Organization and Function of Neuronal Endosomes

项目摘要/摘要。由于神经元是有丝分裂后的，寿命长，而且非常大， 维持蛋白质组尤其重要。毫不奇怪，蛋白质动态平衡的紊乱 (“蛋白质沉积”)与许多神经退行性疾病以及衰老有关。多么 蛋白质的周转，是在时间和空间上调节的，因此是一个基本的细胞生物学问题 对神经功能和疾病的深远影响。树突中的内体转运不是很好 明白了。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、 融合与溶酶体)是由两个小的GTP酶沿着树突协调的，这两个小的GTP酶定位于晚期的内小体和 溶酶体，Rab7和Ar18b，Aim 2)确定两个Rab7效应器在调节蛋白稳定中的作用，并Aim 3)发现中断的内体成熟对神经元健康的功能后果，(包括 膜受体、聚集蛋白的降解、树突的维持和细胞存活)。 内体成熟涉及招募小的GTP酶(Rab7和Ar18b)及其效应物来 调节酸化、运动和融合事件。我们最近的研究表明，Rab7参与了对 降解助熔剂。Rab7与多个效应器结合，但它们如何在空间上调节内体行为 人们对树突知之甚少。值得注意的是，Rab7本身在Charcot-Marie-Tooth病2B和许多 Rab7效应器与疾病有关，突出了正常的内体成熟途径的中心 神经功能。目前取得进展的障碍包括缺少研究生物多样性的“分子句柄”。 晚期内切体和溶酶体的异质性，缺乏可用于跟踪的内源可追踪货物 降解，以及对调节内体通量的许多内体效应器的忽视。 这项应用的前提是我们自己的最新数据： 1)与普遍认为的相反，几种内源性树突状受体在树突中没有降解，但 相反，它们以Rab7依赖的方式逆行运输到胞体/近端树突。降解 因此，不是发生在树突中，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记物，存在于许多隔室中， 不是降解的溶酶体，特别是在树突更远的地方，突出了神经特有的机制。 3)在远端树突中，大多数含有Rab7的晚期内体不含LAMP1，因此与晚期不同 成纤维细胞中绝大多数同时含有Rab7和LAMP1的内涵体。我们把它们称为“早期”。 晚内体，以区别于传统的Rab7/LAMP1晚内体。 这些新的发现使我们现在能够解决一个重要的问题：如何扩大 树枝协调内体成熟和降解，以及蛋白稳定的失败是如何影响的 神经元的细胞功能？我们的长期目标是解开树突蛋白稳定的机制。

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.