Patient-based structural biology of Lewy body dementia using cryo-electron microscopy

摘要 将可溶的单体蛋白转化为不可溶的纤维状淀粉样蛋白聚集体是 许多神经退行性疾病。帕金森病的主要神经病理特征和 路易体痴呆是指α-突触核蛋白在大脑中的积聚。对该事件的调查 从患者脑中分离的tau细丝核心的结构已经阐明了 与阿尔茨海默病(AD)发病机制相关的细丝核心。这些结果 证明存在具有特定分子基序的疾病特异性菌株，如β-螺旋结构 它存在于AD细丝中，并突出了特定的形成所固有的关键特征 淀粉样蛋白结构，如亚基结构中的多态性和亚基之间的差异界面 原丝。最近的结构测定表明，重组tau在存在 肝素采用了与病理不太可能相关的完全不同的折叠，从而突出了 研究蛋白质聚集体在其内生环境中的重要性。通过建立 以高度可重复性的方式从患病的大脑中提取细丝的方法，并利用我们的 在低温电子显微镜(CRYO-EM)方面的技能，我们现在是唯一准备研究其他和高度 相关的病理上相关的纤维形态。因此，当前项目的目标是 α-突触核蛋白(α-SYN)和淀粉样蛋白-β(A-β)微丝的性质和分子结构 路易体痴呆(LBD)大脑对病因、毒性和传播产生新的见解 这种疾病中蛋白质聚集体的数量。具体地说，我们将确定和比较细丝的异质性 在LBD中发现，并评估这些肽共同聚集或相互作用的能力。我们的目标是 使用从每个蛋白质的细丝核心获得的知识来生成健壮的聚集模型 与病理非常相似的体外和体内实验，以及核心特异性抗体和PET配体。我们会 确定新的翻译后修饰(PTM)在疾病大脑中的频率和重要性 通过将冷冻-EM与质谱学、生物化学、分子生物学相结合的综合方法 和生物物理方法。这些方法和见解将为新战略的设计和开发提供信息 诊断和治疗疾病，包括下一代PET配体和抗体，以及发展 能够恰当地概括疾病本身的体外和体外模型。最后，我们将使用低温电子 断层扫描(冷冻-ET)，以绘制存在于其复合体中的这些聚集体的细胞性质 环境以及每种蛋白质的定位，从而使我们能够监测LDB的相互作用 具有其他已知影响α-SYN聚集的细胞因素的细丝，例如特定类型的脂类。

ABSTRACT The conversion of soluble, monomeric protein into insoluble, fibrillar amyloid aggregates is a hallmark of numerous neurodegenerative diseases. The major neuropathological hallmarks of Parkinson’s disease and Dementia with Lewy Bodies is the accumulation of α-synuclein proteins in the brain. Investigations into the structure of the tau filament core isolated from brains of patients have elucidated the molecular composition of the pathologically relevant filament cores implicit to the pathogenesis of Alzheimer’s disease (AD). These results demonstrate the presence of disease-specific strains with specific molecular motifs, such as the β-helix structure that is present in AD filaments, and have highlighted critical features that are innate to the formation of specific amyloid structures, such as polymorphism in the subunit structure and differential interfaces between protofilaments. Recent structural determination has shown that recombinant tau aggregated in the presence of heparin adopts an entirely different fold that is not likely to be relevant to pathology, thereby highlighting the importance of studying the protein aggregates in their endogenous environment. By establishing the methodologies to extract filaments from diseased brains in a highly reproducible manner and by leveraging our skill in cryo-electron microscopy (cryo-EM), we are now uniquely poised to investigate additional and highly relevant pathologically relevant fibrillar forms. Consequently, the objectives of the current project are to characterize the nature and molecular structures of α-synuclein (α-syn) and amyloid-β (Aβ) filaments in the Lewy Body Dementia (LBD) brain to generate novel insight into the etiology, toxicity and spreading of protein aggregates in this disease. Specifically, we will determine and compare the filament heterogeneity found in LBD and assess the ability of these peptides to co-aggregate or interact with one-another. We aim to employ the knowledge gained from the filament cores of each protein to generate robust aggregation models in vitro and in vivo that closely resemble pathology, as well as core-specific antibodies and PET ligands. We will determine the frequency and importance of novel post-translational modifications (PTMs) in the diseased brain through an integrative approach combining cryo-EM with mass-spectrometry, biochemistry, molecular biology and biophysical methods. These methods and insights will inform the design and development of novel strategies to diagnose and treat disease, including next generation PET ligands and antibodies, as well as the development of in vitro and ex vivo models that properly recapitulate the disease itself. Finally, we will employ cryo-electron tomography (cryo-ET) to map the nature of the cell in the presence of these aggregates in their complex environments, as well as where each protein localizes, thereby enabling us to monitor for the interplay of LDB filaments with additional cellular factors known to influence α-syn aggregation, such as specific types of lipids.