Α-syn Degradation Research Articles

Targeting α-Synuclein in Parkinson's Disease: Progress Towards the Development of Disease-Modifying Therapeutics.

Parkinson's disease (PD), the second most common neurodegenerative movement disorder, is characterized by progressive motor and non-motor symptoms [1]. Despite treatment with pharmacologic and surgical therapies, the disease will continue to relentlessly advance. Hence, there is a great deal of interest in potential disease-modifying therapies with the hope that the neurodegenerative process can be slowed or halted. The purpose of this review is to highlight the role toxic α-synuclein (α-syn) plays in PD pathogenesis and critically review the relevant literature about therapeutic modalities targeting α-syn. Toxic α-syn plays a key role in PD pathogenesis, disrupting important cellular functions, and, thus, targeting α-syn is a reasonable disease-modifying strategy. Current approaches under investigation include decreasing α-syn production with RNA interference (RNAi), inhibiting α-syn aggregation, promoting intracellular degradation of α-syn aggregates (via enhancing autophagy and enhancing lysosomal degradation), and promoting extracellular degradation of α-syn via active and passive immunization.

Canthin-6-One Accelerates Alpha-Synuclein Degradation by Enhancing UPS Activity: Drug Target Identification by CRISPR-Cas9 Whole Genome-Wide Screening Technology.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline–inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.

Time-Resolved NMR Analysis of Proteolytic α-Synuclein Processing in vitro and in cellulo.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

Dysregulation of bcl-2 enhanced rotenone-induced α-synuclein aggregation associated with autophagic pathways.

α-Synuclein (α-syn) aggregation has far-reaching implications in the pathogenesis of Parkinson's disease, and the levels of α-syn protein determine its neurotoxic potential. However, the intrinsic pathway of α-syn accumulation and the mode of α-syn degradation remain contentious. Following a stereotactic infusion of rotenone into the substantia nigra and the ventral tegmental area, the chronic rat model of Parkinson's disease was established successfully. In response to the rotenone, increased intracellular α-syn levels and autophagic flux monitored by LC3 II turnover were induced in dopaminergic neurons (TH-positive) of rat substantia nigra and ventral tegmental area. In the cytoplasm, increased immune response of LC3 colocalized with α-syn on the basis of rotenone-mediated neurotoxicity. The immunoreactivity for p62, an adaptor of the autophagy, was upregulated in the cytoplasm and nucleus. The enhancement of autophagy by valproate acid decreased rotenone-induced α-syn aggregation, whereas the inhibition of autophagy by 3-methyladenine increased α-syn aggregation. In addition, the expression of bcl-2 was reduced in rotenone-induced neurotoxicity, accompanied by the enhancement of autophagy. Small interfering RNA-mediated knockdown of bcl-2 expression facilitated the expression of p62 protein and autophagy. Moreover, the inhibition of bcl-2 increased rotenone-based α-syn aggregation. In short, in rotenone-based models, dowregulation of bcl-2 negatively controlled rotenone-induced autophagy and α-syn aggregation.

Import and Export of Misfolded α-Synuclein.

In Parkinson's disease, intracellular α-synuclein (α-syn) inclusions form in neurons and are referred to as Lewy bodies. These aggregates spread through the brain following a specific pattern leading to the hypothesis that neuron-to-neuron transfer is critical for the propagation of Lewy body pathology. Here we review recent studies employing pre-formed fibrils generated from recombinant α-syn to evaluate the uptake, trafficking, and release of α-syn fibrils. We outline methods of internalization as well as cell surface receptors that have been described in the literature as regulating α-syn fibril uptake. Pharmacological and genetic studies indicate endocytosis is the primary method of α-syn internalization. Once α-syn fibrils have crossed the plasma membrane they are typically trafficked through the endo-lysosomal system with autophagy acting as the dominant method of α-syn clearance. Interestingly, both chaperone-mediated autophagy and macroautophagy have been implicated in the degradation of α-syn, although it remains unclear which system is chiefly responsible for the removal of α-syn fibrils. The major hallmark of α-syn spreading is the templating of misfolded properties onto healthy protein resulting in a conformational change; we summarize the evidence indicating misfolded α-syn can seed endogenous α-syn to form new aggregates. Finally, recent studies demonstrate that cells release misfolded and aggregated α-syn and that these processes may involve different chaperones. Nonetheless, the exact mechanism for the release of fibrillar α-syn remains unclear. This review highlights what is known, and what requires further clarification, regarding each step of α-syn transmission.

