Ubiquitination of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function

Lewy bodies are intracellular fibrillar inclusions composed of alpha-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble alpha-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic alpha-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22-24-kDa alpha-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22-24-kDa alpha-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified alpha-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22-24-kDa alpha-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated alpha-synuclein, presumably the result of aberrant proteolysis, is found only in association with alpha-synuclein aggregates.     

p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies

Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25alpha as a candidate that preferentially binds to alpha-synuclein in its aggregated state. Functionally, purified recombinant human p25alpha strongly stimulates the aggregation of alpha-synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25alpha is normally only expressed in oligodendrocytes in contrast to alpha-synuclein, which is normally only expressed in neurons. This expression pattern is changed in alpha-synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25alpha and dystopically expressed alpha-synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25alpha was detectable in the neuronal Lewy body inclusions along with alpha-synuclein. The localization in alpha-synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25alpha plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by alpha-synuclein aggregates.     

Could a loss of α‐synuclein function put dopaminergic neurons at risk?

The alpha-synuclein gene is implicated in Parkinson's disease, the symptoms of which occur after a marked loss of substantia nigra dopamine neurons. While the function of alpha-synuclein is not entirely elucidated, one function appears to be as a normal regulatory protein that can bind to and inhibit tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Soluble alpha-synuclein levels may be diminished in Parkinson's disease substantia nigra dopamine neurons both by reduced expression and by alpha-synuclein aggregation as Lewy bodies and Lewy neurites form. The loss of functional alpha-synuclein may then result in dysregulation of tyrosine hydroxylase, dopamine transport and dopamine storage, resulting in excess cytosolic dopamine. Because dopamine and its metabolites are reactive molecules capable of generating highly reactive quinones and reactive oxygen species, a failure to package dopamine into vesicles could cause irreversible damage to cellular macromolecules and contribute to resultant neurotoxicity. This review focuses on how a loss of normal alpha-synuclein function may contribute to the dopamine-related loss of substantia nigra neurons during Parkinson's disease pathogenesis.     

Overexpression of Parkinson's disease-associated alpha-synucleinA53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP

Mutations in the alpha-synuclein gene are linked to a rare dominant form of familial Parkinson's disease, and alpha-synuclein is aggregated in Lewy bodies of both sporadic and dominant Parkinson's disease. It has been proposed that mutated alpha-synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test this hypothesis in vivo, we overexpressed human alpha-synuclein(A53T) in the substantia nigra of normal and MPTP-treated mice by rAAV-mediated gene transfer. Determination of dopaminergic neuron survival, striatal tyrosine hydroxylase fiber density, and striatal content of dopamine and its metabolites in rAAV-injected and uninjected hemispheres demonstrated that alpha-synuclein(A53T) does not increase the susceptibility of dopaminergic neurons to MPTP. Our findings argue against a direct detrimental role for (mutant) alpha-synuclein in oxidative stress and/or apoptotic pathways triggered by MPTP, but do not rule out the possibility that alpha-synuclein aggregation in neurons exposed to oxidative stress for long periods of time may be neurotoxic.     

Role of cytochrome c as a stimulator of alpha-synuclein aggregation in Lewy body disease.

alpha-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which alpha-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in alpha-synuclein aggregation. When recombinant alpha-synuclein was coincubated with cytochrome c/hydrogen peroxide, alpha-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such as N-acetyl-L-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of alpha-synuclein, and both cytochrome c/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of alpha-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting alpha-synuclein aggregation. Furthermore, double labeling studies for cytochrome c/alpha-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson's disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of alpha-synuclein in Parkinson's disease and related disorders.     

The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease

Oxidative stress appears to be directly involved in the pathogenesis of Parkinson disease. Several different pathways have been identified for the production of oxidative stress conditions in nigral dopaminergic neurons, including a pathological accumulation of cytosolic dopamine with the subsequent production of toxic reactive oxygen species or the formation of highly reactive quinone species. On these premises, tyrosinase, a key copper enzyme known for its role in the synthesis of melanin in skin and hair, has been proposed to take part in the oxidative chemistry related to Parkinson disease. A study is herein presented of the in vitro reactivity of tyrosinase with alpha-synuclein, aimed at defining the molecular basis of their synergistic toxic effect. The results presented here indicate that, in conformity with the stringent specificity of tyrosinase, the exposed tyrosine side-chains are the reactive centers of alpha-synuclein. The reactivity of alpha-synuclein depends on whether it is free or membrane bound, and the chemical modifications on the tyrosinase-treated alpha-synuclein strongly influence its aggregation properties. On the basis of our results, we propose a cytotoxic model which includes a possible new toxic role for alpha-synuclein exacerbated by its direct chemical modification by tyrosinase.     

