characteristics of wild type and PD mutants of alpha-synuclein in cultured cells. In cells treated with high concentrations of fatty acids, wild type alpha-synuclein accumulated on phospholipid monolayers surrounding triglyceride-rich lipid droplets and was able to protect stored triglycerides from hydrolysis. PD mutant synucleins showed variable distributions on lipid droplets and were less effective in regulating triglyceride turnover. Chemical cross-linking demonstrated that synuclein formed small oligomers within cells, primarily dimers and trimers, that preferentially associated with lipid droplets and cell membranes. Our results suggest that the initial phases of synuclein aggregation may occur on the surfaces of membranes and that pathological conditions that induce cross-linking of synuclein may enhance the propensity for subsequent synuclein aggregation.     

rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra.

Results

We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants.

Conclusions

In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7.

     

Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease

The mechanism by which dopaminergic neurons are selectively lost in Parkinson disease (PD) is unknown. Here we show that accumulation of alpha-synuclein in cultured human dopaminergic neurons results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species. In contrast, alpha-synuclein is not toxic in non-dopaminergic human cortical neurons, but rather exhibits neuroprotective activity. Dopamine-dependent neurotoxicity is mediated by 54 83-kD soluble protein complexes that contain alpha-synuclein and 14-3-3 protein, which are elevated selectively in the substantia nigra in PD. Thus, accumulation of soluble alpha-synuclein protein complexes can render endogenous dopamine toxic, suggesting a potential mechanism for the selectivity of neuronal loss in PD.     

GM1 specifically interacts with alpha-synuclein and inhibits fibrillation

The aggregation of alpha-synuclein is believed to be a key step in the etiology of Parkinson's disease. Alpha-synuclein is found in the cytosol and is associated with membranes in the presynaptic region of neurons and has recently been reported to be associated with lipid rafts and caveolae. We examined the interactions between several brain sphingolipids and alpha-synuclein and found that alpha-synuclein specifically binds to ganglioside GM1-containing small unilamellar vesicles (SUVs). This results in the induction of substantial alpha-helical structure and inhibition or elimination of alpha-synuclein fibril formation, depending on the amount of GM1 present. SUVs containing total brain gangliosides, gangliosides GM2 or GM3, or asialo-GM1 had weak inhibitory effects on alpha-synuclein fibrillation and induced some alpha-helical structure, while all other sphingolipids studied showed negligible interaction with alpha-synuclein. alpha-Synuclein binding to GM1-containing SUVs was accompanied by formation of oligomers of alpha-synuclein. The familial mutant A53T alpha-synuclein interacted with GM1-containing SUVs in an analogous manner to wild type, whereas the A30P mutant showed minimal interaction. This is the first detailed report showing a direct association between GM1 and alpha-synuclein, which is attributed to specific interaction between helical alpha-synuclein and both the sialic acid and carbohydrate moieties of GM1. The recruitment of alpha-synuclein by GM1 to caveolae and lipid raft regions in membranes could explain alpha-synuclein's localization to presynaptic membranes and raises the possibility that perturbation of GM1/raft association could induce changes in alpha-synuclein that contribute to the pathogenesis of PD.     

Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson's disease?

Parkinson's disease (PD) is one of many neurodegenerative diseases that are characterized by amyloid fibril formation. Alpha-synuclein is a primary component of the fibrillar neuronal inclusions, known as Lewy bodies, that are diagnostic of PD. In addition, the alpha-synuclein gene is linked to familial PD. Fibril formation by alpha-synuclein proceeds via discrete beta-sheet-rich oligomers, or protofibrils, that are consumed as fibrils grow. Both FPD mutations accelerate formation of protofibrils, suggesting that these intermediates, rather than the fibril product, trigger neuronal loss. In idiopathic PD, other factors may be responsible for accelerating protofibril formation by wild-type alpha-synuclein. One possible factor could be molecular crowding in the neuronal cytoplasm. We demonstrate here that crowding using inert polymers significantly reduced the lag time for protofibril formation and the conversion of the protofibril to the fibril, but did not affect the morphology of either species. Physiologically realistic changes in the degree of in vitro crowding have significant kinetic consequences. Thus, nonspecific changes in the total cytoplasmic protein concentration, induced by cell volume changes and/or altered protein degradation, could promote formation of and stabilize the alpha-synuclein protofibril.     

Impairment of redox state and dopamine level induced by alpha-synuclein aggregation and the prevention effect of hsp70

One hypothesis for the etiology of Parkinson's disease (PD) is that the formation of proteinaceous inclusion, which is mainly composed of alpha-synuclein, may contribute to the selective loss of dopaminergic neurons. To further explore the role of alpha-synuclein in neurodegeneration of PD, we examined the possible effects of aggregated alpha-synuclein on the intracellular redox state, dopamine level, and cell death of SK-N-SH cells. Our present studies show that alpha-synuclein aggregation gives rise to both elevated intracellular oxidative state and dopamine level in SK-N-SH cells. Moreover, alpha-synuclein aggregation results in a higher ratio of apoptosis population (55.8%+/-SEM) in cells overexpressing alpha-synuclein aggregation, compared to their normal control groups (8.0%+/-SEM). In contrast, coexpression of hsp70 with alpha-synuclein suppresses the oxidative state shift, restores the normal dopamine levels and blocks neuron cell loss. Therefore, our data provided one possible mechanism by which the alpha-synuclein aggregation may lead to the neurodegeneration in PD via regulating the level of cytoplasmic dopamine and then disturbing the intracellular redox homeostasis. On the other hand, hsp70 can mitigate the degenerative effect conferred by alpha-synuclein, acting as a protective factor in treatment of PD.