Role of oligomeric alpha-synuclein in synucleinopathies

描述(申请人提供)：不可溶蛋白质聚集体的存在是神经退行性疾病的病理特征，如帕金森氏病(PD)、路易体痴呆(DLB)、阿尔茨海默病、亨廷顿病和普恩蛋白紊乱。在PD和DLB等共核病中，路易体是在易感神经元群体的细胞质中发现的病理性蛋白质聚集体。路易小体是含有大量聚集蛋白的嗜酸性包涵体，包括α-突触核蛋白(1syn)，这表明该蛋白在这些疾病中起到了致病作用。1syn的错误折叠和聚集被认为是与疾病发展相关的关键辅助因素。特别是，作为纤维路易体形成中间体的非纤维二聚体和1syn寡聚体的积累被认为是导致神经退化的原因。由于1syn的寡聚体被认为对细胞有毒性，一个重要的治疗策略将是减少寡聚体的形成或完全阻止它。在这一应用中，我们建议研究寡聚体1syn在共核病中的作用，并研究特定的修饰如何影响毒性和寡聚体的形成。为了能够直接研究1syn齐聚，我们应用了新的策略，包括蛋白质互补分析(PCAs)、大小排除色谱(SEC)和荧光相关光谱(FCS)，通过这些策略可以识别和跟踪1syn寡聚体的形成，从而提供了一个强大的技术工具箱，用于研究影响1syn诱导毒性的操作。这项应用分为三个相互协调和相关的目标，这三个目标将表征1syn寡聚体，并研究1syn寡聚体在疾病发病机制中的作用，以确定疾病相关修饰和分子伴侣对1syn寡聚体形成和亚细胞定位的影响(目标1和2)。我们还建议通过立体定向注射重组腺相关病毒在体内重述1syn寡聚，并检测1syn寡聚体在体内的毒性和亚细胞定位，以及Aim 2(Aim 3)中确定的分子伴侣干预的效果。我们认为，这一应用提供了一种有效的策略，通过它来识别和调节1syn寡聚体的形成，并将治疗靶向于纤维化的前毒性阶段，在该阶段，拯救可溶的1syn单体形式可以显著影响疾病的进展。如果成功，所描述的实验将大大推进1syn诱导的毒性和相关低聚物形成的研究，并有助于开发成功的治疗策略来治疗帕金森病和相关疾病。因此，这项研究符合NINDS的使命--减轻神经系统疾病的负担。

DESCRIPTION (provided by applicant): The presence of insoluble protein aggregates is a pathological hallmark of neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease, Huntington's disease and prion protein disorders. In synucleinopathies such as PD and DLB, the Lewy body is the pathological protein aggregate that is found in the cytoplasm of susceptible neuronal populations. Lewy bodies are eosinophilic inclusions containing numerous aggregated proteins, including alpha-synuclein (1syn), which suggests a causative role for this protein in these disorders. Misfolding and aggregation of 1syn are thought to be critical cofactors linked to disease development. In particular, the accumulation of nonfibrillar dimers and oligomers of 1syn, which serve as intermediates for fibrillar Lewy body formation, is thought to cause neurodegeneration. Because oligomeric forms of 1syn are believed toxic to cells, an important therapeutic strategy will be to reduce oligomer formation or prevent it altogether. In this application we propose to study the role of oligomeric 1syn in synucleinopathies and examine how specific modifications affect toxicity and oligomer formation. To enable the direct study of 1syn oligomerization, we have applied novel strategies including protein complementation assays (PCAs), size exclusion chromatography (SEC) and fluorescence correlation spectroscopy (FCS) by which to identify and follow 1syn oligomer formation, thus providing a powerful toolbox of techniques with which to study manipulations affecting 1syn-induced toxicity. This application is divided into three coordinated and related aims that will characterize 1syn oligomers and investigate the role of 1syn oligomers in disease pathogenesis with a view to determining the effect of disease- related modifications and molecular chaperones on 1syn oligomer formation and subcellular location (aim 1 & 2). We also propose to recapitulate 1syn oligomerization in vivo via stereotactic injection of recombinant adeno-associated virus and examine the resulting toxicity and subcellular localization of 1syn oligomers in vivo, as well as the effect of molecular chaperone interventions identified in aim 2 (aim 3). We propose that this application offers an effective strategy by which to identify and modulate 1syn oligomer formation, and targets therapeutics to a pre-toxic stage of fibrillization, where rescue of the soluble, monomeric forms of 1syn can substantially impact disease progression. If successful, the experiments described will significantly advance the study of 1syn-induced toxicity and related oligomer formation, and contribute to the development of successful therapeutic strategies to treat PD and related disorders. Thus, this study is in accordance with the mission of NINDS - to reduce the burden of neurological disease.