Α-syn Toxicity Research Articles

Lack of additive role of ageing in nigrostriatal neurodegeneration triggered by α-synuclein overexpression.

IntroductionParkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons as well as the presence of proteinaceous inclusions named Lewy bodies. α-synuclein (α-syn) is a major constituent of Lewy bodies, and the first disease-causing protein characterized in PD. Several α-syn-based animal models of PD have been developed to investigate the pathophysiology of PD, but none of them recapitulate the full picture of the disease. Ageing is the most compelling and major risk factor for developing PD but its impact on α-syn toxicity remains however unexplored. In this study, we developed and exploited a recombinant adeno-associated viral (AAV) vector of serotype 9 overexpressing mutated α-syn to elucidate the influence of ageing on the dynamics of PD-related neurodegeneration associated with α-syn pathology in different mammalian species.ResultsIdentical AAV pseudotype 2/9 vectors carrying the DNA for human mutant p.A53T α-syn were injected into the substantia nigra to induce neurodegeneration and synucleinopathy in mice, rats and monkeys. Rats were used first to validate the ability of this serotype to replicate α-syn pathology and second to investigate the relationship between the kinetics of α-syn-induced nigrostriatal degeneration and the progressive onset of motor dysfunctions, strikingly reminiscent of the impairments observed in PD patients. In mice, AAV2/9-hα-syn injection into the substantia nigra was associated with accumulation of α-syn and phosphorylated hα-syn, regardless of mouse strain. However, phenotypic mutants with either accelerated senescence or resistance to senescence did not display differential susceptibility to hα-syn overexpression. Of note, p-α-syn levels correlated with nigrostriatal degeneration in mice. In monkeys, hα-syn-induced degeneration of the nigrostriatal pathway was not affected by the age of the animals. Unlike mice, monkeys did not exhibit correlations between levels of phosphorylated α-syn and neurodegeneration.ConclusionsIn conclusion, AAV2/9-mediated hα-syn induces robust nigrostriatal neurodegeneration in mice, rats and monkeys, allowing translational comparisons among species. Ageing, however, neither exacerbated nigrostriatal neurodegeneration nor α-syn pathology per se. Our unprecedented multi-species investigation thus favours the multiple-hit hypothesis for PD wherein ageing would merely be an aggravating, additive, factor superimposed upon an independent disease process.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0222-2) contains supplementary material, which is available to authorized users.

α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease

Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.

Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation.

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1-β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-β peptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1-β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.

Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease

Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP+ (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP+ induced toxicity in vitro. However, only CBE-induced enhancement of MPP+ toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration.

Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial Regulation and Toxicity in Parkinson Disease

Alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is implicated in the etiology of Parkinson disease (PD) and a number of other neurodegenerative dementias. Though recent research demonstrates the involvement of α-syn in a variety of mitochondrial dysfunction in neurodegeneration, the molecular mechanism of α-syn toxicity and its effect on neuronal mitochondria remain vague. We demonstrate a functional interaction between α-syn and the voltage-dependent anion channel (VDAC), the major conduit for ATP and other bioenergetics metabolites in the mitochondrial outer membrane. We found that at nanomolar concentrations, the full-length α-syn, its 45 amino acid C-terminal truncated mutant, as well as β- and α-synuclein isoforms induce reversible and highly voltage-dependent blockage of VDAC reconstituted into planar lipid bilayers. Binding parameters varied with each isoform, revealing the key role of the negatively charged C-terminal of synucleins in blocking a positively charged pore of VDAC. Synuclein-blocked states of VDAC differ in ionic selectivity, implying the existence of several conformation states of synuclein molecules in the channel pore. We propose a model of α-syn interaction with VDAC, in which the negatively charged C-terminus of α-syn enters the net-positive channel pore, providing a steric block for ATP flux. Experiments with a yeast strain deficient in VDAC1 demonstrate that α-syn toxicity in yeast depends on VDAC, revealing α-syn interaction with the channel in living cells. Thus, our findings show the long-sought physiological and pathophysiological roles for monomeric α-syn, which reconcile previous observations of various synuclein effects on mitochondrial bioenergetics.

