The many faces of α-synuclein: from structure and toxicity to therapeutic target

Disorders characterized by α-synuclein (α-syn) accumulation, Lewy body formation and parkinsonism (and in some cases dementia) are collectively known as Lewy body diseases. The molecular mechanism (or mechanisms) through which α-syn abnormally accumulates and contributes to neurodegeneration in these disorders remains unknown. Here, we provide an overview of current knowledge and prevailing hypotheses regarding the conformational, oligomerization and aggregation states of α-syn and their role in regulating α-syn function in health and disease. Understanding the nature of the various α-syn structures, how they are formed and their relative contributions to α-syn-mediated toxicity may inform future studies aiming to develop therapeutic prevention and intervention

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

Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease

Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD). In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies

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 P

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

Implication des systèmes à acides aminés excitateurs dans la maladie de Parkison et ses traitements

L expression des symptômes moteurs de la maladie de Parkinson (MP) est associée à une activation anormale des systèmes glutamatergiques dans les ganglions de la base (GB), incluant le noyau subthalamique (NST). De fait, le traitement chirurgical de la MP par stimulation à haute fréquence (SHF) du NST s est imposé au cours de ces dernières années comme une option thérapeutique de choix pour les patients à des stades avancés de la maladie chez lesquels le traitement de référence à la L-DOPA induit à long terme des effets indésirables sévères. Cependant, les bases cellulaires de ses effets thérapeutiques restent méconnues et son application présente des limitations importantes. Dans la recherche de traitements pharmacologiques alternatifs, les récepteurs métabotropiques du glutamate (mGluRs) ont été identifiés comme des cibles moléculaires d intérêt. Dans ce contexte, le premier objectif de mes travaux de thèse a été d analyser l impact de la SHF du NST appliquée de façon prolongée sur le fonctionnement physiopathologique des GB et de caractériser son interaction avec les effets de la LDOPA dans un modèle des stades tardifs de la MP, chez le rat. Nous avons montré que 5 jours de SHF continue du NST sont nécessaires pour corriger les déficits akinétiques induits par la lésion dopaminergique extensive et normaliser l activité métabolique neuronale au niveau du cortex moteur. De plus, nous avons identifié le striatum comme un site majeur d interaction entre les deux traitements. En effet, l association de la SHF prolongée du NST avec un traitement chronique à la L-DOPA potentialise les dyskinésies DOPA-induites ainsi que les réponses cellulaires striatales qui leur sont associées. Ce travail montre que la SHF du NST n a pas d effet anti-dyskinétique direct et suggère que la SHF du NST agisse principalement au niveau du striatum au travers d une voie de signalisation activée par la L-DOPA. Dans le deuxième axe, nous avons caractérisé les substrats cellulaires des effets bénéfiques du blocage pharmacologique du sous-type 5 des mGluRs (mGluR5), par un antagoniste sélectif, le MPEP, dans un modèle des stades symptomatiques précoces de la MP, chez le rat. Nos résultats montrent que, contrairement aux effets extensifs de la lésion dopaminergique totale, la lésion partielle a des effets focalisés dans le réseau des GB : une augmentation de l activité métabolique neuronale dans le NST et la SNr, associés une hypoactivité du cortex moteur. L effet anti-akinétique du traitement au MPEP est corrélé à la normalisation de l activité neuronale dans ces structures. Ces données, montrent l implication majeure d un sous-circuit subthalamo-nigro-cortical dans l expression de l akinésie dans les stades précoces de la MP et soulignent le potentiel thérapeutique du blocage des mGluR5. L ensemble de ce travail représente une contribution à l avancée des connaissances sur les bases neurales et moléculaires des symptômes parkinsoniens et de l action de différents traitements ciblant les systèmes glutamatergiques sur le contrôle moteur, avec des retombées potentielles pour améliorer le traitement symptomatique de la MP.The abnormal activation of glutamate systems, notably subthalamic nucleus (STN), in response to dopamine denervation in the basal ganglia (BG), plays a major role in the expression of the symptoms of Parkinson s disease (PD). The surgical treatment by high frequency stimulation (HFS) of STN has emerged as an efficient therapeutic option for PD patients suffering from severe side effects produced by long-term L-DOPA treatment. However, the action mechanisms of this stimulation remain poorly understood and its application presents important limitations. In the research of alternative pharmacological treatment, metabotropic glutamate receptors (mGluRs) have been recently stressed as interesting molecular targets. In this context, the first objective of my PhD work was to characterize the neural bases of the therapeutic action of STN HFS and its interaction with L-DOPA medication in a rat model of late PD. Our results show that prolonged STN HFS for several days is required to significantly alleviate the severe akinetic deficits produced by extensive dopamine lesion. Such prolonged HFS did not alleviate but even exacerbated the L-DOPA-induced dyskinesias (LID). We further identified striatum as a main site for the interactions between the two treatments. Indeed, when applied together or after chronic dyskinesiogenic L-DOPA treatment, the surgical treatment potentiated the L-DOPA-mediated cellular responses, including those linked to LID. These data argue against a direct anti-dyskinetic action of STN-HFS and suggest that this surgical treatment acts primarily at striatal level through L-DOPA signalling pathway. The second objective was to identify the cellular substrates of the antiparkinsonian benefits provided by the pharmacological blockade of mGluR5 using the antagonist MPEP in a rat model of early symptomatic stages of PD based on restricted denervation of the motor striatal territory. In this model, the expression of akinetic deficits was associated with focused metabolic changes in the corticobasal ganglia-cortical loop: neuronal metabolic activity was increased in the STN and SNr and decreased in the frontal cortex. The anti-akinetic action of MPEP was correlated with the reversal of these lesion-induced cellular changes. These data point to the major role of a subthalamo-nigro-cortical sub-circuit in the expression of akinesia in early PD stages, and to mGluR5 negative modulation as a promising antiparkinsonian strategy. Overall, this work contributes in advancing our knowledge of the neural and cellular bases of PD symptoms and of the action of different treatments targeting glutamate systems on motor control, with potential insights onto the symptomatic treatment of PD.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson’s disease

Summary: Studying Parkinson’s disease (PD) is complex due to a lack of cellular models mimicking key aspects of protein pathology. Here, we present a protocol for inducing and monitoring α-synuclein aggregation in living cells using optogenetics. We describe steps for plasmid transduction, biochemical validation, immunocytochemistry, and live-cell confocal imaging. These induced aggregates fulfill the cardinal features of authentic protein inclusions observed in PD-diseased brains and offer a tool to study the role of protein aggregation in neurodegeneration.For complete details on the use and execution of this protocol, please refer to Bérard et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Optimized protocol for the generation of functional human induced-pluripotent-stem-cell-derived dopaminergic neurons

Summary: Generation of functional human dopaminergic (DA) neurons from human induced pluripotent stem cells (hiPSCs) is a crucial tool for modeling dopamine-related human diseases and cell replacement therapies. Here, we present a protocol to combine neuralizing transcription factor (NGN2) programming and DA patterning to differentiate hiPSCs into mature and functional induced DA (iDA) neurons. We describe steps from transduction of hiPSCs and neural induction through to differentiation and maturation of near-pure, fully functional iDA neurons within 3 weeks.For complete details on the use and execution of this protocol, please refer to Sheta et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Alpha-Synuclein and the Endolysosomal System in Parkinson’s Disease: Guilty by Association

Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson’s disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders