Typage moléculaire des maladies neurodégénératives dues à l’agrégation de la protéine alpha synucléine

The aggregation of α-synuclein protein has been shown to be associated with Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, called synucleinopathies. Increasing amount of evidences suggest that synucleinopathies are prion diseases. Some aspects are missing for α-synuclein to be recognized as a prion, such as the existence of strains associated to synucleinopathies. During my thesis I set up a reliable method to amplify α-synuclein-rich deposits from patients tissues. I validated the method using all synucleinopathies tissues. This should allow the identification of α-synuclein strain related to each synucleinopathy. In addition, I also documented cleaning procedures for materials soiled with various amyloid fibers, in order to reduce the risk of contamination. Finally, I was associated to a study that shows the propagation abilities of different α-synuclein assemblies in a neuronal network mimicking human cortico-cortical connections. These results open the way to structural and functional studies of the amplified deposits.Les synucléinopathies regroupent les maladies neurodégénératives de Parkinson, les démences à corps de Lewy et l'atrophie multi-systématisée. Des études suggèrent que les synucléinopathies seraient des maladies à prion. Aujourd'hui, certains aspects manquent pour que l'α-synucléine soit reconnue comme un prion. Par exemple, il est à démontrer que chaque synucléinopathie est causée par une souche précise d'α-synucléine. Durant ma thèse j’ai mis au point une méthode d'amplification fiable des dépôts présents dans le cerveau des patients atteints de synucléinopathies. J’ai aussi documenté les procédures de nettoyage à adopter envers des matériels souillés, par diverses fibres amyloïdes, afin de réduire le risque de contamination. Finalement, j’ai été associé à une étude montrant les capacités de propagation d'assemblages d'α synucléine, dans un réseau de neurones humains en culture. Ces résultats permettront des études structurales, et fonctionnelles, des souches d’α-synucléine dans les synucléinopathies

Molecular Typing of Neurodegenerative Diseases Due to the Aggregation of the Protein Alpha Synuclein

Les synucléinopathies regroupent les maladies neurodégénératives de Parkinson, les démences à corps de Lewy et l'atrophie multi-systématisée. Des études suggèrent que les synucléinopathies seraient des maladies à prion. Aujourd'hui, certains aspects manquent pour que l'α-synucléine soit reconnue comme un prion. Par exemple, il est à démontrer que chaque synucléinopathie est causée par une souche précise d'α-synucléine. Durant ma thèse j’ai mis au point une méthode d'amplification fiable des dépôts présents dans le cerveau des patients atteints de synucléinopathies. J’ai aussi documenté les procédures de nettoyage à adopter envers des matériels souillés, par diverses fibres amyloïdes, afin de réduire le risque de contamination. Finalement, j’ai été associé à une étude montrant les capacités de propagation d'assemblages d'α synucléine, dans un réseau de neurones humains en culture. Ces résultats permettront des études structurales, et fonctionnelles, des souches d’α-synucléine dans les synucléinopathies.The aggregation of α-synuclein protein has been shown to be associated with Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, called synucleinopathies. Increasing amount of evidences suggest that synucleinopathies are prion diseases. Some aspects are missing for α-synuclein to be recognized as a prion, such as the existence of strains associated to synucleinopathies. During my thesis I set up a reliable method to amplify α-synuclein-rich deposits from patients tissues. I validated the method using all synucleinopathies tissues. This should allow the identification of α-synuclein strain related to each synucleinopathy. In addition, I also documented cleaning procedures for materials soiled with various amyloid fibers, in order to reduce the risk of contamination. Finally, I was associated to a study that shows the propagation abilities of different α-synuclein assemblies in a neuronal network mimicking human cortico-cortical connections. These results open the way to structural and functional studies of the amplified deposits

Assessment of the efficacy of different procedures that remove and disassemble alpha-synuclein, tau and A-beta fibrils from laboratory material and surfaces