The Exosomal/Total α-Synuclein Ratio in Plasma Is Associated With Glucocerebrosidase Activity and Correlates With Measures of Disease Severity in PD Patients.

Intensive research efforts in the field of Parkinson’s disease (PD) are focusing on identifying reliable biomarkers which possibly help physicians in predicting disease onset, diagnosis, and progression as well as evaluating the response to disease-modifying treatments. Given that abnormal alpha-synuclein (α-syn) accumulation is a primary component of PD pathology, this protein has attracted considerable interest as a potential biomarker for PD. Alpha-synuclein can be detected in several body fluids, including plasma, where it can be found as free form or in association with exosomes, small membranous vesicles secreted by virtually all cell types. Together with α-syn accumulation, lysosomal dysfunctions seem to play a central role in the pathogenesis of PD, given the crucial role of lysosomes in the α-syn degradation. In particular, heterozygous mutations in the GBA1 gene encoding lysosomal enzyme glucocerebrosidase (GCase) are currently considered as the most important risk factor for PD. Different studies have found that GCase deficiency leads to accumulation of α-syn; whereas at the same time, increased α-syn may inhibit GCase function, thus inducing a bidirectional pathogenic loop. In this study, we investigated whether changes in plasma total and exosome-associated α-syn could correlate with disease status and clinical parameters in PD and their relationship with GCase activity. We studied 39 PD patients (mean age: 65.2 ± 8.9; men: 25), without GBA1 mutations, and 33 age-matched controls (mean age: 61.9 ± 6.2; men: 15). Our results showed that exosomes from PD patients contain a greater amount of α-syn compared to healthy subjects (25.2 vs. 12.3 pg/mL, p < 0.001) whereas no differences were found in plasma total α-syn levels (15.7 vs. 14.8 ng/mL, p = 0.53). Moreover, we highlighted a significant increase of plasma exosomal α-syn/total α-syn ratio in PD patients (1.69 vs. 0.89, p < 0.001), which negatively correlates with disease severity (p = 0.014). Intriguingly, a significant inverse correlation between GCase activity and this ratio in PD subjects was found (p = 0.006). Additional and large-scale studies comparing GCase activity and pathological protein levels will be clearly needed to corroborate these data and determine whether the association between key players in the lysosomal system and α-syn can be used as diagnostic or prognostic biomarkers for PD.

RETRACTED ARTICLE: 27-Hydroxycholesterol increases α-synuclein protein levels through proteasomal inhibition in human dopaminergic neurons

BackgroundAccumulation of the α-synuclein (α-syn) protein is a hallmark of a group of brain disorders collectively known as synucleinopathies. The mechanisms responsible for α-syn accumulation are not well understood. Several studies suggest a link between synucleinopathies and the cholesterol metabolite 27-hydroxycholesterol (27-OHC). 27-OHC is the major cholesterol metabolite in the blood that crosses the blood brain barrier, and its levels can increase following hypercholesterolemia, aging, and oxidative stress, which are all factors for increased synucleinopathy risk. In this study, we determined the extent to which 27-OHC regulates α-syn levels in human dopaminergic neurons, the cell type in which α-syn accumulates in PD, a major synucleinopathy disorder.ResultsOur results show that 27-OHC significantly increases the protein levels, not the mRNA expression of α-syn. The effects of 27-OHC appear to be independent of an action through liver X receptors (LXR), its cognate receptors, as the LXR agonist, GW3965, or the LXR antagonist ECHS did not affect α-syn protein or mRNA levels. Furthermore, our data strongly suggest that the 27-OHC-induced increase in α-syn protein levels emanates from inhibition of the proteasomal degradation of this protein and a decrease in the heat shock protein 70 (HSP70).ConclusionsIdentifying 27-OHC as a factor that can increase α-syn levels and the inhibition of the proteasomal function and reduction in HSP70 levels as potential cellular mechanisms involved in regulation of α-syn. This may help in targeting the correct degradation of α-syn as a potential avenue to preclude α-syn accumulation.

