Investigating mechanisms of microvesicle release and function by GDE2 and GDE3

项目摘要/摘要 细胞外小泡是新近出现的细胞释放的小的膜结合的小泡。 作为人类健康许多方面的中心参与者，包括阿尔茨海默病。然而，这些初步研究提出了 关于EV是神经保护还是致病的数据相互矛盾。电动汽车大致可以分为 外体和微囊泡(MVS)，以及在其他领域的研究表明，EV亚型可以发挥特化 角色。因此，需要更多的工作来确定中枢神经系统(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人- 跨膜酶GDE2和GDE3作为MVS的新型释放剂。初步和已发表的研究归因于 GDE2和GDE3在调节神经元存活中的作用，并表明GDE2的分布和功能 患有阿尔茨海默氏症(AD)。这一提议将检验GDE2和GDE3利用的中心假设 一种独特的生物合成途径来释放具有神经保护功能的MVS，而这一释放途径 在公元后被打乱。具体地说，该提案将分别研究GDE3和GDE3的MV生物合成途径。 GDE2(目标1)，并确定GDE2和GDE3 EV的蛋白质和RNA货物(目标2)。目标3中的实验将 研究阿尔茨海默病患者GDE2定位和MV释放的紊乱，并评价其神经保护作用 体外培养GDE2MVS的能力。该项目的成功完成将使生物合成和 研究新亚型EV在中枢神经系统中的功能，并确定它们在AD中是否受到干扰。拟议中的项目 将为我的研究提供极好的培训。具体地说，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隔离和 生化分析、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，我将拥有多个 有机会指导和教授，并通过演讲和演讲发展我的科学演讲技能 出版物。总体而言，这项Kirschstein-NRSA拨款不仅将为基础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.