Ubiquitination of alpha-synuclein and autophagy in Parkinson's disease

alpha-Synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of alpha-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of alpha-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates alpha-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of alpha-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated alpha-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated alpha-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated alpha-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing alpha- synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.     

Ubiquitination of α-synuclein and autophagy in Parkinson's disease

α-synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of α-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of α-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates α-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of α-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated α-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated α-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated α-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing α-synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.

Addendum to: Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S. Monoubiquitination of α-synuclein by SIAH promotes its aggregation in dopaminergic cells. J Biol Chem 2007; Epub ahead of print.     

Glycogen synthase kinase 3beta modulates synphilin-1 ubiquitylation and cellular inclusion formation by SIAH: implications for proteasomal function and Lewy body formation

alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.     

Alpha-Synuclein is degraded by both autophagy and the proteasome

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. alpha-Synuclein is the major protein in Lewy bodies and rare mutations in alpha-synuclein cause early-onset PD. Consequently, alpha-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of alpha-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T alpha-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed alpha-synuclein degradation in these cells. We found that not only is alpha-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of alpha-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of alpha-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans.     

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.     

Oxidized glutathione stimulated the amyloid formation of alpha-synuclein

alpha-Synuclein is the major filamentous constituent of Lewy bodies found in Parkinson's disease (PD). The amyloid formation of alpha-synuclein was significantly facilitated by oxidized glutathione (GSSG) as the lag period of the aggregation kinetics was shortened by 2.5-fold from its absence. Reduced glutathione (GSH), on the other hand, did not influence the lag phase although it increased the final amyloid formation. The GSSG stimulation was specific for not only alpha-synuclein but also its intactness. The preferred GSSG interaction of alpha-synuclein to GSH was also demonstrated with dissociation constants of 0.53 and 43.5 mM, respectively. It is suggested that the oxidative stress favoring the GSSG generation from GSH could result in the augmented amyloid formation of alpha-synuclein, which ought to be related to the pathogenesis of PD.     

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.     

Molecular mechanisms of alpha-synuclein neurodegeneration

alpha-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynaptic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the alpha-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. alpha-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson's disease. Although there is substantial evidence supporting the toxic nature of alpha-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of alpha-synuclein toxicity is presented and discussed.     

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.     

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.     

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.     

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.     

Mechanisms of Suppression of {alpha}-Synuclein Neurotoxicity by Geldanamycin in Drosophila

Parkinson's disease is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of the protein alpha-synuclein into aggregates called Lewy bodies and Lewy neurites. Parkinson's disease can be modeled in Drosophila where directed expression of alpha-synuclein induces compromise of dopaminergic neurons and the formation of Lewy body-like aggregates. The molecular chaperone Hsp70 protects cells from the deleterious effects of alpha-synuclein, indicating a potential therapeutic approach to enhance neuron survival in Parkinson's disease. We have now investigated the molecular mechanisms by which the drug geldanamycin protects neurons against alpha-synuclein toxicity. Our studies show that geldanamycin sensitizes the stress response within normal physiological parameters to enhance chaperone activation, offering protection against alpha-synuclein neurotoxicity. Further, geldanamycin uncouples neuronal toxicity from Lewy body and Lewy neurite formation such that dopaminergic neurons are protected from the effects of alpha-synuclein expression despite the continued presence of (and even increase in) inclusion pathology. These studies indicate that compounds that modulate the stress response are a promising approach to treat Parkinson's disease.     

Synphilin-1 degradation by the ubiquitin-proteasome pathway and effects on cell survival

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.     

Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity

The deposition of alpha-synuclein and other cellular proteins in Lewy bodies in midbrain dopamine neurons is a pathological hallmark of Parkinson's disease. Nitrative and oxidative stress can induce alpha-synuclein protein aggregation, possibly initiated by the formation of stable cross-linking dimers. To determine whether enhanced dimer formation can accelerate protein aggregation and increase cellular toxicity, we have substituted cysteine for tyrosine at positions 39, 125, 133, and 136 in human wild-type (WT) alpha-synuclein, and in A53T and A30P mutant alpha-synuclein. To reduce the likelihood of cross-linking, phenylalanine was substituted for tyrosine at the same sites. We have found that overexpression of Y39C or Y125C mutant proteins leads to increased intracellular inclusions and apoptosis in a rat dopaminergic cell line (N27 cells) and in human embryonic kidney 293 cells. Expression of Y133C, Y136C, and all four Tyr-to-Phe mutations were not more cytotoxic than WT control. Exposure to oxidative stress increased Y39C and Y125C alpha-synuclein aggregation and toxicity. Dimers and oligomers were found in Triton X-100-soluble fractions from adenovirus-mediated overexpression of Y39C and Y125C in N27 cells. In contrast, WT beta-synuclein and all four Tyr-to-Cys mutant beta-synucleins did not cause protein aggregation and cell death. We conclude that cysteine substitution at critical positions in the alpha-synuclein molecule can increase dimer formation and accelerate protein aggregation and cellular toxicity of alpha-synuclein.     