International audienceα-synuclein fibrillar polymorphs, Tau and Aß 1-42 fibrillar assemblies have been shown to propagate, amplify and trigger the formation of protein deposits reminiscent of those present within the central nervous system of patients developing synucleinopathies, tauopathies and amyloid plaques after injection intracerebrally, intramuscularly, intraperitoneally or within the blood stream of model animals. They are thus hazardous and there is need for decontamination and inactivation procedures for laboratory surfaces and non-disposable material. We assessed the effectiveness of different reagents to clean and disassemble potentially pathogenic assemblies adsorbed on non-disposable materials in laboratories. We show that commercial detergents and SDS are way more suited to detach α-synuclein fibrillar polymorphs, Tau and Aß 1-42 fibrillar assemblies from contaminated surfaces and disassemble the fibrils than methods designed to decrease PrP prion infectivity. Our observations reveal that the choice of the most adapted cleaning procedure for one given protein assembly or fibrillar polymorph should integrate detergent's cleaning efficiency, material compatibility and capacity to dismantle assemblies. We provide an integrated representation where desorption and neutralization efficacy and surface compatibility are combined to facilitate the choice of the most adapted decontamination procedure. This representation, together with good laboratory practices, contributes to reducing potential health hazards associated to manipulating protein assemblies with prion-like properties

α-Synuclein liquid condensates fuel fibrillar α-synuclein growth

International audienceα-Synuclein (α-Syn) aggregation into fibrils with prion-like features is intimately associated with Lewy pathology and various synucleinopathies. Emerging studies suggest that α-Syn could form liquid condensates through phase separation. The role of these condensates in aggregation and disease remains elusive and the interplay between α-Syn fibrils and α-Syn condensates remains unexplored, possibly due to difficulties in triggering the formation of α-Syn condensates in cells. To address this gap, we developed an assay allowing the controlled assembly/disassembly of α-Syn condensates in cells and studied them upon exposure to preformed α-Syn fibrillar polymorphs. Fibrils triggered the evolution of liquid α-Syn condensates into solid-like structures displaying growing needle-like extensions and exhibiting pathological amyloid hallmarks. No such changes were elicited on α-Syn that did not undergo phase separation. We, therefore, propose a model where α-Syn within condensates fuels exogenous fibrillar seeds growth, thus speeding up the prion-like propagation of pathogenic aggregates

Aptamer binding footprints discriminate α-synuclein fibrillar polymorphs from different synucleinopathies

International audienceAbstract Synucleinopathies, including dementia with Lewy bodies (DLB), Parkinson's disease (PD), and multiple system atrophy (MSA), are characterized by the presence of α-synuclein (α-syn) aggregates in the central nervous system. Recent evidence suggests that the heterogeneity of synucleinopathies may be partly explained by the fact that patients may have different α-syn fibrillar polymorphs with structural differences. In this study, we identify nuclease resistant 2′fluoro-pyrimidine RNA aptamers that can differentially bind to structurally distinct α-syn fibrillar polymorphs. Moreover, we introduce a method, AptaFOOT-Seq, designed to rapidly assess the affinity of a mixture of these aptamers for different α-SYN fibrillar polymorphs using next-generation sequencing. Our findings reveal that the binding behavior of aptamers can be very different when they are tested separately or in the presence of other aptamers. In this case, competition and cooperation can occur, providing a higher level of information, which can be exploited to obtain specific ‘footprints’ for different α-Syn fibrillar polymorphs. Notably, these footprints can distinguish polymorphs obtained from patients with PD, DLB or MSA. This result suggests that aptaFOOT-Seq could be used for the detection of misfolded or abnormal protein conformations to improve the diagnosis of synucleinopathies

Seeding Propensity and Characteristics of Pathogenic α\alphaSyn Assemblies in Formalin-Fixed Human Tissue from the Enteric Nervous System, Olfactory Bulb, and Brainstem in Cases Staged for Parkinson’s Disease

International audienceWe investigated $\alpha$-synuclein’s ($\alpha$Syn) seeding activity in tissue from the brain and enteric nervous system. Specifically, we assessed the seeding propensity of pathogenic $\alpha$Syn in formalinfixed tissue from the gastric cardia and five brain regions of 29 individuals (12 Parkinson’s disease,8 incidental Lewy body disease, 9 controls) using a protein misfolding cyclic amplification assay. The tructural characteristics of the resultant $\alpha$Syn assemblies were determined by limited proteolysis and transmission electron microscopy. We show that fixed tissue from Parkinson’s disease (PD) and incidental Lewy body disease (ILBD) seeds the aggregation of monomeric $\alpha$Syn into fibrillar assemblies. Significant variations in the characteristics of fibrillar assemblies derived from different regions even within the same individual were observed. This finding suggests that fixation stabilizes seeds with an otherwise limited seeding propensity, that yield assemblies with different intrinsic structures (i.e., trains). The lag phase preceding fibril assembly for patients ≥80 was significantly shorter than in other age groups, suggesting the existence of increased numbers of seeds or a higher seeding potential of pathogenic $\alpha$Syn with time. Seeding activity did not diminish in late-stage disease. No statistically significant difference in the seeding efficiency of specific regions was found, nor was there a relationship between seeding efficiency and the load of pathogenic $\alpha$Syn in a particular region at a given neuropathological stage

