Organization and Function of Neuronal Endosomes

项目总结/摘要。由于神经元是有丝分裂后的，寿命长，而且非常大， 维持蛋白质组是特别重要的。毫不奇怪，蛋白质稳态的紊乱 蛋白质稳态（"proteostasis"）与许多神经退行性疾病以及衰老有关。如何 因此，蛋白质周转在时间和空间上受到调节是一个基本的细胞生物学问题， 对神经元功能和疾病的深远影响。树突内的内体运输不好 明白我们的具体目标是：目标1）发现树枝状降解通量（即运输，酸化， 与溶酶体融合）由位于晚期内体的两个小GTP酶沿着树突沿着协调， 溶酶体，Rab7和Arl8b，Aim 2）鉴定了两种Rab7效应物在调节蛋白质稳态中的作用，并且Aim 3)发现内体成熟中断对神经元健康的功能后果，（包括 膜受体的降解、聚集蛋白的降解、树突的维持和细胞存活）。 内体成熟涉及募集小GTPases（Rab 7和Arl8b）及其效应子， 调节酸化、运动和融合事件。我们最近的工作表明Rab7参与了空间调控， 降解通量Rab7与多种效应物结合，但它们如何在空间上调节内体行为， 对树突了解甚少。值得注意的是，Rab7本身在Charcot-Marie-Tooth病2B中发生突变，并且许多 Rab7效应子与疾病有关，突出了内体成熟途径对正常发育的中心作用。 神经元功能目前的进展障碍包括缺少研究这些蛋白质的"分子把手"。 晚期内体和溶酶体的异质性，缺乏可用于追踪的内源性可追踪货物 降解，以及对调节内体通量的许多内体效应物的忽视。 这个应用程序的前提是基于我们自己最近的数据： 1)与普遍看法相反，一些内源性树突受体在树突中不会降解，但 而是以Rab7依赖的方式逆行转运到索马/近端树突。降解 因此不发生在树突中，而是发生在体细胞溶酶体中。 2)LAMP1是成纤维细胞中最常用的溶酶体标志物，存在于许多区室中， 不是降解性溶酶体，特别是在树突的更远端，突出了神经特异性机制。 3)远端树突中大多数含有Rab7的晚期内体不含LAMP 1，因此与晚期内体不同。 成纤维细胞中的核内体，其绝大多数含有Rab7和LAMP 1。我们称之为"早期" 晚期内体以将它们与常规Rab7 +/LAMP 1+晚期内体区分开。 这些新发现使我们现在能够解决一个重要的首要问题： 树突状乔木协调内体成熟和降解，以及蛋白质稳定的失败如何影响 神经元的细胞功能我们的长期目标是解开树突中蛋白质稳态的机制。

PROJECT SUMMARY/ABSTRACT. Since neurons are post-mitotic, long-lived, and extraordinarily large, maintaining the proteome is of particular importance. Not surprisingly, disturbances in protein homeostasis (“proteostasis”) have been associated with numerous neurodegenerative disorders, as well as aging. How protein turnover, is regulated in time and space is thus an essential cell biological question with profound implications for neuronal function and disease. Endosome transport in dendrites is not well understood. Our specific aims are to: Aim 1) discover how dendritic degradative flux (i.e. transport, acidification, fusion with lysosomes) is coordinated along dendrites by two small GTPases localized to late endosomes and lysosomes, Rab7 and Arl8b, Aim 2) identify the roles of two Rab7 effectors in regulating proteostasis, and Aim 3) discover functional consequences of disrupted endosomal maturation for neuronal health, (including degradation of membrane receptors, of aggregated proteins, maintenance of dendrites, and cell survival). Endosome maturation involves recruitment of small GTPases (Rab7 and Arl8b) and their effectors to regulate acidification, motility, and fusion events. Our recent work has implicated Rab7 in spatial regulation of degradative flux. Rab7 binds to multiple effectors, but how they spatially regulate endosome behavior in dendrites is poorly understood. Notably, Rab7 itself is mutated in Charcot-Marie-Tooth disease 2B and many Rab7 effectors are linked to diseases, highlighting the centrality of endosomal maturation pathways for normal neuronal function. Current barriers to progress include missing “molecular handles” for studying the heterogeneity of late endosomes and lysosomes, lack of endogenous trackable cargos that can be used to follow degradation, and ignorance of the many endosomal effectors which regulate endosomal flux. The premise for this application rests on our own recent data: 1) Contrary to common belief, several endogenous dendritic receptors are not degraded in dendrites, but instead are retrogradely transported to the soma/proximal dendrite in a Rab7-dependent manner. Degradation thus does not take place in dendrites, but in somatic lysosomes. 2) LAMP1, the most commonly used lysosome marker in fibroblasts, is present in many compartments which are not degradative lysosomes, especially more distally in dendrites, highlighting neural-specific mechanisms. 3) Most Rab7-containing late endosomes in distal dendrites contain no LAMP1, and are thus different from late endosomes in fibroblasts which overwhelmingly contain both Rab7 and LAMP1. We refer to them as “early” late endosomes to distinguish them from the conventional Rab7+/LAMP1+ late endosomes. These new discoveries allow us to now address a significant overarching question: How does the expansive dendritic arbor coordinate endosome maturation and degradation, and how do failures of proteostasis affect cellular function of neurons? Our long-term goal is to unravel the mechanisms of proteostasis in dendrites.