Investigating mechanisms of microvesicle release and function by GDE2 and GDE3

项目总结/摘要 细胞外囊泡（EV）是由新近出现的细胞释放的小的膜结合囊泡 作为人类健康许多方面的核心参与者，包括AD。然而，这些初步研究已经提出， 关于EV是神经保护性的还是致病性的数据相互矛盾。电动汽车可大致分为 外泌体和微泡（MV），以及其他领域的工作表明EV亚型可以发挥专门的作用， 角色因此，需要更多的工作来表征中枢神经系统（CNS）中EV的亚型。 并确定它们是否具有神经保护或致病功能。我们的实验室已经确认了六个- 跨膜酶GDE 2和GDE 3作为MV的新型释放剂。初步和已发表的研究认为， GDE 2和GDE 3在调节神经元存活中的作用，并表明GDE 2的分布和功能与神经元的存活有关。 在阿尔茨海默病（AD）中被破坏。本提案将检验GDE 2和GDE 3利用 一种独特的生物合成途径来释放具有神经保护功能的MV， 在AD中被破坏。具体地，该提议将分别研究GDE 3的MV生物合成途径和 GDE 2（目标1）和鉴定GDE 2和GDE 3 EV的蛋白质和RNA货物（目标2）。目标3的实验将 表征AD中GDE 2定位和MV释放的破坏，并评估AD中GDE 2的神经保护作用。 GDE 2 MV的体外能力。该项目的成功完成将表征生物合成和 新亚型EV在CNS中的功能，并确定它们是否在AD中受到干扰。拟建项目 将为我的研究提供很好的培训。具体来说，我将在电动汽车领域获得新的经验 生物学和神经科学，并获得多种技术的新专业知识，包括EV隔离， 生物化学分析，蛋白质组学和深度测序，生物信息学，小鼠遗传学，原代细胞培养， 病毒转导和人类疾病。研究环境是高度协作的，我将有多个 有机会指导和教学，并通过演示文稿发展我的科学演示技巧， 出版物。总的来说，Kirschstein-NRSA的资助不仅将为基础EV生物学提供新的见解， 并探索与AD的相关性，但也将为我未来作为独立调查员的职业生涯做好准备。

Project Summary / Abstract Extracellular vesicles (EVs) are small membrane bound vesicles released by cells that have recently emerged as central players for many aspects of human health, including AD. However, these initial studies have raised conflicting data about whether EVs are neuroprotective or pathogenetic. EVs can be broadly divided into exosome and microvesicles (MVs), and work in other fields suggests that EV subtypes can play specialized roles. Accordingly, more work is needed to characterize subtypes of EVs in the central nervous system (CNS) and determine if these have neuroprotective or pathogenetic functions. Our lab has identified the six- transmembrane enzymes GDE2 and GDE3 as novel releasers of MVs. Preliminary and published studies ascribe GDE2 and GDE3 roles in regulating neuronal survival and indicate that GDE2 distribution and function are disrupted in Alzheimer's Disease (AD). This proposal will test the central hypothesis that GDE2 and GDE3 utilize a unique biosynthesis pathway to release MVs that have neuroprotective function, and that this release pathway is disrupted in AD. Specifically, this proposal will separately examine the MV biosynthesis pathway of GDE3 and GDE2 (Aim 1) and identify protein and RNA cargo of GDE2 and GDE3 EVs (Aim 2). Experiments in Aim 3 will characterize the disruption of GDE2 localization and MV release in AD, and asses the neuroprotective capabilities of GDE2 MVs in vitro. Successful completion of this project will characterize the biosynthesis and function of novel subtypes of EVs in the CNS and determine if they are perturbed in AD. The proposed project will provide excellent training for me in research. Specifically, I will gain new experiences in the fields of EV biology and neuroscience and gain new expertise across multiple techniques that include EV isolation and biochemical analysis, proteomics and deep sequencing, bioinformatics, mouse genetics, primary cell-culture, viral transduction, and human disease. The research environment is highly collaborative, I will have multiple opportunities to mentor and teach, and develop my scientific presentation skills though presentations and publications. Overall, this Kirschstein-NRSA grant will not only provide new insight into fundamental EV biology and explore relevance to AD, but will also prepare me for my future career as an independent investigator.