High-Affinity Interaction with VDAC Links Cytosolic Proteins to Mitochondrial Regulation in Health, Cancer, and Neurodegeneration

We address the question of how mitochondria control cell survival and cell death by studying the voltage-dependent anion channel (VDAC). VDAC, the major channel of the mitochondrial outer membrane (MOM), is a well-recognized key conduit for ATP and other bioenergetics metabolites fluxes across MOM. We have found that dimeric tubulin, the subunit of microtubule, induces highly efficient reversible blockage of VDAC reconstituted into planar lipid membranes. Although the tubulin-blocked state still conducts small ions, it is virtually impermeable to ATP. We propose that by modulating VDAC permeability for ATP and other respiratory substrates, tubulin controls mitochondrial respiration. These findings are supported by experiments with isolated mitochondria and human hepatoma cells, thus uncovering a mechanism of regulation of mitochondrial energetics by free tubulin and also suggesting how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (the Warburg effect).We also found a functional interaction between VDAC and α-synuclein (α-syn), an intrinsically disordered neuronal protein intimately associated with Parkinson disease (PD) pathogenesis. Importantly, in addition to regulation of VDAC permeability by α-syn, our data indicate that VDAC facilitates translocation of α-syn across MOM where it could target complexes of the mitochondrial respiratory chain in the inner membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. Considering that VDAC is a major conduit for respiratory substrates across the mitochondrial outer membrane, we conclude that the α-syn/VDAC functional interaction reveals the elusive physiological and pathophysiological roles for monomeric α-syn in PD and also in general neurodegeneration.

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons

Mutual exacerbation of peroxisome proliferator‐activated receptor γ coactivator 1α deregulation and α‐synuclein oligomerization

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression. We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay. In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity. Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies.

The Parkinson's Disease-Related Protein DJ-1 Protects Dopaminergic Neurons in vivo and Cultured Cells from Alpha-Synuclein and 6-Hydroxydopamine Toxicity

Background: Dopaminergic degeneration is a major finding in brains of patients with Parkinson's disease (PD), together with Lewy bodies, intraneuronal inclusions mainly composed of the fibrillogenic protein α-synuclein (α-syn). The familial-PD-related protein DJ-1 was reported to reduce dopaminergic degeneration triggered by α-syn or by the dopaminergic-selective neurotoxin 6-hydroxydopamine (6-OHDA). Objective: The aim was to further investigate the role of DJ-1 in dopaminergic degeneration and to see whether a cell-permeable recombinant form of DJ-1 (TAT-DJ-1) could restore dopamine depletion in vivo, thus representing an innovative therapeutic approach. Methods: We developed in vitro (PC12/TetOn cells and mouse primary mesencephalic neurons) and in vivo models [including DJ-1 knockout (-/-) mice] to investigate DJ-1 in dopaminergic degeneration. Results: We found that in PC12/TetOn cells overexpressing α-syn with the familial-PD linked mutation A30P, DJ-1 silencing increased α-syn (A30P) toxicity. Primary mesencephalic neurons from DJ-1 (-/-) mice were more vulnerable to a cell-permeable form of α-syn (TAT-α-syn) and to 6-OHDA. Intrastriatally administered TAT-DJ-1 reduced 6-OHDA toxicity in vivo in C57BL/6 mice. Finally, when we injected TAT-α-syn (A30P) in the striatum of DJ-1 (-/-) animals, dopamine was depleted more than in the control strain. Conclusion: DJ-1 appears to have a protective role against dopaminergic degeneration triggered by α-syn or 6-OHDA, reinforcing the possible therapeutic importance of this protein in PD.

Protective role of olesoxime against wild-type α-synuclein-induced toxicity in human neuronally differentiated SHSY-5Y cells.

Parkinson's disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity. We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways. Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation. Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD.

(Poly)phenols protect from α-synuclein toxicity by reducing oxidative stress and promoting autophagy.

Parkinson's disease (PD) is the most common movement neurodegenerative disorder and is associated with the aggregation of α-synuclein (αSyn) and oxidative stress, hallmarks of the disease. Although the precise molecular events underlying αSyn aggregation are still unclear, oxidative stress is known to contribute to this process. Therefore, agents that either prevent oxidative stress or inhibit αSyn toxicity are expected to constitute potential drug leads for PD. Both pre-clinical and clinical studies provided evidence that (poly)phenols, pure or in extracts, might protect against neurodegenerative disorders associated with oxidative stress in the brain. In this study, we analyzed, for the first time, a (poly)phenol-enriched fraction (PEF) from leaves of Corema album, and used in vitro and cellular models to evaluate its effects on αSyn toxicity and aggregation. Interestingly, the PEF promoted the formation of non-toxic αSyn species in vitro, and inhibited its toxicity and aggregation in cells, by promoting the autophagic flux and reducing oxidative stress. Thus, C. album (poly)phenols appear as promising cytoprotective compounds, modulating central events in the pathogenesis of PD, such as αSyn aggregation and the impairment of autophagy. Ultimately, the understanding of the molecular effects of (poly)phenols will open novel opportunities for the exploitation of their beneficial effects and for drug development.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no β-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human α-synuclein.

