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.

Public Health Relevance

A stroke occurs when blood flow to a particular region of the brain is interrupted, thereby preventing oxygen and nutrient supply from reaching that area. According to the Center for Disease Control and Prevention, 800,000 individuals in the United States die each year after having suffered a stroke, with many more suffering from lifelong disabilities. An understanding of the mechanisms behind stroke-induced damage is necessary in order to begin to alleviate the significant individual and societal burden that strokes, as one f the leading causes of death, create. About 85% of strokes are classified as ischemic, or due to a clot that has formed in a major vessel of the brain. Therefore, in order to elucidate the mechanisms of cell death at the site of injury, it is important to understand ischemic signaling within neural cells. The study of transglutaminase 2 in astrocytes, which have been shown to be involved in cell death and survival following ischemia, will allow for a greater understanding of some of these mechanisms, with the potential to lead to the development of therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31NS081894-02
Application #
8587443
Study Section
Special Emphasis Panel (ZRG1-F03A-N (20))
Program Officer
Bosetti, Francesca
Project Start
2012-09-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$42,232
Indirect Cost
Name
University of Rochester
Department
Anesthesiology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627