Novel Endosomal Pathways Underlying NGF/TrkA Dependent Dendrite Development

项目摘要/摘要：长距离信号是外周神经元的一个关键特征。在发展中 在交感神经系统中，通过神经营养素受体对NGF-TrkA的长距离信号是 在信号内体（SE）中从远端轴突转运回索马。抵达索马后， SE发出生存信号，并且引人注目地进入树突，在那里它组织突触后密度。 然而，运输机制的基础SE运输到树突是未知的。因此有 在理解SE的贩运和信号传递如何联系起来以创造 功能性突触连接由NGF-TrkA引起的信号级联是很好理解的。 然而，一旦它到达索马，它所经历的贩运事件却严重不足。的 SE的运输影响其信号传导，反之亦然。因此，揭示SE如何被贩运到 使用高分辨率细胞生物学工具的树突将越过目前机械连接的障碍， 信号生理学与内体运输和成熟的细胞生物学。我们的长期目标是 了解逆行信号是如何组织突触形成的。 促使这一提议的基本原理是，逆行运输的NGF-TrkA能够运输到 树突，并启动突触后密度的聚集。此外，抑制MEK/MAPK通路 在细胞体中的特异性抑制SE进入树突，表明SE与树突细胞之间存在着重要的联系。 贩运和信号。我们的实验室最近表明，SE经历了一种新的贩运事件， 转胞吞作用，其中SE在索马膜上外化，随后再内化。的 该建议的中心概念是SE调节多个下游结果（生存与PSD 聚类）通过经历不同的、受调节的内体转化以使它们的信号转导多样化 容量我们的初步数据表明，树突状SE是长寿和非 （目的1和2），并且MEK/MAPK途径的抑制阻止NGF-TrkA的转变 进入这种可以迁移到树突的长寿命SE（Aim 3）。我将使用创新的方法，包括敲入 小鼠、微流体装置和定量单囊泡活体成像，以解决三个具体目标： 目的1）确定树枝状SE的身份。什么样的转运蛋白（Rabs）与树突状SE相关？ 目的2）确定树枝状SE是如何产生的。SE通过什么途径到达树突？ 目的3）确定干扰MEK/MAPK通路如何破坏SE向树突的运输。如何 TrkA信号传导是否影响其在索马中的运输特性？ 这项研究具有重要意义，因为它将揭示长距离NGF信号传导的机制 用于树突发育。这项工作中发现的机制也将与其他长期- 距离信号级联通过其他生长因子在神经发育。

Project Summary/Abstract: Long-distance signaling is a key feature of peripheral neurons. In the developing sympathetic nervous system, long-distance signals via the neurotrophin-receptor pair NGF-TrkA are transported in a signaling endosome (SE) from the distal axon back to the soma. Upon arrival in the soma, the SE signals for survival and, strikingly, traffics into the dendrite where it organizes post-synaptic densities. However, the trafficking mechanisms underlying SE transport into dendrites is unknown. There is thus a fundamental gap in the understanding of how trafficking and signaling of the SE are linked to create functional synaptic connections. The signaling cascades elicited by NGF-TrkA are well-understood. However, the trafficking events that it undergoes once it reaches the soma are severely understudied. The trafficking of the SE affects its signaling and vice versa. Therefore, uncovering how the SE is trafficked into dendrites using high resolution cell biological tools will move past the current barriers of mechanistically linking the physiology of signaling with the cell biology of endosome traffic and maturation. Our long-term goal is to understand how a retrograde signal is able to organize synapse formation. The rationale motivating this proposal is that retrogradely trafficked NGF-TrkA is able to transport into dendrites and initiate the clustering of post-synaptic densities. Additionally, inhibiting the MEK/MAPK pathway exclusively in the cell body prohibits SEs from entering dendrites, indicating a significant link between trafficking and signaling. Our lab recently showed that SEs undergo a novel trafficking event, signaling transcytosis, where the SE is externalized on the soma membrane and is subsequently re-internalized. The central concept of this proposal is that SEs regulate multiple downstream outcomes (survival vs PSD clustering) by undergoing distinct, regulated endosomal conversions in order to diversify their signaling capacity. Our preliminary data suggest the specific hypotheses that dendritic SEs are long-lived and non- degradative (Aims 1 and 2), and that the inhibition of the MEK/MAPK pathway prevents NGF-TrkA’s transition into this long-lived SE that can travel to dendrites (Aim 3). I will use innovative approaches including knockin mice, microfluidic devices, and quantitative single vesicle live imaging to address three specific aims: Aim 1) Determine the identity of dendritic SEs. What trafficking proteins (Rabs) associate with dendritic SEs? Aim 2) Determine how dendritic SEs are generated. What trafficking route do SEs take to reach the dendrites? Aim 3) Determine how interference with the MEK/MAPK pathway disrupts SE trafficking into dendrites. How does TrkA signaling affect its trafficking properties in the soma? The proposed research is significant because it will uncover the mechanisms of long-distance NGF signaling for dendrite development. The mechanisms uncovered in this work will have relevance as well to other long- distance signaling cascades via other growth factors in neurodevelopment.