Endogenous inhibitors of inflammation in CNS function

炎症是涉及细胞毒性、死亡和生存的核心稳态过程。我的研究计划的长期目标是研究炎症机制以及这些机制如何影响不同类型的行为活动。为了实现这一目标，我使用Nlrx 1，一种内源性线粒体蛋白，是NOD样受体家族的一部分。具体而言，Nlrx 1限制中枢神经系统（CNS）炎症，并减少谷氨酸的细胞毒性和细胞死亡。我将操纵Nlrx 1来调节炎症，同时记录行为。在接下来的5年里，我的研究计划分为三个短期目标：1.表征操纵Nlrx 1基因如何影响Nlrx 1蛋白在不同细胞类型中的细胞定位。 nlrx 1抑制植物和动物的免疫反应。利用现代细胞和分子生物学，我们将定义Nlrx 1的原型结构域负责其在星形胶质细胞中的抗炎和促生存特性。具体来说，我们将使用CRISPR/Cas9与腺病毒转导配对来剖析Nlrx 1的分子结构与细胞内定位之间的关系。 2.表征体外不同细胞类型中Nlrx 1突变后的炎症、细胞死亡和/或谷氨酸稳态。 我们发表的结果和初步数据表明，Nlrx 1通过改善谷氨酸平衡和减少炎症和细胞死亡来支持CNS稳态过程。我们将描述Nlrx 1的突变和细胞定位如何影响谷氨酸的摄取和释放，促炎细胞因子和趋化因子的产生，以及细胞死亡和存活。在CNS中，负责炎症反应的主要细胞类型是小胶质细胞和星形胶质细胞。因此，所有实验将在小鼠原代小胶质细胞和星形胶质细胞以及人星形胶质细胞系中进行。3.表征Nlrx 1缺失在不同类型的体内行为活动中的影响。 我将使用专门的软件和机器学习来研究Nlrx 1-/-小鼠的连续行为特征。基于我最近的进展，我将使用探索性统计方法算法，包括层次聚类和因子分析。此外，我将使用人工神经网络来搜索与Nlrx 1表达相关的行为活动和昼夜节律模式。我的初步研究表明，Nlrx 1显著影响睡眠的快速眼动阶段，与谷氨酸稳态的作用一致。总之，拟议的研究计划将确定新的机制，通过中枢神经系统的稳态过程调节炎症和谷氨酸平衡，以塑造行为。高素质的人员将在多学科环境中使用复杂的细胞，分子和行为工具工作。

Inflammation is a central homeostatic process involved in cell toxicity, death and survival. The long-term goal of my research program is to investigate inflammatory mechanisms and how these affect different types of behavioral activities. To accomplish this goal, I use Nlrx1, an endogenous mitochondrial protein that is part of the NOD-like receptor family. Specifically, Nlrx1 limits central nervous system (CNS) inflammation and reduces glutamate cytotoxicity and cell death. I will manipulate Nlrx1 to modulate inflammation while recording behavior. In the next 5 years, my research program is divided into three short-term objectives: 1.Characterize how manipulation of the Nlrx1 gene affects the cellular localization of the Nlrx1 protein in different cell types. Nlrx1 inhibits immune responses in plants and animals. Using modern cellular and molecular biology, we will define the archetypal domains of Nlrx1 responsible for its anti-inflammatory and pro-survival properties in astrocytes. Specifically, we will dissect the relationship between Nlrx1's molecular structure and intracellular localization using CRISPR/Cas9 paired with adenoviral transduction. 2.Characterize inflammation, cell death and/or glutamate homeostasis following Nlrx1 mutation in different cell types in vitro. Our published results and preliminary data suggest that Nlrx1 supports CNS homeostatic processes by improving glutamate balance and reducing inflammation and cell death. We will characterize how mutation and cellular localization of Nlrx1 affect glutamate uptake and release, production of pro-inflammatory cytokines and chemokines, and cell death and survival. In the CNS, the main cell types responsible for the inflammatory response are microglia and astrocytes. Therefore, all experiments will be performed in mouse primary microglia and astrocytes, and a human astrocytic cell line. 3.Characterize the effects of Nlrx1 deletion in different types of behavioural activities in vivo. I will study the continuous behavioral profiles of Nlrx1-/- mice using specialized software and machine learning. Building on my recent progress, I will use exploratory statistical method algorithms, including hierarchical clustering and factor analysis. Also, I will use artificial neural networks to search for patterns of behavioral activities and circadian rhythms that relate to Nlrx1 expression. My preliminary studies suggest that Nlrx1 significantly affects the rapid eye movement phase of sleep, consistent with a role in glutamate homeostasis. In conclusion, the proposed research program will identify novel mechanisms through which CNS homeostatic processes modulate inflammation and glutamate balance to shape behavior. Highly qualified personnel will work in a multidisciplinary environment with sophisticated cellular, molecular, and behavioral tools.