W Michael Zawada - US grants

DESCRIPTION (provided by applicant): Brain dopamine is postulated to modulate alcohol consumption. We hypothesize that reduction in extracellular dopamine (DA) levels would reduce ethanol intake. The main goal of this proposal is to examine if transplantation of neural stem cells (NSCs) modified to overexpress the human DA transporter (hDAT) into specific brain sites can reduce extracellular DA levels and alter ethanol?s behavioral actions. NSCs are pluripotent cells that exist in the developing and adult brain. NSCs have a capacity to differentiate into all known neural type cells including neurons, astrocytes and oligodendrocytes. Unprecedented plasticity of NSCs makes them ideal candidates for genetic modification and transplantation into the central nervous system (CNS). This proposal will explore a novel strategy using stem cell transplantation for moderating alcohol effects and intake. The specific aims of this proposal are: (1) Generation of neural stem cell lines expressing human DAT (hDAT) under control of an inducible promoter. Because cell lines expressing high levels of hDAT suitable for neural transplantation do not exist, we propose to generate a hDAT-expressing high neural stem cell line (C17.hDAT) for cell transplantation. We will use mouse v-myc immortalized neural stem cells (C17.2) obtained from Dr. Evan Snyder as the cell line development platform. (2) Examine the hDAT expression and function in C17.hDAT stem cell-derived neurons and glia. We propose to examine hDAT expression and function in stem cells differentiated into neurons or glia. This aim will also examine the effectiveness of regulating Tet-On system-driven hDAT expression and function with doxycycline. (3) Determine if transplantation of C17.hDAT stem cells into mouse brain can reduce extracellular dopamine in vivo and alter ethanol?s actions. We will first transplant C17.hDAT cells developed in the aim 1 into the brains of wildtype (wt) and DAT knockout mice provided by Dr. Marc Caron and into the brains of wt mice with high (C57BL/6) and low (DBA) alcohol preference. Target areas for grafting will include nucleus accumbens, prefrontal cortex, amygdala and dorsal striatum. Grafting into DAT knockout mice should reverse the behavioral and biochemical consequences of reduction in DAT levels. Survival of grafted cells and function of transgenic hDAT will be examined. Behavioral testing will examine ethanol-induced locomotor activity and the mice will be exported to other sites in the consortium to test for alcohol preference. These studies will generate hDAT-expressing stem cells for transplantation in the CNS and examine whether stem cell therapy can reduce alcohol?s actions. Such findings will provide the first evidence for use of stem cells in the treatment of drug dependence.

DESCRIPTION (provided by applicant): 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.

[unreadable] DESCRIPTION (provided by applicant): The original five-year INIA West has identified a large number of candidate genes that are differentially expressed in the brains of mice selectively bred for either high- or low-preference for alcohol. Additional conserved genes were identified in screens for alcohol tolerance in Drosophila. Manipulating the expression of these genes has naturally become a critical component of studies designed to further define their role in the development of alcohol preference or tolerance. The RNA Interference Core (RNAi Core) will provide a means for systematic modification of the target genes' expression in selected and precisely-defined brain areas. Two technological platforms for in vivo RNA interference will be employed: (1) small interfering RNA (siRNA) delivered by a direct injection or infusion into the CNS and (2) short hairpin RNA (shRNA) delivered via a lentiviral vector-based transduction. Initially, these methods will be standardized using transcripts identified in HAP and LAP mice as contributing to alcohol preference drinking behavior, and the functional consequences of the RNAi treatment will be assessed in these mice. The successful implementation of the methodology will provide an important resource for all INIA investigators, including those working with rat models. At the time of this submission, laboratories from California, Colorado, Indiana, Oregon and Texas are already collaborating in both Binge and Dependence Domains. The core will also perform RNAi treatments for INIA investigators, who will develop need for RNAi services with the emergence of additional target genes. Proposed projects will be evaluated for integration with INIA West goals by a Project Evaluation Committee composed of members of the INIA Steering Committee and an independent consultant. The core will capitalize on our ongoing collaboration with Dharmacon Corporation, a leader in the field of siRNA development. Dharmacon will provide many of the necessary reagents and work with us on improving the efficiency of gene silencing in the CNS. The core's goals will be accomplished by successful completion of the following aims: Aim 1. Perform high throughput in vitro screening of the RNAi sequences targeting the genes of interest. Aim 2. Silence expression of selected genes in vivo employing RNAi within precise neuroanatomical targets. Aim 3. Examine behavioral and transcriptional effects of gene silencing. The creation of the RNAi core is a logical extension of the work already completed by the INIA. The core will allow for systematic and high throughput manipulation of genes in the mammalian CNS, facilitating functional studies of these genes in alcohol preference. [unreadable] [unreadable] [unreadable]