Robert W. Keane - US grants

neural plasticity; cell transformation; early embryonic stage; cell differentiation; melanocyte; neurons; cell type; astrocytes; morphology; molecular biology;

DESCRIPTION (provided by applicant): The overall goal of this project is to determine whether the induction of the inflammatory response involving activation of caspase-1 and processing of interleukin-12 (IL-12) following moderate cervical spinal cord injury (SCI) in the rat is mediated by the formation of multiprotein inflammatory caspase-activating platforms termed inflammasomes. Our recent work has shown that the NLRP1 inflammasome is expressed in rat spinal cord neurons and regulates IL-12 and IL-18 production. We have found that in SCI neutralization of the adaptor protein apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) reduced inflammation and resulted in significant tissue sparing and functional improvement. The Specific Aims of this proposal will extend these results to determine the cellular and molecular mechanisms regulating inflammasome activation in glia. Aim 1 will identify the structural components and protein associations of inflammasomes in primary cultures of neurons, astrocytes, and microglia damaged by stretch injury. These studies will delineate which cell types express inflammasomes and will provide insight into the mechanisms leading to cytokine secretion caused by caspase-1 after injury. Aim 2 will identify key inflammasome signaling components in distinct CNS cell types using lentiviral short hairpin (shRNA) vectors that target inflammasome genes and proteins. Related experiments will determine the cellular distribution and temporal sequence of protein expression of inflammasome proteins in spinal cords of normal and injured rats. Experiments in Specific Aim 4 will determine if disruption of inflammasome signaling by antibody neutralization of inflammasome components or lentiviral shRNA silencing of inflammasome genes results in inhibition of inflammasome signaling, thus leading to improved histopathological and behavioral outcomes. These studies will provide critical information about temporal and spatial activation patterns of the inflammasome and identify relevant therapeutic targets to control inflammation following SCI and other neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Spinal cord injury (SCI) occurs throughout the world with an annual incidence of 15 to 40 million people per year and results in lifelong disabilities. The main cause of SCI in young males is motor-vehicle accidents and most spinal cord traumas occur at the cervical spinal cord level. The proposed research seeks to reduce cell death by inflammation that occurs within days of SCI, and to devise therapies to treat SCI and help in long-term recovery of function. The development of new treatments that can be given in the first hours after SCI may prevent or reverse much of the spinal cord damage resulting from SCI with a goal of restoring normal functioning in behavior.

PROJECT SUMMARY The overall goal of this project is to determine whether TBI-induced inflammatory changes result in peripheral organ damage that is dependent on activation of the inflammasome by extracellular vesicles (EV) and ASC specks. Our recent work has shown that a crucial part of the TBI-induced systemic inflammatory response involves release of extracellular vesicles containing a cargo of innate immune proteins that are secreted from damaged central nervous system (CNS) tissue. Importantly, EV and ASC specks from TBI animals induce inflammasome activation in peripheral organs. The specific aims of this proposal will determine the cellular and molecular mechanisms regulating EV- and ASC speck-mediated innate immune inflammatory reactions in peripheral organs after TBI. The hypothesis of this study is that EV and ASC specks play a central role in innate immune signaling by carrying inflammasome proteins to peripheral organs after TBI, thus causing tissue injury. Moreover, neutralization of secreted ASC with a monoclonal antibody decreases peripheral organ damage after TBI, resulting in improved histopathological outcomes. Aim 1 will determine if TBI alters the composition of EV proteins in brain, peripheral organs and bodily fluids. These studies will delineate a protein profile of EV proteins after TBI. Aim 2 will establish if ASC specks accumulate in brain and peripheral organs after TBI and induce an inflammatory responses leading to pyroptosis. We will investigate whether the activation of inflammasomes in vivo leads to the appearance of ASC specks and whether the deposition of ASC specks in tissues induces the recruitment of neutrophils and lymphocytes, thus contributing to the inflammatory milieu in peripheral organs. Aim 3 will determine the therapeutic effects of antibody neutralization of the inflammasome on histopathological outcomes after TBI. These studies will provide critical information about the activation patterns of innate immunity regulated by EV and ASC specks in the CNS, and identify relevant therapeutic targets to control inflammation following TBI and other neurodegenerative disorders.