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重要特征。在发展中 交感神经系统，通过神经营养因子受体对NGF-TrkA的远程信号 在信号内体(SE)中从远端轴突运输回胞体。在抵达索马岛后， SE是生存的信号，令人惊讶的是，它进入树突，在那里组织突触后的密度。 然而，SE转运到树突的运输机制尚不清楚。因此，有一个 对贩运和SE信号如何联系在一起的理解存在根本差距， 功能性突触连接。NGF-TrkA引起的信号级联是众所周知的。 然而，一旦它到达索马，它所经历的贩运事件就被严重地忽视了。这个 SE的贩运会影响其信号传递，反之亦然。因此，发现SE是如何被贩运到 使用高分辨率细胞生物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 理解逆行信号是如何组织突触形成的。 这项提议的基本原理是，退行性传输的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 仅在细胞体内禁止SE进入树突，这表明 贩卖和发信号。我们的实验室最近发现，SE经历了一种新的贩运事件，即信号 跨细胞作用，SE外化在胞体膜上，随后被重新内化。这个 这一建议的核心概念是SE调节多个下游结果(生存与PSD 群集)通过经历不同的、受调控的内体转换以使其信号多样化 容量。我们的初步数据表明，树枝状SE是长期存在的，而且不是 降解性(目标1和2)，并且抑制MEK/MAPK通路阻止NGF-TrkA的转变 进入这个长寿的SE，可以到达树突(目标3)。我将使用包括敲门在内的创新方法 小鼠、微流控设备和定量单泡活体成像，以满足三个特定目标： 目的1)确定树突状SES的特性。运输蛋白(RAB)与树突状SE有什么关系？ 目的2)确定树枝状Se是如何产生的。SE通过什么运输路线到达树枝状突起？ 目的3)确定干扰MEK/MAPK通路是如何干扰SE转运到树突的。多么 TrkA信号是否影响其在Soma的贩运特性？ 这项拟议的研究具有重要意义，因为它将揭示长距离NGF信号的机制 用于树枝晶的发育。这项工作中发现的机制也将对其他长期的 在神经发育中，距离信号通过其他生长因子级联。

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.