Glutamine promotes Hsp70 and inhibits α-Synuclein accumulation in pheochromocytoma PC12 cells.

Hsp70 regulates α-Synuclein (α-Syn) degeneration in Parkinson's disease (PD), indicating that Hsp70 promotion may be able to prevent or reverse α-Syn-induced toxicity in PD. Additionally, it has been demonstrated that glutamine (Gln) enhances Hsp70 expression. In the present study, Gln-induced Hsp70 promotion in pheochromocytoma was investigated with reverse transcription- quantitative polymerase chain reaction and western blotting methods. Then it was observed whether heat shock factor (HSF)-1 was required for this phenomenon with an RNA interference strategy. The regulatory role of Gln on α-Syn degeneration was also determined in the α-Syn-overexpressed PC12 [PC12 (α-Syn+)] cells, which were treated with or without the proteasomal inhibitor lactacystin (Lac). The results demonstrated that treatment with ≥10 mM Gln significantly increased Hsp70 mRNA and protein levels (P<0.05) and that this promotion was HSF-1-dependent, as HSF-1 knockout with HSF-1-specific small interfering RNA abrogated Hsp70 promotion in PC12 (α-Syn+) cells. Furthermore, Gln treatment markedly upregulated α-Syn degeneration in PC12 (α-Syn+) cells, which was significantly reduced (P<0.05) in the presence of Lac. Therefore, the present study suggests that Gln is able to induce the promotion of Hsp70 expression in PC12 cells in an HSF-1-dependent manner and that Gln-mediated Hsp70 promotion may increase α-Syn degradation even in the presence of proteasomal inhibitor. Thus, glutamine may be a potential therapeutic agent to prevent α-Syn aggregation in PD.

Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation

Increasing lines of evidence support the causal link between α-synuclein (α-syn) accumulation in the brain and Parkinson's disease (PD) pathogenesis. Therefore, lowering α-syn protein levels may represent a viable therapeutic strategy for the treatment of PD and related disorders. We recently described a novel selective α-syn degradation pathway, catalyzed by the activity of the Polo-like kinase 2 (PLK2), capable of reducing α-syn protein expression and suppressing its toxicity in vivo However, the exact molecular mechanisms underlying this degradation route remain elusive. In the present study we report that among PLK family members, PLK3 is also able to catalyze α-syn phosphorylation and degradation in living cells. Using pharmacological and genetic approaches, we confirmed the implication of the macroautophagy on PLK2-mediated α-syn turnover, and our observations suggest a concomitant co-degradation of these two proteins. Moreover, we showed that the N-terminal region of α-syn is important for PLK2-mediated α-syn phosphorylation and degradation and is implicated in the physical interaction between the two proteins. We also demonstrated that PLK2 polyubiquitination is important for PLK2·α-syn protein complex degradation, and we hypothesize that this post-translational modification may act as a signal for the selective recognition by the macroautophagy machinery. Finally, we observed that the PD-linked mutation E46K enhances PLK2-mediated α-syn degradation, suggesting that this mutated form is a bona fide substrate of this degradation pathway. In conclusion, our study provides a detailed description of the new degradation route of α-syn and offers new opportunities for the development of therapeutic strategies aiming to reduce α-syn protein accumulation and toxicity.