Propagation of α-Synuclein Strains within Human Reconstructed Neuronal Network

International audienceReappraisal of neuropathological studies suggests that pathological hallmarks of Alzheimer’s disease and Parkinson’s disease (PD) spread progressively along predictable neuronal pathways in the human brain through unknown mechanisms. Although there is much evidence supporting the prion-like propagation and amplification of α-synuclein (α-Syn) in vitro and in rodent models, whether this scenario occurs in the human brain remains to be substantiated. Here we reconstructed in microfluidic devices corticocortical neuronal networks using human induced pluripotent stem cells derived from a healthy donor. We provide unique experimental evidence that different strains of human α-Syn disseminate in “wild-type” human neuronal networks in a prion-like manner. We show that two distinct α-Syn strains we named fibrils and ribbons are transported, traffic between neurons, and trigger to different extents, in a dose- and structure-dependent manner, the progressive accumulation of PD-like pathological hallmarks. We further demonstrate that seeded aggregation of endogenous soluble α-Syn affects synaptic integrity and mitochondria morphology

Detection of alpha-synuclein aggregates in gastrointestinal biopsies by protein misfolding cyclic amplification

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Parkinson’s disease-derived α-Synuclein assemblies combined with chronic-type inflammatory cues promote a neurotoxic microglial phenotype

Parkinson’s disease (PD) is a common age-related neurodegenerative disorder characterized by the aggregation of $\alpha$-synuclein ($\alpha$SYN) building up intraneuronal inclusions termed Lewy pathology. Mounting evidence suggests that neuron-released $\alpha$SYN aggregates could be central to microglial activation, which in turn mounts and orchestrates neuroinflammatory processes potentially harmful to neurons. Therefore, understanding the mechanisms that drive microglial cell activation, polarization and function in PD might have important therapeutic implications. Here, using primary microglia, we investigated the inflammatory potential of pure $\alpha$SYN fibrils derived from PD patients. We further explored and characterized microglial cell responses to a chronic-type inflammatory stimulation combining PD patient-derived $\alpha$SYN fibrils (F$^{PD}$ ), Tumor necrosis factor-$\alpha$ (TNF$\alpha$) and prostaglandin E$_2$ (PGE$_2$ ) (TPF $^{PD}$ ). We showed that F$^{PD}$ hold stronger inflammatory potency than pure $\alpha$SYN fibrils generated de novo . When combined with TNF$\alpha$ and PGE$_2$ , F$^{PD}$ polarizes microglia toward a particular functional phenotype departing from F$^{PD}$ -treated cells and featuring lower inflammatory cytokine and higher glutamate release. Whereas metabolomic studies showed that TPF$^{PD}$ -exposed microglia were closely related to classically activated M1 proinflammatory cells, notably with similar tricarboxylic acid cycle disruption, transcriptomic analysis revealed that TPF$^{PD}$ -activated microglia assume a unique molecular signature highlighting upregulation of genes involved in glutathione and iron metabolisms. In particular, TPF$^{PD}$ -specific upregulation of $Slc7a11$ (which encodes the cystine-glutamate antiporter xCT) was consistent with the increased glutamate response and cytotoxic activity of these cells toward midbrain dopaminergic neurons in vitro . Together, these data further extend the structure-pathological relationship of $\alpha$SYN fibrillar polymorphs to their innate immune properties and demonstrate that PD-derived αSYN fibrils, TNF$\alpha$and PGE$_2$ act in concert to drive microglial cell activation toward a specific and highly neurotoxic chronic-type inflammatory phenotype characterized by robust glutamate release and iron retention