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.

项目摘要 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＆ tau在阿尔茨海默氏病中，tau在额叶痴呆症中，亨廷顿病中的亨廷顿和α- Lewy身体痴呆症（LBD）和帕金森氏病（PD）中的综合蛋白。治疗目前不存在 疾病进展，部分是因为驱动蛋白质聚集的潜在细胞生物学机制和 清除知识很少。 DLB＆PD中越来越多的证据表明α-核蛋白 神经元内的蛋白质聚集会导致神经元在跨越信号的能力中功能障碍 突触产生了患者中发现的认知和运动符号。 α-核蛋白蛋白是 Lewy Bodies的主要骨料组成部分，即定义神经元内的标志性病变病变 这些“突触性疾病”疾病。有趣的证据表明，聚集始于突触终端 并可能单向扩散到细胞体。特定点突变或翻译后修改 与疾病相关的可能在α-核蛋白的聚集中起关键作用，进而影响轴突 和终端功能，包括轴突运输。了解α-核蛋白涉及的机制 聚集需要剖析不同疾病的点突变和磷酸化的作用 它们的事件和/或潜在组合，并确定聚合形成的模式/s 理想情况下，在实验范式中传播，该范式允许在生活神经系统中实时研究。 Weissman Lab开发了一种新的斑马鱼模型和实验方法来研究α-核蛋白 体内单个神经元内的聚集和功能。实验室可以轻松表达不同形式的α- 具有特定点突变的综合蛋白，可视化整个轴突轴和体内母体细胞体，然后 测量蛋白质迁移率，蛋白质聚集和轴突功能。最近发布了来自 实验室强烈建议以前认为是关键的磷酸化事件 帕金森氏病发病机理（丝氨酸-129磷酸化）似乎并未起作用以驱动 聚合。相反，可能存在更复杂的“磷酸化代码”，其中多种磷酸化 必须简单地发生几个残留事件。研究这种机制，复杂的参与 各种关键残留物和细胞内聚集模式需要一个易于手工系统 用于可视化和检测对α-突触核蛋白蛋白的多个简单变化的影响。 实验室使用生活，透明的斑马鱼神经系统的方法非常适合这些问题。 拟议的项目将结合体内荧光成像，定位点突变体，处理 具有小分子激酶抑制剂，免疫组织化学和三维图像重建。这 从实验设计到动手实验，数据分析和写作的工作都是由 刘易斯与克拉克学院的魏斯曼实验室的本科生。