The Role of Astrocytic Transglutaminase 2 in Mediating Ischemic Stroke Damage

University Of Rochester, Rochester NY

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Abstract

DESCRIPTION (provided by applicant): Ischemic strokes lead to widespread damage within the brain, which can result in death or debilitating disability. This damage is the outcome of various cellular cascades that contribute to cell death processes. Astrocytes are a significant variable in ischemic injury; not only is their survival a factor, but their ability to protect neurns is also critically important. Transglutaminase 2 (TG2) is one molecular factor that has been identified as a likely mediator of ischemic cell death processes. TG2 exhibits widespread functionality throughout the central nervous system, and its expression in neurons has been shown to be protective against ischemic injury. Knockout of TG2 in astrocytes, on the other hand, has been shown to significantly improve astrocytic survival, as well as enhance their ability to protect neurons from conditions of oxygen glucose deprivation (OGD). The exact functions of TG2 in these particular processes, however, are largely unknown. The goal of my proposed research is to elucidate the role that astrocytic TG2 plays in mediating stroke-induced damage. To this end, I have formulated three specific aims to test the hypotheses that: 1) TG2 localization, activity state and molecular interactions are altered in astrocytes under hypoxic conditions, 2) the absence of TG2 in astrocytes both protects astrocytes from ischemia-induced cell death, and facilitates their ability to confer protection for neurons under ischemic condition, and 3) astrocyte-specific knockout of TG2 will reduce the infarct volume after a middle cerebral artery ligation (MCAL) in vivo. For the first two aims, I will use cell culture models. I will determine TG2 localization and function in wild-type astrocytes that are subjected to hypoxic conditions. I will also perform knockdown experiments using shRNA against TG2 and assess toxicity after OGD treatment. Using these conditions, I will also examine potential molecular factors that TG2 may be interacting with, such as matrix metalloproteinases (MMPs) and the NF-¿B pathway, to mediate its toxic effects in astrocytes. Using astrocyte-neuron co-cultures, as well as conditioned media from wild type and TG2-/- astrocytes, I will determine how the presence of TG2 affects the ability of astrocytes to protect neurons against OGD-induced cell death. In aim 3 I will perform MCALs on mice in which TG2 is knocked out selectively in astrocytes (GFAP-Cre/TG2fl/fl) and compare infarct volume and other biochemical markers of cell death and survival to those seen in control animals (GFAP-Cre/TG2WT/WT). Through these experiments, I hope to make a significant contribution to the understanding of molecular cascades that contribute to the damage that occurs subsequent to ischemic injury. Knowledge of these pathways will thus allow for the development of possible treatments that can target the molecular factors that are involved in stroke-induced cellular toxicity.