Mechanisms and regulation of extracellular vesicle traffic in the nervous system

本项目的目标是了解细胞外小泡(EV)运输的分子机制。 体内的神经系统。EV是由供体细胞分泌的小囊泡，可以携带不同阵列的 作为一种新的蜂窝间交通和通信模式，最近已经走到了前列 大脑。EV被认为有助于许多人类健康状况，包括病理性疾病的传播 神经退行性疾病中的蛋白质。然而，因为大多数对电动汽车的研究都是在细胞中进行的 对于EV在不同的相互作用的细胞类型中的释放、摄取和命运，人们知之甚少 完整的神经系统。我们已经开发了一个系统来研究内源性神经元EV的交通 利用果蝇可获得的尖端遗传和细胞生物学工具，在活动物中运送货物。vbl.使用 在这个系统中，我们发现了突触周围区(PAZ)膜重构的一个意想不到的作用 电动汽车货物分拣、稳定性和果蝇幼虫轴突终末释放的机械 神经肌肉接头。我们还发现，内源性电动汽车货物的释放受到以下动态调节 神经元活动。最后，我们发现了一个令人惊讶的发现，发布的电动汽车部分受到保护，不受 靶细胞细胞质，这表明额外的监管步骤可能会管理它们的暴露 和/或在内化到受体细胞时降解。我们提议的研究将阐明细胞如何 膜运输机械控制电动汽车货物的释放、吸收和命运。为了实现这些目标，我们 将使用先进的果蝇遗传学，活细胞成像技术，结构照明显微镜， 电子显微镜，以及对供受者细胞中EV货物的组织特异性检测和操作。 具体地说，我们建议：1)阐明PAZ蛋白转运和释放EV的体内机制。2)至 确定活动如何调节内源性EV货物的神经元交通，以及3)决定 NMJ EV货物，在休息时和对神经元活动的反应。鉴于突触的保守性 膜转运机制，我们的发现和工具将为对内源的新见解奠定基础 EV流量在神经系统功能的许多方面，包括在人类神经系统疾病中。

The goal of this project is to understand the molecular mechanisms of extracellular vesicle (EV) trafficking in the nervous system in vivo. EVs are small vesicles secreted from donor cells that can carry a diverse array of cargoes, and have recently come to the forefront as a novel mode of intercellular traffic and communication in the brain. EVs are thought to contribute to many human health conditions, including the spread of pathological proteins in neurodegenerative disease. However, because most studies of EVs are conducted with cells in culture, little is known about the release, uptake and fate of EVs in the diverse interacting cell types of the intact nervous system. We have developed a system in which to study traffic of endogenous neuronal EV cargoes in a living animal, using cutting edge genetic and cell biological tools available in Drosophila. Using this system, we discovered an unexpected role for synaptic periactive zone (PAZ) membrane remodeling machinery in EV cargo sorting, stability and release from axon terminals at the Drosophila larval neuromuscular junction. We also found that endogenous EV cargo release is dynamically regulated by neuronal activity. Finally, we made the surprising discovery that released EVs are partially protected from the target cell cytoplasm, suggesting the possibility that additional regulatory steps may govern their exposure and/or degradation upon internalization to recipient cells. Our proposed research will elucidate how cellular membrane traffic machinery controls the release, uptake, and fate of EV cargoes. To achieve these goals we will use advanced Drosophila genetics, live cell imaging techniques, structured illumination microscopy, electron microscopy, and tissue-specific detection and manipulation of EV cargoes in donor and recipient cells. Specifically, we propose: 1) To elucidate in vivo mechanisms of EV traffic and release by PAZ proteins. 2) to determine how activity regulates neuronal traffic of endogenous EV cargoes and 3) to determine the fate of NMJ EV cargoes, at rest and in response to neuronal activity. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into endogenous EV traffic in many aspects of nervous system function, including in human neurological disease.