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

使用冷冻电子显微镜进行路易体痴呆的基于患者的结构生物学

• 批准号: 10478192
• 负责人: Anthony William Paul Fitzpatrick
• 金额: $ 54.38万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10478192
• 关键词: Adopted; Alzheimer' s Disease; Alzheimer' s disease brain; Amyloid; Amyloid beta-Protein; Antibodies; Biochemistry; Biological Assay; Brain; Brain Diseases; Cells; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Dementia with Lewy Bodies; Detection; Development; Diagnosis; Diagnostic; Disease; Environment; Etiology; Filament; Frequencies; Genetic Polymorphism; Heparin; Heterogeneity; In Vitro; Investigation; Knowledge; Lewy Bodies; Lewy Body Dementia; Ligands; Link; Lipids; Location; Maps; Mass Spectrum Analysis; Methodology; Methods; Modeling; Molecular; Molecular Biology; Molecular Structure; Monitor; Nature; Neurodegenerative Disorders; Parkinson' s Dementia; Pathogenesis; Pathologic; Pathology; Patients; Peptides; Phase; Phenotype; Play; Positron-Emission Tomography; Post-Translational Protein Processing; Process; Property; Proteins; Reagent; Recombinants; Reproducibility; Role; Senile Plaques; Structure; System; Tauopathies; Toxic effect; abeta deposition; alpha synuclein; amyloid structure; base; biophysical techniques; brain tissue; design; in vitro Model; in vivo; insight; next generation; novel; novel strategies; protein aggregation; skills; structural biology; tau Proteins; tau aggregation; tomography

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.

摘要 可溶性单体蛋白质转化为不溶性纤维状淀粉样蛋白聚集体是淀粉样变性的标志。 许多神经退行性疾病。帕金森病的主要神经病理学特征， 路易体痴呆是α-突触核蛋白在大脑中的积累。的调查 从患者脑中分离的tau细丝核心的结构已经阐明了 病理相关的细丝核心隐含的阿尔茨海默病（AD）的发病机制。这些结果 证明存在具有特定分子基序（如β-螺旋结构）的疾病特异性菌株 存在于AD细丝中，并强调了特异性的形成所固有的关键特征， 淀粉样蛋白结构，例如亚基结构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蛋白在存在下聚集， 肝素采用完全不同的折叠，不太可能与病理学相关，从而突出了 研究蛋白质聚集体在其内源性环境中的重要性。通过建立 以高度可重复的方式从患病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）的技能，我们现在独特地准备研究额外的和高度 相关的病理相关的纤维状形式。因此，本项目的目标是 表征α-突触核蛋白（α-syn）和淀粉样蛋白-β（Aβ）丝的性质和分子结构， 路易体痴呆症（LBD）的大脑产生新的见解的病因，毒性和传播 蛋白质聚集体的数量具体来说，我们将确定和比较细丝的异质性 发现在LBD和评估这些肽的能力，共聚集或相互作用。我们的目标是 利用从每种蛋白质的细丝核心获得的知识来生成鲁棒的聚集模型， 与病理学非常相似的体外和体内，以及核心特异性抗体和PET配体。我们将 确定患病大脑中新型翻译后修饰（PTM）的频率和重要性 通过将冷冻电镜与质谱、生物化学、分子生物学相结合的综合方法， 和生物物理学方法。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，以及开发 的体外和离体模型，适当地概括疾病本身。最后，我们将采用低温电子 断层扫描（cryo-ET）来绘制在这些聚集体以其复合物存在的情况下细胞的性质 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 具有已知影响α-syn聚集的额外细胞因子的细丝，例如特定类型的脂质。