Alcohol, Glial Gene Expression and the Heat Shock Pathway

描述（由申请人提供）：饮酒导致各种短期行为变化和长期生理适应，可导致耐受性和身体依赖性。据报道，长期接触酒精会导致脑内神经元和白色物质的损失。已知酒精会触发星形胶质细胞释放细胞因子和活性氧（ROS），并且可能在小胶质细胞（MCG）中具有类似的作用，以及减少少突胶质细胞（OLG）的髓鞘合成。酒精调节基因表达，不同的酒精响应基因（ARG）组被急性酒精激活。我们最近在Gabra 4基因中发现了酒精诱导基因转录所必需的11个碱基对序列元件-酒精反应元件（ARE）。酒精对Gabra 4的刺激涉及热休克途径，导致热休克因子1（HSF 1）与ARE结合（Pignataro等人，2007年）。ARE存在于大量其他神经元ARG中。酒精还激活星形胶质细胞中的热休克通路。我们现在建议将这些研究扩展到OLG和MCG细胞。我们的总体假设是，酒精通过激活热休克途径改变了对神经胶质细胞功能至关重要的基因的表达。目的1）研究酒精对星形胶质细胞热休克通路的激活作用。我们将研究小鼠星形胶质细胞系和小鼠星形胶质细胞原代培养物中酒精对热休克通路的激活作用。我们将检测酒精对星形胶质细胞核内热休克蛋白（Hsps）表达的影响，并研究HSF 1的转位，以及HSF 1与ARE之间的相互作用。目的2）鉴定星形胶质细胞中的酒精反应基因。我们将使用生物信息学和基因芯片的组合，以确定新的ARGs在星形胶质细胞，然后检查的ARGs的子集依赖于ARE通过热休克途径。我们将使用热休克途径的药理学抑制剂和RNAi来确定HSF 1的参与。目的3）研究乙醇对少突胶质细胞基因表达的影响。我们将研究酒精对OLG细胞系中髓鞘相关基因表达的影响，研究酒精对OLG中Hsps表达的影响，并使用药理学抑制剂研究HSF 1在OLG中ARG调节中的作用。我们将使用生物信息学、微阵列和Q-PCR来鉴定OLG中的ARG。目的4）研究酒精对小胶质细胞基因表达的影响。我们将研究酒精对MCG细胞系中细胞因子IL-1b（IL 1b）、细胞因子受体IL-1 R1（IL 1 r1）和TLR 4（TLR 4）以及iNOS（Nos 2）和考克斯-2（COX 2）的表达的影响。我们将研究酒精对MCG中Hsps表达的影响，并使用药理学抑制剂来评估HSF 1的作用。 公共卫生相关性：酗酒是美国的一个主要公共卫生问题。从长远来看，长期饮酒会导致生理变化，对人类健康有害，并产生社会后果。酗酒者的大脑变得“重新连接”，这一过程的一部分是由于对酒精的反应而发生的基因表达的变化。我们已经证明，一种被称为热休克途径的细胞防御机制被酒精激活。在这项提案中，我们计划将这项工作扩展到研究被称为神经胶质细胞的细胞内的变化，神经胶质细胞是支持大脑内神经活动的脑细胞。星形胶质细胞控制神经细胞的化学环境，并在大脑和血液之间形成屏障。少突胶质细胞在神经细胞周围形成鞘，加速大脑内的信号传导。小胶质细胞是大脑的免疫组成部分，并消除外来入侵者。酒精可能通过改变基因表达来改变所有三种类型的神经胶质细胞的活性，这样做可能会损害神经细胞。

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.