Sex dependent astrocyte activation and alcohol-induced neurotoxicity

描述（由申请人提供）：本提案探讨慢性酒精（乙醇；乙醇）暴露和戒除对性别特异性星形胶质细胞激活、表达和功能的影响。慢性EtOH滥用与神经毒性和反应性星形胶质增生有关，但其机制和涉及的细胞类型仍不清楚。此外，与酒精中毒相关的脑损伤是两性二态的，但慢性乙基羟基乙酸暴露的性别特异性影响尚未得到充分探讨。该建议扩展了我们之前的分析，使用基因表达转录谱来比较男性和女性，以表征与慢性EtOH暴露戒除相关的神经适应性变化。值得注意的是，这些研究表明，与基因型和/或戒断严重程度表型相比，性别是慢性暴露后神经适应和转录反应的更强大决定因素。基因表达差异的生物学证实表明，女性更容易受到酒精引起的脑损伤，这与一些（但不是全部）临床研究一致。在前额皮质慢性EtOH显著调控的基因中，有一些主要或只由星形胶质细胞表达。此外，etoh诱导的脑损伤与谷氨酸释放增加引起的兴奋性毒性有关，可能来自星形胶质细胞。GABA受体也是慢性EtOH滥用的神经适应靶点，并与星形胶质细胞功能有关。由于星形胶质细胞能够调节神经元活动和突触神经传递（兴奋性和抑制性信号）来影响神经毒性，并且星形胶质细胞的激活可以在酒精滥用中观察到，因此研究慢性EtOH对星形胶质细胞功能的影响是及时和重要的。鉴于有证据表明EtOH戒断对女性和男性大脑的损害不同，本研究旨在验证慢性酒精暴露和戒断以性别特异性方式靶向星形胶质细胞功能的假设，从而导致基因表达和功能的改变，从而导致兴奋毒性信号的升高。目的1描述体外慢性乙醇对雄性和雌性皮质星形胶质细胞培养的细胞效应。本研究的目的是发展初级单性别皮质星形胶质细胞培养方法，直接测试在受控环境下，男性和女性星形胶质细胞群体对长期接触和戒除EtOH的反应。本研究还研究了慢性乙醇暴露和戒断后星形胶质细胞增殖、凋亡、谷氨酸摄取和释放、gaba能信号以及钙储存的贡献。目的2将定义慢性EtOH损伤后星形胶质细胞的性别特异性基因表达差异。方法包括对谷氨酸能和gaba能信号重要转录本进行qRT-PCR分析，并用Western或免疫细胞化学分析来确认表达差异。利用原始皮层星形胶质细胞男性与女性离体培养方法和慢性EtOH治疗范式，这些研究将更好地了解酒精对星形胶质细胞功能的具体影响，从而潜在地确定性别特异性治疗与慢性EtOH滥用相关的脑损伤的治疗靶点。

DESCRIPTION (provided by applicant): This proposal explores consequences of chronic alcohol (ethanol; EtOH) exposure and withdrawal on sex- specific astrocyte activation, expression and function. Chronic EtOH abuse is associated with neurotoxicity and reactive astrogliosis, but the mechanisms and cell types involved remain poorly characterized. Furthermore, brain damage associated with alcoholism is sexually dimorphic, yet the sex-specific effects of chronic EtOH exposure have not been fully explored. This proposal extends our previous analysis that compared males vs. females using gene expression transcriptional profiling, to characterize neuroadaptive changes that are associated with withdrawal from chronic EtOH exposure. Notably, these studies demonstrated that sex is a more powerful determinant of neuroadaptation and the transcriptional response after chronic exposure than genotype and/or withdrawal severity phenotype. Biological confirmation of gene expression differences revealed that females were more vulnerable to the resulting alcohol-induced brain damage, consistent with some (but not all) clinical studies. Among the genes significantly regulated by chronic EtOH in the prefrontal cortex are some that are predominantly or exclusively expressed by astrocytes. Furthermore, EtOH-induced brain damage is associated with excitotoxicity resulting from increased glutamate release, likely from astrocyte sources. GABA receptors are also a target of neuroadaptation to chronic EtOH abuse, and have been implicated in astrocyte function. Because astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse, investigations into the effects of chronic EtOH on astrocyte function are timely and important. Given the evidence that EtOH withdrawal differentially damages female vs. male brain, the present application examines the hypothesis that chronic alcohol exposure and withdrawal targets astrocyte function in a sex- specific fashion, resulting in changes in gene expression and function, resulting in elevated excitotoxic signaling. Aim 1 characterizes the cellular effects of chronic ethanol ex vivo on cortical astrocyte cultures from males vs. females. This aim will develop primary single sex cortical astrocyte culture methodologies to directly test the response to chronic EtOH exposure and withdrawal in male vs. female astrocyte populations in a controlled environment. This aim also characterizes astrocyte proliferation, apoptosis, glutamate uptake and release, GABAergic signaling and the contribution of calcium stores after chronic ethanol exposure and withdrawal. Aim 2 will define sex-specific gene expression differences in astrocytes after chronic EtOH insult. Methods include qRT-PCR analysis of transcripts important in glutamatergic and GABAergic signaling with Western or immunocytochemical analysis for confirmation of expression differences. Utilizing the primary cortical astrocyte male vs. female ex vivo culture methodologies and chronic EtOH treatment paradigms developed in this developmental R21 application, these studies will lead to a better understanding of the specific effects of alcohol on astrocyte function to potentially identify therapeutic targets for the sex-specific treatment of brain damage associated with chronic EtOH abuse in both males and females. PUBLIC HEALTH RELEVANCE: Chronic alcohol abuse is a major public health problem. Although many abused drugs, including alcohol, cause neurotoxicity and brain damage, the mechanisms and the contribution of specific cell types involved are poorly characterized. In addition, there are many differences between males and females with regard to EtOH behaviors and sex-specific brain damage has been reported, but there is little analysis of the pathways involved. Astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse. However, the influence of chronic alcohol exposure and withdrawal on astrocyte expression and function has not been determined. The present application utilizes sex-specific primary cortical astrocyte cell culture as an ex vivo model to elucidate how alcohol influences astrocyte viability, gene expression and function in males vs. females, with a focus on glutamatergic and GABAergic signaling. These studies will provide for a better understanding of the consequences of chronic alcohol exposure and withdrawal on astrocyte function in both males and females, and may lead to novel targets for the sex-specific treatment or amelioration of brain damage associated with chronic ethanol abuse.