Unexpected Function of Inflammasomes in Axon Pruning: Focus on NLRP1

项目摘要/摘要 神经元能够激活通路，通过以下方式诱导整个细胞退化 或选择性地仅使轴突退化。生理性轴突特异性变性，称为轴突 修剪很重要，因为它允许神经元移除过多或错误引导的轴突，并允许在 神经元连接。在几种神经退行性疾病中观察到异常修剪，包括 阿尔茨海默病(AD)。然而，神经元究竟是如何激活这一途径以退化轴突的 阿尔茨海默病的生理或病理状态尚不清楚。 我们在一个基于微流体室的模型中研究了细胞凋亡和轴突修剪途径。 利用交感神经元。在神经生长因子(NGF)被剥夺后，这些神经元可以诱导 细胞凋亡(当NGF从胞体和轴突之间被剥夺时)或轴突修剪(当NGF 仅从轴突隔室被剥夺)。我们的研究发现了大量的重叠，但也有明显的不同 细胞凋亡和轴突修剪途径的差异。例如，caspase-9(CASP9)和 Caspase-3(Caspase-3)是这两条途径所必需的，它们的激活依赖于过程中的凋亡体 细胞凋亡，但令人惊讶的是，在轴突修剪过程中不依赖于凋亡体。 在研究caspase在修剪过程中被激活的机制时，我们意外地发现 炎性小体途径在轴突修剪中起着重要作用。炎性小体一直是 主要研究病原体信号背景下的免疫细胞。这些是多蛋白质复合体 对致病或危险刺激的反应形成的，这些刺激导致促炎症半胱氨酸天冬氨酸酶的激活， Caspase-1(CASP1)。令人惊讶的是，我们发现CASP1和NLRP1(特定的 炎性小体)对轴突的修剪都是必不可少的。这些结果令人惊讶，因为轴突修剪并没有 与病原体接触有关。在这项提案中，我们将确定特定的炎症体途径组件 对轴突修剪至关重要，并进行了机械实验来定义NLRP1的这一新功能 在神经元中。在目标1中，我们将定义对轴突修剪至关重要的特定炎症体蛋白， 并确定它们在已知的修剪途径中的作用位置。在目标2中，我们将定义IL-1β/IL-18的作用 在轴突修剪中。我们在目标3中的重点将是进行机械实验来检验NLRP1是如何 在轴突修剪的背景下激活。重要的是，在目标4中，我们将重点关注AD并调查 NLRP1参与了AD突触和轴突的病理性变性。我们将在一个 β诱导的体外轴突变性的微流体模型以及在体内的AD小鼠模型 我们将研究NLRP1缺乏是否会减少AD的病理和行为缺陷。此项目将打开一个 NLRP1炎症体在神经元中的这一意想不到的功能的令人兴奋的新研究途径。

Project Summary/Abstract Neurons are capable of activating pathways that induce either the degeneration of the entire cell by apoptosis or to selectively degenerate only the axons. Physiological axon-specific degeneration, known as axon pruning, is important as it allows neurons to remove excessive or misguided axons and permit plasticity in neuronal connections. Aberrant pruning is observed in several neurodegenerative diseases, including Alzheimer’s Disease (AD). However, exactly how neurons activate this pathway to degenerate axons in physiological or pathological situations of AD is unclear. We have investigated the apoptosis and axon pruning pathways in a microfluidic chamber-based model utilizing sympathetic neurons. Upon nerve growth factor (NGF) deprivation, these neurons 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). Our research has identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. For example, while caspase-9 (Casp9) and caspase-3 (Casp3) are required for both pathways, their activation is dependent on the apoptosome during apoptosis but is surprisingly independent of the apoptosome during axon pruning. While investigating the mechanism by which caspases are activated during pruning, we unexpectedly found that the inflammasome pathway plays an important function in axon pruning. Inflammasomes have been studied primarily in immune cells in the context of pathogen signaling. These are multi-protein complexes formed in response to pathogenic or danger stimuli, which result in activation of the proinflammatory caspase, caspase-1 (Casp1). Strikingly, we found that Casp1 and NLRP1 (a key component of a particular inflammasome) are both essential for axon pruning. These results are surprising as axon pruning does not involve pathogen exposure. In this proposal, we will identify the specific inflammasome pathway components that are critical for axon pruning and conduct mechanistic experiments to define this novel function of NLRP1 in neurons. In Aim 1, we will define the specific inflammasome proteins that are essential for axon pruning, and determine where they act in the known pruning pathway. In Aim 2, we will define the role of IL-1β/IL-18 in axon pruning. Our focus in Aim 3 will be to conduct mechanistic experiments to examine how NLRP1 is activated in the context of axon pruning. Importantly, in Aim 4 we will focus on AD and investigate whether the pathological degeneration of synapses and axons in AD are mediated by NLRP1. We will examine this in a microfluidic model of Aβ-induced axon degeneration in vitro as well as in a mouse model of AD in vivo where we will examine if NLRP1 deficiency reduces AD pathology and behavioral defects. This project opens an exciting new avenue of research into this unexpected function of the NLRP1 inflammasome in neurons.