Role of oligomeric alpha-synuclein in synucleinopathies

描述（由申请人提供）：不溶性蛋白聚集物的存在是神经退行性疾病的病理标志，如帕金森病（PD）、路易体痴呆（DLB）、阿尔茨海默病、亨廷顿氏病和朊蛋白疾病。在突触核蛋白病如PD和DLB中，路易体是在易感神经元群体的细胞质中发现的病理蛋白聚集体。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致病作用。错误折叠和1syn聚集被认为是与疾病发展相关的关键辅助因素。特别是，非纤原二聚体和1syn低聚体的积累，作为纤原路易体形成的中间体，被认为是导致神经变性的原因。由于1syn的低聚体形式被认为对细胞有毒，一个重要的治疗策略将是减少低聚体的形成或完全阻止它。在这个应用中，我们建议研究寡聚物1syn在突触核蛋白病中的作用，并检查特异性修饰如何影响毒性和寡聚物的形成。为了能够直接研究1syn寡聚化，我们应用了新的策略，包括蛋白质互补分析（PCAs），大小排斥色谱（SEC）和荧光相关光谱（FCS）来识别和跟踪1syn寡聚物的形成，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。该应用分为三个协调和相关的目标，将表征1syn低聚物并研究1syn低聚物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低聚物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来概括体内1syn寡聚化，并检查体内1syn寡聚物的毒性和亚细胞定位，以及在目标2（目标3）中确定的分子伴侣干预的效果。我们提出，该应用提供了一种有效的策略，通过该策略可以识别和调节1syn低聚物的形成，并将治疗靶向到纤维化的毒性前阶段，在此阶段，可溶性单体形式的1syn的挽救可以显著影响疾病进展。如果成功，上述实验将显著推进1syn诱导的毒性和相关寡聚物形成的研究，并有助于开发成功的治疗PD和相关疾病的治疗策略。因此，这项研究符合NINDS的使命-减轻神经系统疾病的负担。公共卫生相关性：神经退行性疾病的一个共同特征是“错误折叠”蛋白质的过度积累和细胞功能的破坏。有强有力的证据表明α -突触核蛋白（1syn）积累是散发性和遗传性帕金森病（PD）发展的早期步骤。由于1syn在神经元中积累和聚集，我们假设对1syn错误折叠、寡聚化和聚集的全面了解将对开发治疗PD和相关突触核蛋白病的新治疗策略至关重要。当前应用的目标是了解1syn低聚物的形成及其与人类疾病发展的关系，并进一步探索修饰和挽救1syn聚集的因素。

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. PUBLIC HEALTH RELEVANCE: A common feature of neurodegenerative diseases is the excessive accumulation of "misfolded" proteins and a disruption in cellular function. There is strong evidence to suggest that alpha-synuclein (1syn) accumulation is an early step in the development of both sporadic and inherited Parkinson disease (PD). Because 1syn accumulates and aggregates in neurons, we hypothesize that a complete understanding of 1syn misfolding, oligomerization and aggregation will be crucial to the development of new therapeutic strategies for the treatment of PD and related synucleinopathies. The goals of the current application are to understand 1syn oligomer formation and how it relates to disease development in humans and to further explore factors that modify and rescue 1syn aggregation.