Neuroprotective Effects of Paeoniflorin on 6-OHDA-Lesioned Rat Model of Parkinson's Disease.

Paeoniflorin (PF) is the main active component extracted from the roots of Paeonialactiflora, a traditional Chinese medicine used for the treatment of neurodegenerative disorders, especially Parkinson's disease (PD). The degeneration of dopaminergic (DA-) neurons in PD may be caused by pathological activation of acid-sensing ion channels (ASICs). Thus, we designed a series of experiments to evaluate the therapeutic effects of PF and to test whether its effects are related to its inhibitory effect on ASIC1a. We found that systemic administration of PF or ASICs blockers (psalmotoxin-1 and amiloride) improved behavioral symptoms, delayed DA-neuronal loss and attenuated the reduction of dopamine (DA) and its metabolites in a rat model of 6-hydroxydopamine (6-OHDA)-induced PD. In addition, our data showed that PF, like ASICs blockers, regulated the expression of ASIC1a, decreased the level of α-synuclein (α-SYN), and improved autophagic dysfunction. Further experiments showed that ASIC1a knockdown down-regulated the α-SYN level and alleviated the autophagic injury in the 6-OHDA-treated ASIC1a-silenced PC12 cells. In summary, these findings indicate that PF enhanced the autophagic degradation of α-SYN and, thus, protected DA-neurons against the neurotoxicity caused by 6-OHDA. These findings also provide experimental evidence that PF may be a neuroprotectant for PD by acting on ASIC1a and that ASIC1a may be involved in the pathogenesis of PD.

β-asarone increases MEF2D and TH levels and reduces α-synuclein level in 6-OHDA-induced rats via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway: Chaperone-mediated autophagy activation, macroautophagy inhibition and HSP70 up-expression

Inactive myocyte enhancer factor 2D (MEF2D) and alpha-synuclein (α-syn) aggregation will cause neuronal death. MEF2D or α-syn degradation is also associated with macroautophagy, chaperone-mediated autophagy (CMA) and heat-shock protein 70 (HSP70). We found that β-asarone had positive effects on treating 6-hydroxydopamine (6-OHDA)-induced rats, but mechanisms of β-asarone affecting on MEF2D and α-syn via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway remain unclear. Unilateral 6-OHDA injection into the medial forebrain bundle was used to create PD rats, which were divided into four groups and administered for 30days: 6-OHDA model group, MEF2D inhibitor-treated group (SB203580, 0.5mg/kg, i.p.), MEF2D activator-treated group (LiCl, 100mg/kg, i.p.), β-asarone-treated group (15mg/kg, p.o.). Expressions of tyrosine hydroxylase (TH), α-syn, heat-shock cognate protein 70 (HSC70), lysosome-associated membrane protein type 2a (LAMP-2A), MEF2D, HSP70, Beclin-1, light chain 3B (LC3B) and p62 in the mesencephalon were measured after 30-day administration. α-syn, Beclin-1 and LC3B levels were higher in the 6-OHDA model group, while TH, MEF2D, HSC70, LAMP-2A, p62 levels were lower compared to the sham-operated group. Our results also showed thatβ-asarone treatment reduced protein and mRNA levels of α-syn, Beclin-1 and LC3B, but increased HSP70, TH, MEF2D, HSC70, LAMP-2A and p62 levels compared to the 6-OHDA model group. Additionally, certain correlations among α-syn, TH, Beclin-1, LC3B, p62, HSP70, LAMP-2A and MEF2D were also discovered in this study. These findings suggested that β-asarone treatment could increase MEF2D and TH as well as reduce α-syn to protect against 6-OHDA induced damage in PD rat mesencephalon via modulating the HSP70/MAPK/MEF2D/Beclin-1 pathway.

