Exploring Apoptosome-Independent Mechanisms for Casp9 activation in Axon Pruning

项目摘要/摘要 神经元有能力通过以下方式激活导致整个细胞退化的通路 细胞凋亡或者只是轴突。生理性轴突特异性退行性变，也称为修剪，重要的是 它允许神经元移除过多或被误导的轴突分支，并允许神经元的可塑性 关系。然而，神经元到底是如何激活和分割这条通路的，从而使其退化 在没有将细胞的其余部分置于危险之中的情况下，轴突的情况尚不清楚。这一点特别有趣，因为最近的研究 确定了凋亡途径中的关键蛋白bax和caspase，以也调节轴突修剪 我们正在使用一种基于微流室的交感神经元模型，在该模型中，剥夺神经 生长因子(NGF)可以诱导任何一种细胞凋亡(当NGF从胞体和轴突中去除时 室)或轴突修剪(当NGF仅从轴突室被剥夺时)。使用这个模型， 我们发现细胞凋亡和轴突修剪之间有很大的重叠，但也有明显的不同。 小路。具体地说，我们发现，虽然caspase-9(CASP9)对于两个途径都是必需的，但CASP9的激活 在凋亡过程中依赖于APAF-1，但令人惊讶的是，在轴突修剪过程中不依赖于APAF-1。这些 结果是意想不到的，并指出了轴突修剪过程中CASP9激活的新机制。 在这项提案中，我们的目标是研究CASP9在轴突修剪过程中如何被激活的特定方面。 在目标1中，我们将重点介绍Bax在轴突修剪过程中的作用。由于APAF-1不是轴突修剪所必需的，我们 提出Bax在轴突修剪过程中的基本功能不是释放细胞色素c(激活APAF-1)。 1在细胞凋亡的背景下)，而是从线粒体释放XIAP的抑制物Smac。 在目标2中，我们将研究CASP9在修剪过程中是如何被激活的。我们的重点是确定 对轴突修剪很重要的CASP9功能。此外，我们还将检查 依赖体样复合体在轴突修剪过程中对激活CASP9起重要作用。这些研究将会有所帮助 揭示神经元如何利用许多相同的成分进行细胞凋亡和轴突修剪的关键方面 但也有明显的不同。

Project Summary/Abstract Neurons have the capability to activate pathways that cause degeneration of either the entire cell by apoptosis or only the axons. Physiological axon-specific degeneration, also known as pruning, is important as it allows neurons to remove excessive or misguided axon branches and permit plasticity in neuronal connections. However, exactly how a neuron can activate and compartmentalize this pathway to degenerate its axon without putting the rest of the cell at risk is unclear. This is particularly interesting since recent studies identified Bax and caspases, key proteins in the apoptosis pathway, to also regulate axon pruning We are using a microfluidic chamber-based model of sympathetic neurons where deprivation of nerve growth factor (NGF) can induce either apoptosis (when NGF is deprived from both soma and axon compartments) or axon pruning (when NGF is deprived from only the axon compartment). Using this model, we identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. Specifically, we found that while caspase-9 (Casp9) is required for both pathways, Casp9 activation is dependent of Apaf-1 during apoptosis but, surprisingly, independent of Apaf-1 during axon pruning. These results were unexpected and point to a novel mechanism of Casp9 activation during axon pruning. In this proposal, our goals are to examine specific aspects of how Casp9 is activated during axon pruning. In Aim 1, we will focus on Bax function during axon pruning. Since Apaf-1 is not needed for axon pruning, we propose that the essential function of Bax during axon pruning is not the release of cyt c (which activates Apaf- 1 in the context of apoptosis), but instead is the release of the Smac, an inhibitor of XIAP, from mitochondria. In Aim 2, we will examine how Casp9 is activated during pruning. Our focus is on identifying key features of Casp9 function that are important for axon pruning. Additionally, we will examine whether components of a dependosome-like complex are important for activating Casp9 during axon pruning. These studies will help uncover critical aspects of how neurons utilize many of the same components for apoptosis and axon pruning yet with distinct differences.