Roles of Recycling Endosomes in Neuronal Extracellular Vesicle Cargo Traffic

项目总结 本提案的目的是了解细胞膜运输机械如何控制包装。 以及体内突触释放细胞外小泡(EV)货物。电动汽车是一种小型的薄膜汽车。 囊泡由多种细胞类型释放，包括神经元，携带对信号和信号至关重要的货物 疾病。然而，我们对电动汽车货运如何在空间和时间上进行调控知之甚少。 在神经元的极化和复杂的形态中。我们已经开发了跟踪和操纵电动汽车的工具 在体内的果蝇突触前终末的交通，并发现货物流量通过血浆 膜回收内涵体的路线决定了它们是否被本地分拣包装并在 电动汽车，而不是通过逆行运输从突触中耗尽。回收内含体主要是 在非神经细胞中研究，对它们的寿命、功能或突触前动力学知之甚少 终点站。我们知道循环内小体在信号传递、神经元形态发生、EV等方面起着关键作用 交通和突触传输。对这些重要过程的理解和治疗干预 将需要对神经元循环内体功能的机制有更深入的了解。在这份提案中， 我们将在活体内阐明突触EV货物和循环内小体运输的机制。要实现 为了实现这些目标，我们将使用果蝇遗传学、生物化学、高分辨率显微镜和活细胞成像。 1)我们将确定不同类型突触再循环的功能、动力学和调节 内噬菌体。为此，我们将开发新的工具和方法来定义和控制不同的功能 在突触处回收隔间。使用这些工具，我们将测试新的机械论假设如何 膜运输机械在突触回收车厢对货物进行分拣。2)我们将确定电动汽车如何 货物运输依赖于不同的突触内吞作用模式：胞膜蛋白介导的内吞作用 在低神经元活动和活动依赖的大量内吞作用下，在高强度神经元 活动。这些实验将确定EV的命运是否由不同的内化模式决定，如何 循环内吞体内有助于这些功能，并提供了新的机制来连接活性，内吞 交通，和电动汽车的释放。鉴于突触膜运输机制的保守性，我们的发现 和工具将为在神经系统功能的许多方面对电动汽车交通的新见解奠定基础， 包括人类神经疾病。

PROJECT SUMMARY The goal of this proposal is to understand how cellular membrane trafficking machinery controls the packaging and release of extracellular vesicle (EV) cargoes from synapses in vivo. EVs are small membrane-bound vesicles released by numerous cell types, including neurons, carrying cargoes critical for signaling and disease. However, we understand very little about how EV cargo traffic is spatially and temporally regulated within the polarized and complex morphology of neurons. We have developed tools to track and manipulate EV traffic at Drosophila presynaptic terminals in vivo, and discovered that flux of cargoes through a plasma membrane-recycling endosome route determines whether they are locally sorted for packaging and release in EVs, rather than depleted from synapses by retrograde transport. Recycling endosomes have primarily been studied in non-neuronal cells, and very little is known about their lifetime, functions, or dynamics at presynaptic terminals. We do know that recycling endosomes play critical roles in signaling, neuronal morphogenesis, EV traffic, and synaptic transmission. Understanding and therapeutically intervening in these important processes will require a deeper knowledge of the mechanisms of neuronal recycling endosome function. In this proposal, we will elucidate the mechanisms of synaptic EV cargo and recycling endosome traffic in vivo. To achieve these goals, we will use Drosophila genetics, biochemistry, high-resolution microscopy, and live cell imaging. 1) We will determine the functions, dynamics, and regulation of different types of synaptic recycling endosomes. To this end, we will develop new tools and approaches to define and control functionally distinct recycling compartments at synapses. Using these tools, we will test novel mechanistic hypotheses for how membrane traffic machinery sorts cargoes at synaptic recycling compartments. 2) We will determine how EV cargo traffic depends on distinct modes of synaptic endocytosis: clathrin-mediated endocytosis, which operates under low neuronal activity and activity-dependent bulk endocytosis, which operates during intense neuronal activity. These experiments will ascertain if EV fate is determined by different modes of internalization, how recycling endosomes contribute to these functions, and provide new mechanisms to link activity, endosomal traffic, and EV release. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into EV traffic in many aspects of nervous system function, including in human neurological disease.