What can trigger the onset of Parkinson's disease – A modeling study based on a compartmental model of α-synuclein transport and aggregation in neurons

The aim of this paper is to develop a minimal model describing events leading to the onset of Parkinson's disease (PD). The model accounts for α-synuclein (α-syn) production in the soma, transport toward the synapse, misfolding, and aggregation. The production and aggregation of polymeric α-syn is simulated using a minimalistic 2-step Finke–Watzky model. We utilized the developed model to analyze what changes in a healthy neuron are likely to lead to the onset of α-syn aggregation. We checked the effects of interruption of α-syn transport toward the synapse, entry of misfolded (infectious) α-syn into the somatic and synaptic compartments, increasing the rate of α-syn synthesis in the soma, and failure of α-syn degradation machinery. Our model suggests that failure of α-syn degradation machinery is probably the most likely cause for the onset of α-syn aggregation leading to PD.

Fasudil, a Rho kinase inhibitor, promotes the autophagic degradation of A53T α-synuclein by activating the JNK 1/Bcl-2/beclin 1 pathway

Accumulation of α-synuclein (α-syn) is pivotally implicated in the pathogenesis of Parkinson׳s disease (PD), and enhancing its clearance might be a promising strategy in PD treatment. It has recently been shown that Rho kinase (ROCK) activation is involved in many neurodegenerative diseases, and some ROCK inhibitors might promote the degradation of abnormal protein aggregates. However, it is not known if fasudil, the only ROCK inhibitor available in clinical setting, could promote the degradation of α-syn, and ameliorate the α-syn induced neurotoxicity. In this regard, we investigated the effect of fasudil on neurite injury caused by A53T α-syn overexpression and the implicated pathway it might mediate. In the current study, we found that under the condition of A53T α-syn overexpression, the neurite outgrowth decreased significantly with the increasing expression of ROCK2. Fasudil, the ROCK inhibitor, ameliorated such neurotoxicity and promoted the clearance of A53T α-syn. Its underlying mechanism was supported by that fasudil could increase the macroautophagy activation via JNK 1 and Bcl-2 phosphorylation and beclin 1/Vps34 complex formation. Taken together, fasudil might be able to provide a novel and promising strategy for PD treatment by enhancing α-syn clearance and activating the JNK 1/Bcl-2/beclin 1 pathway.

The Interplay between Alpha-Synuclein Clearance and Spreading.

Parkinson’s Disease (PD) is a complex neurodegenerative disorder classically characterized by movement impairment. Pathologically, the most striking features of PD are the loss of dopaminergic neurons and the presence of intraneuronal protein inclusions primarily composed of alpha-synuclein (α-syn) that are known as Lewy bodies and Lewy neurites in surviving neurons. Though the mechanisms underlying the progression of PD pathology are unclear, accumulating evidence suggests a prion-like spreading of α-syn pathology. The intracellular homeostasis of α-syn requires the proper degradation of the protein by three mechanisms: chaperone-mediated autophagy, macroautophagy and ubiquitin-proteasome. Impairment of these pathways might drive the system towards an alternative clearance mechanism that could involve its release from the cell. This increased release to the extracellular space could be the basis for α-syn propagation to different brain areas and, ultimately, for the spreading of pathology and disease progression. Here, we review the interplay between α-syn degradation pathways and its intercellular spreading. The understanding of this interplay is indispensable for obtaining a better knowledge of the molecular basis of PD and, consequently, for the design of novel avenues for therapeutic intervention.

Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy

Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.

Proteasome inhibitor-induced autophagy in PC12 cells overexpressing A53T mutant α-synuclein.

