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

项目总结 蛋白质聚集在许多重要的神经退行性疾病中起着关键作用，如β-淀粉样蛋白和 Tau在阿尔茨海默病中，tau在额颞叶痴呆中，Huntingtin在亨廷顿病中，以及阿尔法- 路易体痴呆(LBD)和帕金森病(PD)中的突触核蛋白。目前还不存在停止治疗的方法 疾病进展，部分原因是驱动蛋白质聚集和 人们对通关的了解很少。越来越多的DLB和PD证据表明，α-突触核蛋白 神经元内的蛋白质聚集导致神经元传递信号的能力障碍 突触，引起患者的认知和运动症状。α-突触核蛋白是 路易小体的主要聚集成分，神经元内的标志性病理损害，定义 这些“联核症”疾病。有趣的证据表明，聚集始于突触终末 并且可以单向扩散到细胞体。特定的点突变或翻译后修饰 与疾病相关的基因可能在α-突触核蛋白的聚集中起关键作用，进而影响轴突 和终末功能，包括轴突运输。了解α-突触核蛋白的作用机制 聚集需要解剖不同疾病相关点突变和磷酸化的作用/S 事件和/或事件的潜在组合，并确定聚集形成和聚集的模式/S 传播，理想的是在实验范式中，允许在活的神经系统中实时研究。这个 魏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 体内单个神经元内的聚集和功能。实验室可以很容易地表达不同形式的阿尔法- 具有特定点突变的突触核蛋白，在体内显示整个轴突和亲本细胞体，以及 测量蛋白质流动性、蛋白质聚集性和轴突功能。最近公布的初步数据来自 该实验室强烈建议，以前被认为是关键的磷酸化事件 帕金森氏病的发病机制(丝氨酸-129磷酸化)似乎不是由自身驱动的 聚合。相反，可能存在更复杂的“磷酸化密码”，即多重磷酸化。 几个残留物上的事件必须同时发生。研究这一机制，复杂的牵连 各种关键残基，以及细胞内聚集的模式需要一个易于操纵的系统 用于可视化和检测对α-突触核蛋白的多个同时变化的影响。这个 Lab使用活的、透明的斑马鱼神经系统的方法非常适合解决这些问题。这个 拟议的项目将结合使用体内荧光成像、定点突变、治疗 用小分子激酶抑制剂、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，工作主要由以下人员完成 刘易斯和克拉克学院魏斯曼实验室的本科生。

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.