Recent evidence shows that neural stem cell (NSC) proliferation and function are affected by alcohol. However, the mechanism of these effects is unknown. Alcohol induces oxidative stress, which will be considered as a potential mechanism for the alcohol's actions on NSCs in this application. Our recent work has shown that oxidative stress causes normally non-immunogenic tissues to express costimulatory molecules capable of activating T cells. The novelty of the present application is derived from the observation that alcohol affects the immunogenicity of neural stem cells. The expression and function of the immunologically important molecules on the surface of NSCs are poorly understood. We have begun addressing these questions and found that immunomodulatory molecules such as CD95 (Fas receptor) and the co-stimulatory molecule B7.1 are present on the surface of NSCs of mouse derivation and are upregulated by oxygen radicals. The key hypothesis behind this application is that alcohol's neurotoxic effects on neural progenitors are mediated by the recognition and response of the immune system to stress-induced changes in NSCs.
The specific aims of this proposal are: (1) Determine whether alcohol kills NSCs and how it affects the expression of immune molecules in NSCs. We will initially examine whether alcohol exposure kills cultured mouse C17.2 NSCs and mouse endogenous NSCs in vivo using well-established assays for cell death and apoptosis. We will then use the C17.2 NSC line to systematically examine the expression of immune recognition molecules in NSCs and determine the effects of alcohol on their expression. The emphasis will be placed on the examination of the expression levels of a death receptor (CD95), primary immune recognition molecules such as major histocompatibility complexes (MHCs) and co-stimulatory molecules such as B7 and CD40. The cell surface molecules will be quantitated using flow cytometry. (2) Examine whether protection from reactive oxygen intermediates promotes NSCs' survival and differentiation following alcohol exposure. In this aim, we will stably overexpress in a regulatable fashion a mouse mitochondrial uncoupling protein 2 (UCP-2) in C17.2 cells to generate C17.UCP-2 cell line. UCP-2 decreases reactive oxygen species inside mitochondria. We hypothesize that expression of UCP-2 will reduce those actions of ethanol that are mediated by oxidative stress. (3) Determine whether transplanted C17.UCP-2 cells are more resistant to alcohol than NSCs expressing only natural levels of UCP-2. To extend the in vitro findings from aim 2, we will examine the effects of alcohol in vivo by transplanting cell lines generated in aim 2 into brains of wild type mice and mice deficient in UCP-2. We will systematically explore the changes in the immune molecules on the surface of transplanted NSCs in animals maintained on ethanol diet. Following transplantation, grafted NSC survival and UCP-2 expression in the grafts will be assayed and compared to these measures in endogenous NSCs. Proposed studies will systematically evaluate the effects of alcohol on the dynamics of immune molecules' expression on NSCs and their function. Such findings will stimulate further research bridging fields of alcohol, stem cells and immunology.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Exploratory/Developmental Grants (R21)
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Health Services Research Review Subcommittee (AA)
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Sorensen, Roger
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University of Colorado Denver
Internal Medicine/Medicine
Schools of Medicine
United States
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