Stabilization of partially folded conformation during alpha-synuclein oligomerization in both purified and cytosolic preparations

Aggregation of alpha-synuclein is tightly associated with many neurodegenerative diseases, such as Parkinson's disease, dementia with Lewy body, Lewy body variant of Alzheimer's disease, multiple system atrophy, and Hallervorden-Spatz disease, implicating a crucial role of aggregated forms of alpha-synuclein in the pathogenesis. Here, we examined the effect of elevated temperature on the oligomerization and structural changes of alpha-synuclein in the early stage of aggregation and show that self-assembly is crucial for the stabilization of a partially folded conformation. The efficiency of alpha-synuclein oligomerization increased proportional to the temperature increase, both in purified form and in crude cytosolic preparation. This oligomerization coincided with a small but reproducible change in the circular dichroism spectrum and an increase in the 1-anilinonaphthalene-8-sulfonic acid binding. The hydrodynamic dimensions of the dimer measured by size exclusion chromatography suggest a pre-molten globule-like structure. These data suggest that partially folded alpha-synuclein, which is unstable in the monomeric form, is stabilized by self-assembly and that these oligomers may evolve into the fibril nucleus.     

Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies

Intracellular proteinaceous aggregates are hallmarks of many common neurodegenerative disorders, and recent studies have shown that alpha-synuclein is a major component of several pathological intracellular inclusions, including Lewy bodies in Parkinson's disease (PD) and glial cell inclusions in multiple system atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into filamentous inclusions remain unknown. Since oxidative and nitrative stresses are potential pathogenic mediators of PD and other neurodegenerative diseases, we asked if oxidative and/or nitrative events alter alpha-synuclein and induce it to aggregate. Here we show that exposure of human recombinant alpha-synuclein to nitrating agents (peroxynitrite/CO(2) or myeloperoxidase/H(2)O(2)/nitrite) induces formation of nitrated alpha-synuclein oligomers that are highly stabilized due to covalent cross-linking via the oxidation of tyrosine to form o,o'-dityrosine. We also demonstrate that oxidation and nitration of pre-assembled alpha-synuclein filaments stabilize these filaments to withstand denaturing conditions and enhance formation of SDS-insoluble, heat-stable high molecular mass aggregates. Thus, these data suggest that oxidative and nitrative stresses are involved in mechanisms underlying the pathogenesis of Lewy bodies and glial cell inclusions in PD and multiple system atrophy, respectively, as well as alpha-synuclein pathologies in other synucleinopathies.     

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.     

Interactions between metals and alpha-synuclein--function or artefact?

alpha-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenerative disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson's disease and Lewy body dementias, alpha-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson's diseases are linked to mutations in the gene for alpha-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins associated with a neurodegenerative disorder, this protein has now been suggested to bind copper. This finding is currently controversial. This review examines the evidence that alpha-synuclein is a copper binding protein and discusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation.     

Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein

The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.     

The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways

Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.     

Sensitization of neuronal cells to oxidative stress with mutated human alpha-synuclein

Linkage of alpha-synuclein (alpha-SN) mutations to familial Parkinson's disease (PD) and presence of alpha-SN as a major constituent of Lewy body in both sporadic and familial PD implicate alpha-SN abnormality in PD pathogenesis. Here we demonstrate that overexpression of wild-type or mutant alpha-SN does not cause any deleterious effect on the growth or continued propagation of transfected human cells, but overproduction of mutant alpha-SN heightens their sensitivity to menadione-induced oxidative injury. Such enhanced vulnerability is more pronounced in neuronal transfectants than in their nonneuronal counterparts and is associated with increased production of reactive oxygen species. The data suggest that mutated alpha-SN, especially with an alanine-to-proline substitution at residue 30, sensitizes neuronal cells to oxidative damage.     

Alpha-synuclein aggregation

Alpha-synuclein is a major component of Lewy bodies in Parkinson's disease and is found associated with several other forms of dementia. As with other neurodegenerative diseases, the ability of alpha-synuclein to aggregate and form fibrillar deposits seems central to its pathology. We have defined a sequence within the NAC region of alpha-synuclein that is necessary for aggregation. Exploitation of chemically modified analogues of this peptide may produce inhibitors of aggregation.     

Evidence for a partially folded intermediate in alpha-synuclein fibril formation

Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.     

Alpha-synuclein and the pathogenesis of Parkinson's disease

Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.     

alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD. alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease.     

Conformational behavior and aggregation of alpha-synuclein in organic solvents: modeling the effects of membranes

Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of alpha-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of alpha-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on alpha-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of alpha-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of beta-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TFE), or because of the appearance of a highly alpha-helical conformation at high TFE and hexafluoro-2-propanol concentrations. At least to some extent, these conformational effects mimic those observed in the presence of phospholipid vesicles, and can explain some of the observed effects of membranes on alpha-synuclein fibrillation.     

The role of JNK pathway in familial Parkinson's disease

Parkinson's disease is a neurodegenerative disorder characterized by a dramatic loss of dopaminergic neurons in the substantia nigra. Among the many pathogenic mechanisms thought to contribute to the demise of these cells in sporadic cases of PD, oxidative stress has taken center stage due to extensive experimental evidence showing that dopamine- or MPTP-derived reactive oxygen species and oxidized dopamine metabolites may trigger toxicity through mitochondrial inhibition or deleterious modifications of biomolecules. In familial forms of PD, however, the involvement of toxic protein aggregation (synuclein), impairment of ubiquitin-proteosome system (parkin. and loss of antioxidative properties (DJ-1) has gained attention. Recently, JNK pathway has come to light that could link malfunction of mutated DJ-1, parkin, PINK1 and alpha-synuclein to the oxidative stress-triggered apoptosis, finally ascribing a common pathogenic mechanism to both the sporadic and familial forms of PD.