Production and Distribution of well-characterized polymorphic variants of alpha-synuclein aggregates

摘要 阿尔茨海默病(AD)是最常见的痴呆症形式，可能导致60%-70%的病例，其次是路易体痴呆(LBD)，占17%。AD和相关的痴呆症被认为是由于错误折叠的蛋白质聚集体在大脑中积累而引起的。在AD中，蛋白质聚集体主要以淀粉样斑块和神经原纤维缠结的形式出现，分别由淀粉样β蛋白(ABeta)和过度磷酸化的Tau组成。在LBD中，聚集体的主要成分是α-突触核蛋白(ASyn)。然而，病理异常有很大的重叠，导致混合病理的出现相对频繁，其特征是在同一大脑中存在多个蛋白质聚集体。事实上，多达30%-40%的AD患者在他们的大脑中显示出Syn Louy小体。尽管已有大量研究了解A~+和Tau聚集体在AD中的作用，但很少有人研究aSyn在AD及相关痴呆中的作用。最近，我们实施并开发了一种高度敏感和特异的分析方法(称为aSyn-PMCA)，以忠实地扩增存在于包括LBD在内的各种联核病患者的脑和生物液中的aSyn种子。使用这项技术，我们能够从患者的样本中扩增出具有播种能力的aSyn聚合体，并识别与不同疾病相关的构象菌株。该项目的主要目标是利用aSyn-PMCA技术的可靠性来大规模扩增aSyn多态变体，以便生产并向科学界分发从患者样本中扩增出的具有良好特征的aSyn聚合体。材料将使用各种体外、细胞和动物模型来表征其大小、生化、生物物理、结构和生物学特性。具体目标包括：(1)通过Syn-PMCA扩增从AD和LBD(以及其他共核病)患者的脑和脑脊液中产生aSyn聚集体，并综合分析这些聚集体的大小、生化和生物物理性质；(2)研究区分不同共核病的结构基础，特别是我们建议阐明与不同疾病有关的aSyn聚集体的结构折叠；(3)使用体外和体内模型研究AD、LBD和其他共核病患者产生的aSyn聚集体的生物学特性。(4)生产并向科学界分发具有良好特性和生物相关性的aSyn聚集体。该项目产生的资源将完全提供给研究人员，以使试剂标准化，并确保研究结果的生物学相关性。

ABSTRACT Alzheimer's disease (AD) is the most common form of dementia and may contribute to 60--70% of cases, followed in prevalence by Lewy body dementia (LBD), responsible for 17% of the cases. AD and related dementias are thought to be caused by the accumulation of misfolded protein aggregates in the brain. In AD, protein aggregates mostly appear in the form of amyloid plaques and neurofibrillary tangles composed of amyloid-beta (ABeta) and hyperphosporylated Tau, respectively. In LBD the main component of the aggregates is the alpha-synuclein (aSyn) protein. However, there is a substantial overlap of pathological abnormalities, leading to the relatively frequent appearance of mixed pathologies, characterized by the presence of multiple protein aggregates in the same brain. Indeed, up to 30-40% of AD patients display aSyn Lewy bodies in their brains. Although, much investigation has been devoted to understand the role of A~ and Tau aggregates in AD, little research has been done to evaluate the contribution of aSyn in AD and related dementias. Recently, we implemented and developed a highly sensitive and specific assay (termed aSyn-PMCA) to faithfully amplify aSyn seeds present in brain and biological fluids of patients affected by various synucleinopathies, including LBD. Using this technology we were able to amplify seeding competent aSyn aggregates from patients' samples and identify conformational strains associated to different diseases. The major goal of this project is to take advantage of the reliability of the aSyn-PMCA technology to massively amplify aSyn polymorphic variants in order to produce and distribute to the scientific community well-characterized aSyn aggregates amplified from patient's samples. Materials will be characterized for their size, biochemical, biophysical, structural and biological properties using various in vitro, cellular and animal models. Specific aims include: (1) Production of aSyn aggregates by aSyn-PMCA amplification from brain and CSF of patients affected by AD and LBD (as well as other synucleinopathies) and comprehensive analysis of the size, biochemical and biophysical properties of these aggregates; (2) Study the structural basis differentiating different synucleinopathies, in particular we propose to elucidate the structural fold at atomic resolution for aSyn aggregates implicated in different diseases; (3) Study the biological properties of aSyn aggregates derived from AD, LBD and other synucleinopathies using in vitro and in vivo models. (4) Produce and distribute to the scientific community well-characterized and biologically relevant aSyn aggregates. The resources generated in this project will be made fully available to researchers in order to standardize reagents and ensure the biological relevance of the findings.