Investigating the role of Amphiphysin in extracellular vesicle cargo uptake in vivo

研究 Amphiphyn 在体内细胞外囊泡货物摄取中的作用

• 批准号: 10285979
• 负责人: Cassandra Blanchette
• 金额: $ 3.47万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285979
• 关键词: Actins; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Area; Automobile Driving; Biological; Biological Models; Biology; Brain; Cell Communication; Cell Culture Techniques; Cell membrane; Cells; Communication; Complex; Cytoplasm; DNA; Disease; Drosophila genus; Electron Microscopy; Endocytosis; Environment; Exhibits; Genetic; Goals; Health; Human; Image; Image Analysis; In Vitro; Intracellular Transport; Knowledge; Label; Late Onset Alzheimer Disease; Leadership; Lighting; Lipids; Medical; Membrane; Membrane Proteins; Mentorship; Microscopy; Modeling; Molecular; Multivesicular Body; Muscle; Muscle Cells; Mutation; Nervous system structure; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Parkinson Disease; Pathogenicity; Pathologic; Pathway interactions; Phagocytosis; Physiological; Play; Postsynaptic Membrane; Process; Proteins; RNA; Recycling; Regulation; Resolution; Role; SH3 Domains; Scientist; Signal Transduction; Structure; System; Testing; Tissues; Training; amphiphysin; base; endosome membrane; exosome; experimental study; extracellular vesicles; genetic risk factor; in vivo; microscopic imaging; microvesicles; mutant; nervous system disorder; novel; postsynaptic; presynaptic; presynaptic neurons; quantitative imaging; responsible research conduct; tool; trafficking; uptake; vesicle transport; vesicular release

Extracellular vesicles (EVs) are small, membrane bound vesicles that transport cargoes such as proteins, RNA, DNA, and lipids between cells. EVs are universally released from tissues within the body, and trafficking of EVs within the nervous system is important for cell-cell communication. EVs can also contribute to the disposal or spread of pathological proteins in neurodegenerative disease, making understanding the biology of EV regulation within the nervous system highly relevant to human health. Our knowledge of EV biology is primarily based on studies carried out using in vitro or cell culture-based systems. While these studies have identified a number of important molecular players that are likely involved in these processes, the molecular mechanisms driving EV cargo uptake by target cells, in a complex, in vivo nervous system remains unstudied. The Drosophila NMJ was the first system to study in vivo trafficking of EV cargoes, and our lab has developed novel tools to track endogenously labeled, neuronally derived EV cargoes as they get transferred to and taken up by the postsynaptic muscle cell. This level of detail within an in vivo complex nervous system cannot be attained in another system, and when combined with its genetic tractability, amenability to imaging, and available cell-biological, and tissue specific manipulations, the Drosophila NMJ is the ideal model system with which to ask direct questions about EV cargo uptake mechanisms. Using this system, we have identified a role for the Drosophila membrane-remodeling protein Amphiphysin in the regulation of postsynaptic EV cargo distribution. The goal of this proposal is to determine how Amph contributes to EV cargo uptake in vivo, and to define the cell-biological pathways that regulate EV cargo uptake in vivo in a complex nervous system. This is particularly intriguing because in humans, mutations in Amphiphysin-2/BIN1 are a very strong genetic risk factor for late onset Alzheimer's disease, and disruption of EV cargo trafficking is implicated in neurodegenerative disease. Together, this makes defining the mechanisms of Amph in EV cargo uptake, and how these are regulated at the cell-biological level, critical for understanding how Amph, and EV cargo uptake may contribute to neurological disease processes. Carrying out the experiments outlined in this proposal will provide me with training in high resolution microscopy, quantitative image analysis, cell- biological manipulations, and advanced Drosophila genetics. Furthermore, it includes concrete plans to enhance my training in leadership, mentorship, the responsible conduct of research, and scientific communication. Together, training in these areas will prepare me to become an independent scientist.

胞外囊泡(EV)是一种小的、结合在膜上的囊泡，可以运输货物，如 蛋白质、RNA、DNA和细胞间的脂类。EV在体内的组织中被普遍释放 EV在神经系统内的运输对于细胞与细胞之间的交流非常重要。电动汽车 也可以促进神经退行性疾病中病理性蛋白质的处置或扩散， 使了解神经系统内EV调节的生物学高度相关 人类健康。我们对EV生物学的知识主要是基于使用体外或 基于细胞培养的系统。虽然这些研究已经确定了一些重要的分子 可能参与这些过程的参与者，驱动电动汽车货运的分子机制 在复杂的活体神经系统中，靶细胞的摄取仍未被研究。果蝇NMJ 是第一个研究电动汽车货物体内运输的系统，我们的实验室开发了新的工具 跟踪内源性标记的、神经来源的电动汽车货物被转移和被接收时的情况 通过突触后肌肉细胞。体内复杂神经系统中的这种详细程度不能 在另一个系统中获得，当与其遗传可控性相结合时，可修改为 成像，以及可用的细胞生物学和组织特异性操作，果蝇NMJ是 理想的模型系统，用于直接询问有关电动汽车货物吸收机制的问题。使用这个 系统中，我们已经确定了果蝇膜重塑蛋白两栖动物蛋白在 突触后EV货物分布的调节。这项提案的目标是确定如何 Amph有助于在体内摄取EV货物，并定义调节EV货物的细胞生物学途径 EV货物在复杂神经系统的活体摄取。这特别耐人寻味，因为在 人类，两性体蛋白-2/BIN1突变是晚发的一个非常强的遗传风险因素 阿尔茨海默病和EV货物运输中断与神经退行性变有关 疾病。总而言之，这就定义了Amph在电动汽车货物吸收中的机制，以及这些机制是如何 在细胞生物水平上受到调控，这对于了解Amph和EV货物的摄取是至关重要的 可能会导致神经系统疾病的进程。执行本文件中概述的实验 提案将为我提供高分辨率显微镜，定量图像分析，细胞- 生物操纵和先进的果蝇遗传学。此外，它还包括具体的计划 加强我在领导、指导、负责任的研究行为和科学研究方面的培训 沟通。总而言之，在这些领域的培训将为我成为一名独立的 科学家。