Alcohol, Glial Gene Expression and the Heat Shock Pathway

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DESCRIPTION (provided by applicant): Drinking alcohol results in a variety of short-term behavioral changes and long-term physiological adaptations that can lead to tolerance and physical dependence. Chronic exposure to alcohol has been reported to produce loss of neurons and white matter within the brain. Alcohol is known to trigger the release of cytokines and reactive oxygen species (ROS) from astrocytes and may have similar effects in microglia (MCG), as well as decreasing the synthesis of myelin by oligodendrocytes (OLGs). Alcohol regulates gene expression, and distinct sets of alcohol-responsive genes (ARGs) are activated by acute alcohol. We recently discovered, in the Gabra4 gene, an 11 base pair sequence element essential for the induction of gene transcription by alcohol - the alcohol response element (ARE). Stimulation of Gabra4 by alcohol involves the heat shock pathway, causing heat shock factor 1 (HSF1) to bind to the ARE (Pignataro et al., 2007). The ARE is present in a large number of other neuronal ARGs. Alcohol also activates the heat shock pathway in astrocytes. We now propose to extend these studies to OLG and MCG cells. Our overall hypothesis is that alcohol alters the expression of genes critical for the function of glial cells via activation of the heat shock pathway. The specific aims are: Aim 1) To study the activation of the heat shock pathway by alcohol in astrocytes. We will investigate activation of the heat shock pathway by alcohol in a mouse astrocyte cell line and in primary cultures of mouse astrocytes. We will measure the effects of alcohol on the expression of heat shock proteins (Hsps) and study the translocation of HSF1, and the interaction between HSF1 and the ARE in astrocyte nuclei. Aim 2) To identify alcohol-responsive genes in astrocytes. We will use a combination of bioinformatics and gene microarrays to identify novel ARGs in astrocytes, and then examine the subset of ARGs dependent on the ARE via the heat shock pathway. We will use pharmacological inhibitors of the heat shock pathway and RNAi to determine the involvement of HSF1. Aim 3) To study the effects of alcohol on gene expression in oligodendrocytes. We will study effects of alcohol on the expression of myelin- associated genes in an OLG cell line, investigate the effects of alcohol on expression of Hsps in OLGs and use pharmacological inhibitors to study the role of of HSF1 in the regulation of ARGs in OLGs. We will use bioinformatics, microarrays and Q-PCR to identify ARGs in OLGs. Aim 4) To study the effects of alcohol on gene expression in microglia. We will study the effects of alcohol on the expression of cytokine IL-1b (Il1b), the cytokine receptors, IL-1R1 (Il1r1) and TLR4 (Tlr4) and the enzymes iNOS (Nos2) and COX-2 (COX2) in a MCG cell line. We will investigate the effects of alcohol on the expression of Hsps in MCG and use pharmacological inhibitors to assess the role of HSF1. PUBLIC HEALTH RELEVANCE: Alcoholism is a major public health problem in the US. In the long-term, chronic drinking results in physiological changes that are detrimental to human health and have societal consequences. The brains of alcoholics become "rewired", and a part of this process results from changes in gene expression that occur in response to alcohol. We have shown that a cellular defense mechanism known as the heat shock pathway is activated by alcohol. In this proposal we plan to extend this work to study the changes within cells known as glia, the brain cells that support the activity of nerves within the brain. Astrocytes control the chemical environment of the nerve cells, and form a barrier between the brain and the blood. Oligodendrocytes form a sheath around nerve cells that speeds signal conduction within the brain. Microglia are the immune component of the brain and eliminate foreign invaders. Alcohol may alter the activity of all three types of glial cell by changing gene expression, and in doing so may harm nerve cells.