Endogenous inhibitors of inflammation in CNS function

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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.