Exploring Apoptosome-Independent Mechanisms for Casp9 activation in Axon Pruning

探索轴突修剪中 Casp9 激活的独立凋亡体机制

• 批准号: 10288453
• 负责人: Mohanish P Deshmukh
• 金额: $ 42.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288453
• 关键词: Active Sites; Adult; Alzheimer' s Disease; Apoptosis; Axon; BIRC4 gene; Binding; Biological; CASP3 gene; CASP9 gene; Caspase; Cell Death; Cells; Cessation of life; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Dependence; Development; Dimerization; Ensure; FHL2 gene; Goals; Huntington Disease; Impairment; Interphase Cell; Knock-out; Mediating; Microfluidics; Mitochondria; Modeling; Molecular; Mus; Mutation; Nerve Growth Factors; Nervous system structure; Neurodegenerative Disorders; Neurons; Outer Mitochondrial Membrane; Parkinson Disease; Pathway interactions; Peripheral; Physiological; Proteins; Reporting; Risk; Signal Transduction; Site; apoptotic protease-activating factor 1; aposome; axonal degeneration; base; clinically relevant; cytochrome c; deprivation; inhibitor/antagonist; knock-down; mimetics; mutant; neuron apoptosis; neuronal cell body; novel; receptor; small hairpin RNA; virtual

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.

项目摘要/摘要 神经元有能力激活通过 凋亡或仅轴突。生理轴突特异性变性，也称为修剪，很重要 它允许神经元去除过多或误导的轴突分支，并允许神经元的可塑性 连接。但是，确切的神经元如何激活并将这种途径划分以变成其途径 轴突不将其余的细胞处于危险之中，尚不清楚。自最近研究以来，这特别有趣 鉴定出Bax和caspases，凋亡途径中的关键蛋白，也调节轴突修剪 我们正在使用基于微流体的交感神经元模型，而神经的剥夺 生长因子（NGF）可以诱导凋亡 隔室）或轴突修剪（仅将NGF从轴突隔室中剥夺时）。使用此模型， 我们确定了大量重叠，但凋亡和轴突修剪之间也有明显的差异 途径。具体而言，我们发现，尽管两种途径都需要Caspase-9（CASP9），但CASP9激活 在凋亡过程中取决于APAF-1，但令人惊讶的是，在修剪轴突修剪过程中与APAF-1无关。这些 结果是出乎意料的，并指出了轴突修剪过程中CASP9激活的新机制。 在此提案中，我们的目标是研究在轴突修剪过程中如何激活CASP9的特定方面。 在AIM 1中，我们将在轴突修剪期间专注于Bax功能。由于轴突修剪不需要APAF-1，我们 提出，轴突修剪过程中BAX的基本功能不是Cyt C的释放（它激活了APAF- 1在凋亡的背景下），但取而代之的是SMAC（XIAP的抑制剂）从线粒体中释放。 在AIM 2中，我们将检查在修剪过程中如何激活CASP9。我们的重点是确定 CASP9功能对于轴突修剪很重要。此外，我们将检查一个组件是否 依赖体样复合物对于在轴突修剪过程中激活CASP9很重要。这些研究将有助于 发现神经元如何利用许多相同成分进行细胞凋亡和轴突修剪的关键方面 但是有明显的差异。