The aim of the present study was to examine the effects of proteasome inhibitor (PI)-induced autophagy on PC12 cells overexpressing A53T mutant α-synuclein (α-syn) by detecting alterations in the levels of microtubule-associated protein 1A/1B light chain (LC3)+ autophagosomes and the lysotracker-positive autolysosomes using immunofluorescence, the expression of LC3-II using western blot analysis and the morphology of PC12 cells using transmission electron microscopy. It was found that the addition of MG132 (500 nmol/l) significantly increased the number of autophagosomes and autolysosomes and upregulated the expression of LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) completely inhibited the autophagy induced by MG132 (500 nmol/l). The autophagy enhancer trehalose significantly increased the number of autophagosomes and autolysosomes and improved the protein level of LC3-II induced by MG132. To examine the effect of PI-induced autophagy on the degradation of A53T mutant α-syn, the expression of α-syn was detected by western blot analysis. It was revealed that MG132 increased the expression of A53T α-syn and trehalose counteracted the increase of A53T α-syn induced by MG132. Combined inhibition of 3-MA and PI significantly increased the accumulation of A53T α-syn as compared with treatment using either single agent. In addition, combination of MG132 (500 nmol/l) with trehalose (50 mmol/l) or 3-MA (2 mmol/l) markedly decreased the cell viability as compared with treatment using either single agent individually as demonstrated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These results suggest that the PI, MG132, could induce autophagy in PC12 cells overexpressing A53T mutant α-syn and this autophagy could be completely inhibited by 3-MA, indicating that PI-induced autophagy is mediated by the upregulation of the macroautophagy class III PI3K pathway. PI-induced autophagy may act as a compensatory degradation system for degradation of A53T α-syn when the ubiquitin-proteasome system is impaired. Autophagy activation may directly contribute to the survival of PC12 cells treated with proteasome inhibitors. The present study may assist in illuminating the association between PI and autophagy in the pathogenesis of Parkinson’s disease.

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.

VPS35 dysfunction impairs lysosomal degradation of α-synuclein and exacerbates neurotoxicity in a Drosophila model of Parkinson's disease

Mutations in vacuolar protein sorting 35 (VPS35) have been linked to familial Parkinson's disease (PD). VPS35, a component of the retromer, mediates the retrograde transport of cargo from the endosome to the trans-Golgi network. Here we showed that retromer depletion increases the lysosomal turnover of the mannose 6-phosphate receptor, thereby affecting the trafficking of cathepsin D (CTSD), a lysosome protease involved in α-synuclein (αSYN) degradation. VPS35 knockdown perturbed the maturation step of CTSD in parallel with the accumulation of αSYN in the lysosomes. Furthermore, we found that the knockdown of Drosophila VPS35 not only induced the accumulation of the detergent-insoluble αSYN species in the brain but also exacerbated both locomotor impairments and mild compound eye disorganization and interommatidial bristle loss in flies expressing human αSYN. These findings indicate that the retromer may play a crucial role in αSYN degradation by modulating the maturation of CTSD and might thereby contribute to the pathogenesis of the disease.

Mimicking Lysosomal Degradation of α-Synuclein

Pathological accumulation of α-synuclein (α-syn) is a feature of Parkinson's disease. This accumulation may be counteracted by mechanisms of protein degradation that involve the proteasome and lysosome. Specifically, the lysosomal protease, Cathepsin D (CatD), has been suggested to be the main enzyme involved in the degradation of α-syn in vivo. In vitro, only C-terminal truncated species are generated, arguing that other mechanisms are needed to fully explain α-syn degradation. Here, we show that N-terminally acetylated α-syn also generates C-terminal as well as N-terminal truncated variants in the presence of CatD. These species are shown to be more aggregation prone. Since α-syn associates with membranes, we have investigated the effects of various glycosphingolipids such as glucosylceramide (GlcCer), on CatD degradation of α-syn. It is known that GlcCer buildup is a hallmark of the lysosomal storage disorder Gaucher disease and that these patients have an increased risk of developing PD. Our data clearly shows that in the presence of GlcCer and CatD, α-syn is completely proteolyzed. These data offer new mechanistic insight into α-syn degradation in the lysosome.

Polo-like kinase 2 regulates selective autophagic α-synuclein clearance and suppresses its toxicity in vivo

An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.