A new in vivo zebrafish model to study alpha-synuclein aggregation in Lewy Body Disease

研究路易体病中α-突触核蛋白聚集的新体内斑马鱼模型

• 批准号: 10731005
• 负责人: Tamily A Weissman
• 金额: $ 41.12万
• 依托单位: LEWIS AND CLARK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731005
• 关键词: Alzheimer' s Disease; Amyloid beta-Protein; Automobile Driving; Axon; Axonal Transport; Biological; Biological Assay; Cells; Code; Cognitive; Color; Communication; Complex; Data; Data Analyses; Dementia with Lewy Bodies; Development; Disease; Disease Progression; Event; Experimental Designs; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Frontotemporal Dementia; Functional disorder; Genetic; Goals; Huntington Disease; Huntington gene; Immunohistochemistry; Impairment; Individual; Interruption; Lead; Lesion; Lewy Bodies; Lewy Body Dementia; Lewy Body Disease; Measures; Methods; Mitochondria; Modeling; Movement; Mutate; Nerve; Nervous System; Neurobehavioral Manifestations; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Parents; Parkinson Disease; Pathogenesis; Pathologic; Patients; Pattern; Phosphorylation; Play; Point Mutation; Post-Translational Protein Processing; Presynaptic Terminals; Proteins; Publishing; Recurrence; Research; Role; Serine; Serine/Threonine Phosphorylation; Signal Transduction; Site; Stains; Structure; Symptoms; Synapses; System; Technology; Testing; Thioflavin S; Three-Dimensional Image; Time; Tyrosine; Visualization; Western Blotting; Work; Writing; Zebrafish; alpha synuclein; brain cell; college; genetic approach; image reconstruction; in vivo; in vivo fluorescence; in vivo fluorescence imaging; interest; kinase inhibitor; mutant; neurotoxicity; nitration; novel strategies; novel therapeutics; pharmacologic; presynaptic; prevent; protein aggregation; reconstruction; screening; small molecule; synucleinopathy; tau Proteins; undergraduate student; vesicle transport

PROJECT SUMMARY Protein aggregation plays a critical role in many important neurodegenerative diseases such as beta-amyloid & tau in Alzheimer's disease, tau in Frontotemporal dementia, huntingtin in Huntington's disease, and alpha- synuclein in Lewy body dementia (LBD) & Parkinson's disease (PD). Treatments do not currently exist to halt disease progression, in part because the underlying cell biological mechanisms driving protein aggregation and clearance are poorly understood. A growing body of evidence in DLB & PD suggests that alpha-synuclein protein aggregation within neurons causes dysfunction in the neuron's ability to communicate signals across the synapse, giving rise to the cognitive and movement symptoms found in patients. Alpha-synuclein protein is the major aggregated component of Lewy bodies, the hallmark pathological lesion within neurons that defines these “synucleinopathy” diseases. Intriguing evidence suggests that aggregation begins in synaptic terminals and may spread unidirectionally to the cell body. Specific point mutations or post-translational modifications associated with disease might play a critical role in alpha-synuclein's aggregation, and in turn influence axonal and terminal function, including axonal transport. Understanding the mechanisms involved in alpha-synuclein aggregation requires dissecting the role/s of different disease-relevant point mutations and phosphorylation events, and/or potential combinations of them, and determining the pattern/s of aggregation formation and spread, ideally in an experimental paradigm that allows for study in real time in the living nervous system. The Weissman Lab has developed a new zebrafish model and experimental approaches to study alpha-synuclein aggregation and function within individual neurons in vivo. The lab can readily express different forms of alpha- synuclein with specific point mutations, visualize whole axonal arbors and parent cell bodies in vivo, and measure protein mobility, protein aggregation, and axonal function. Recently published preliminary data from the lab strongly suggest that what was previously thought to be the critical phosphorylation event in Parkinson's disease pathogenesis (serine-129 phosphorylation) does not appear to act by itself to drive aggregation. Rather, a more elaborate “phosphorylation code” may exist, where multiple phosphorylation events at several residues must occur simultaneously. Investigating this mechanism, the complex involvement of various critical residues, and patterns of aggregation within the cell requires an easily manipulatable system for visualizing and detecting the effects of multiple simultaneous changes to the alpha-synuclein protein. The lab's approach using the living, transparent zebrafish nervous system is ideally suited for these questions. The proposed project will use a combination of in vivo fluorescence imaging, site-directed point mutants, treatment with small molecule kinase inhibitors, immunohistochemistry, and three-dimensional image reconstruction. This work, from experimental design to hands-on experimentation, data analysis and writing, is done primarily by undergraduate students in the Weissman Lab at Lewis & Clark College.

项目概要 蛋白质聚集在许多重要的神经退行性疾病中起着至关重要的作用，例如β-淀粉样蛋白和 阿尔茨海默病中的 tau 蛋白、额颞叶痴呆中的 tau 蛋白、亨廷顿病中的亨廷顿蛋白以及 α- 突触核蛋白在路易体痴呆 (LBD) 和帕金森病 (PD) 中的治疗目前尚不存在。 疾病进展，部分原因是驱动蛋白质聚集的潜在细胞生物学机制和 越来越多的 DLB 和 PD 证据表明 α-突触核蛋白的清除机制尚不清楚。 神经元内的蛋白质聚集导致神经元之间传递信号的能力功能障碍 突触，引起患者的认知和运动症状。 路易体的主要聚集成分，神经元内的标志性病理损伤，定义 这些“突触核蛋白病”疾病表明聚集始于突触末梢。 并可能单向传播到细胞体。 与疾病相关的可能在 α-突触核蛋白的聚集中发挥关键作用，进而影响轴突 和末端功能，包括轴突运输的了解涉及α-突触核蛋白的机制。 聚集需要剖析不同疾病相关点突变和磷酸化的作用 事件和/或它们的潜在组合，并确定聚集形成的模式和 传播，理想情况下是在允许在活体神经系统中进行实时研究的实验范式中。 韦斯曼实验室开发了一种新的斑马鱼模型和实验方法来研究α-突触核蛋白 实验室可以很容易地表达不同形式的α-体内单个神经元的聚集和功能。 具有特定点突变的突触核蛋白，在体内可视化整个轴突乔木和亲代细胞体，以及 测量蛋白质迁移率、蛋白质聚集和轴突功能。 该实验室强烈建议，以前被认为是关键的磷酸化事件 帕金森病发病机制（丝氨酸 129 磷酸化）似乎并不单独驱动 相反，可能存在更复杂的“磷酸化代码”，其中存在多个磷酸化。 多个残基上的事件必须同时发生，研究这一机制的复杂性。 各种关键残基的分析以及细胞内的聚集模式需要一个易于操作的系统 用于可视化和检测 α-突触核蛋白的多个同时变化的影响。 该实验室使用活体透明斑马鱼神经系统的方法非常适合解决这些问题。 拟议的项目将结合体内荧光成像、定点点突变、治疗 与小分子激酶抑制剂、免疫组织化学和三维图像重建。 从实验设计到动手实验、数据分析和写作，工作主要由 刘易斯与克拉克学院韦斯曼实验室的本科生。