The essential ubiquitin ligase Rsp5 is a key enzyme involved in the degradation of abnormal or unfavourable proteins in the yeast Saccharomyces cerevisiae. Overexpression of human α-synuclein (α-syn), a small lipid-binding protein implicated in several neurodegenerative diseases, in S. cerevisiae leads to growth inhibition due to many intracellular defects, including accumulation of reactive oxygen species (ROS). Here, to understand the mechanism of Rsp5-mediated detoxification of α-syn, we isolated novel Rsp5 variants (T255A, D295G, P343S and N427D), which conferred α-syn tolerance to yeast cells. Interestingly, these mutants were phenotypically distinguished from our previously identified RSP5(T357A) mutation, which increases ubiquitination of the general amino acid permease Gap1. Among them, the RSP5(P343S) substitution accelerated the degradation of α-syn, suppressed the accumulation of intracellular ROS and enhanced the interaction with α-syn and its ubiquitination. In contrast, the RSP5(T255A) mutation did not contribute to degradation of α-syn, but improved cell growth under acetate stress conditions, possibly leading to alleviation of the α-syn toxicity. Thus, these novel mutations might be useful not only in elucidating the molecular basis by which disused proteins are specifically recognized and effectively removed but also in screening drug candidates for neurodegenerative diseases or in improving ethanol production under acidic fermentation conditions.

ATP13A2/PARK9 regulates secretion of exosomes and α-synuclein.

Kufor-Rakeb syndrome (KRS) is caused by loss-of-function mutations in ATP13A2 (PARK9) and characterized by juvenile-onset parkinsonism, pyramidal signs, and cognitive decline. Previous studies suggested that PARK9 deficiency causes lysosomal dysfunction and α-synuclein (α-syn) accumulation, whereas PARK9 overexpression suppresses toxicity of α-syn. However, the precise mechanism of PARK9 effect on lysosomes and α-syn has been unknown. Here, we found that overexpressed PARK9 localized to multivesicular bodies (MVBs) in the human H4 cell line. The results from patient fibroblasts showed that loss of PARK9 function leads to decreased number of the intraluminal vesicles in MVBs and diminished release of exosomes into culture media. By contrast, overexpression of PARK9 results in increased release of exosomes in H4 cells and mouse primary cortical neurons. Moreover, loss of PARK9 function resulted in decreased secretion of α-syn into extracellular space, whereas overexpressed PARK9 promotes secretion of α-syn, at least in part via exosomes. Finally, we found that PARK9 regulates exosome biogenesis through functional interaction with the endosomal sorting complex required for transport machinery. Together, these data suggest the involvement of PARK9 in the biogenesis of exosomes and α-syn secretion and raise a possibility that disruption of these pathways in patients with KRS contributes to the disease pathogenesis.

Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour.

A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD.

Structure activity relationship of phenolic acid inhibitors of α-synuclein fibril formation and toxicity.

The aggregation of α-synuclein (α-syn) is considered the key pathogenic event in many neurological disorders such as Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, giving rise to a whole category of neurodegenerative diseases known as synucleinopathies. Although the molecular basis of α-syn toxicity has not been precisely elucidated, a great deal of effort has been put into identifying compounds that could inhibit or even reverse the aggregation process. Previous reports indicated that many phenolic compounds are potent inhibitors of α-syn aggregation. The aim of the present study was to assess the anti-aggregating effect of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a benzoic acid derivative that belongs to a group of phenolic compounds known as phenolic acids. By employing an array of biophysical and biochemical techniques and a cell-viability assay, GA was shown not only to inhibit α-syn fibrillation and toxicity but also to disaggregate preformed α-syn amyloid fibrils. Interestingly, GA was found to bind to soluble, non-toxic oligomers with no β-sheet content, and to stabilize their structure. The binding of GA to the oligomers may represent a potential mechanism of action. Additionally, by using structure activity relationship data obtained from fourteen structurally similar benzoic acid derivatives, it was determined that the inhibition of α-syn fibrillation by GA is related to the number of hydroxyl moieties and their position on the phenyl ring. GA may represent the starting point for designing new molecules that could be used for the treatment of PD and related disorders.

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

Sequestration of a β‐Hairpin for Control of α‐Synuclein Aggregation

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson's disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β-wrapins (β-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:β-wrapin complex reveals a β-hairpin of α-syn comprising the sequence region α-syn(37-54). The β-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.

C-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.