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.     

Magnesium inhibits spontaneous and iron-induced aggregation of alpha-synuclein

Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to alpha-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in alpha-synuclein as indicators of metal-related conformational changes in alpha-synuclein, we show that iron and magnesium both interact with alpha-synuclein. alpha-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of alpha-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits alpha-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases alpha-synuclein aggregation, while magnesium at concentrations >0.75 mm inhibits the aggregation of alpha-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of alpha-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation.     

A single amino acid substitution differentiates Hsp70-dependent effects on alpha-synuclein degradation and toxicity

alpha-Synuclein aggregation and toxicity play a major role in Parkinson's disease and dementia with Lewy bodies. Hsp70 is a multipurpose stress response chaperone protein that mediates both refolding and degradation of misfolded proteins. We have shown that Hsp70 is able to block both alpha-synuclein toxicity and aggregation. Here we introduce a mutation into the ATPase domain of Hsp70 (K71S) and demonstrate that this abolishes Hsp70 refolding activity. Nonetheless, Hsp70K71S continues to mediate alpha-synuclein degradation and blocks aggregate formation. In contrast to wild type Hsp70, the ATPase domain mutant mediates alpha-synuclein degradation through a non-proteasome inhibitor sensitive pathway. Although Hsp70K71S can diminish levels of alpha-synuclein to an even greater extent than Hsp70, HSP70K71S does not protect against alpha-synuclein toxicity. The Hsp70K71S mutant appears to dissociate the formation of aggregates, which it blocks, and toxicity, which it does not block. These data suggest that the ability of Hsp70 to prevent toxicity is distinct from degradation of alpha-synuclein and is dependent on its ATPase domain.     

Dequalinium-induced protofibril formation of alpha-synuclein

alpha-Synuclein is the major constituent of Lewy bodies, a pathological signature of Parkinson disease, found in the degenerating dopaminergic neurons of the substantia nigra pars compacta. Amyloidosis generating the insoluble fibrillar protein deposition has been considered to be responsible for the cell death observed in the neurodegenerative disorder. In order to develop a controlling strategy toward the amyloid formation, 1,1'-(1,10-decanediyl)-bis-[4-a-mino-2-methylquinolinium] (dequalinium), was selected and examined in terms of its specific molecular interaction with alpha-synuclein. The protein was self-oligomerized by dequalinium, which gave rise to the ladder formation on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-PAGE in the presence of a coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The double-headed structure of dequalinium with the two cationic 4-aminoquinaldinium rings was demonstrated to be critical for the protein self-oligomerization. The dequalinium-binding site was located on the acidic C-terminal region of the protein with an approximate dissociation constant of 5.5 mum. The protein self-oligomerization induced by the compound has resulted in the protofibril formation of alpha-synuclein before it has developed into amyloids. The protofibrils were demonstrated to affect the membrane intactness of liposomes, and they have also been shown to influence cell viability of human neuroblastoma cells. In addition, dequalinium treatment of the alpha-synuclein-overexpressing cells exerted a significant cell death. Therefore, it is pertinent to consider that dequalinium could be used as a molecular probe to assess toxic mechanisms related to the amyloid formation of alpha-synuclein. Ultimately, the compound could be employed to develop therapeutic and preventive strategies toward alpha-synucleinopathies including Parkinson disease.     

Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate alpha-synuclein fibrilization

Oxidative stress, inflammation and alpha-synuclein overexpression confer risk for development of alpha-synucleinopathies-neurodegenerative diseases that include Parkinson disease and Lewy body dementia. Dopaminergic neurons undergo degeneration in these diseases and are particularly susceptible to oxidative stress because dopamine metabolism itself creates reactive oxygen species. Intraneuronal deposition of alpha-synuclein as amyloid fibrils or Lewy bodies is the hallmark of these diseases. Herein, we demonstrate that concentrations of oxidative cholesterol metabolites derived from reactive oxygen species are elevated in the cortices of individuals with Lewy body dementia relative to those of age-matched controls, and we show that these metabolites accelerate alpha-synuclein aggregation in vitro. The increase in the production of these cytotoxic cholesterol metabolites is also observed in a dopaminergic cell line that overexpresses alpha-synuclein. By extension, these data lead to the hypothesis that oxidative stress produces cholesterol aldehydes that enable alpha-synuclein aggregation, leading to a pathologic cycle.