Sex dependent astrocyte activation and alcohol-induced neurotoxicity

性别依赖性星形胶质细胞激活和酒精诱导的神经毒性

• 批准号: 7936066
• 负责人: KRISTINE M. WIREN
• 金额: $ 17.47万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936066
• 关键词: Agonist; Alcohol abuse; Alcohol-Induced Neurotoxicity; Alcoholism; Alcohols; Anions; Apoptosis; Astrocytes; Biological; Brain; Brain Injuries; Calcium; Cell Culture Techniques; Chronic; Clinical Research; Coculture Techniques; Complex Mixtures; Controlled Environment; Development; Drug Exposure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Estrogens; Ethanol; Exploratory/Developmental Grant; Exposure to; Female; Future; GABA Receptor; Gender; Gene Expression; Genes; Genetic Models; Genotype; Glutamate Receptor; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Gonadal Steroid Hormones; Growth; Health; In Vitro; Intervention; Investigation; Lead; Lentivirus Vector; Mediating; Metabolism; Metabotropic Glutamate Receptors; Methodology; Methods; Microarray Analysis; Microglia; Modeling; Molecular Target; Neuraxis; Neuroglia; Neurons; Oligodendroglia; Pathway Analysis; Pathway interactions; Phenotype; Polymerase Chain Reaction; Population; Prefrontal Cortex; Prevention; Progesterone; Public Health; Publishing; Radial; Reporting; Resistance; Reverse Transcription; Severities; Sex Behavior; Signal Transduction; Signaling Molecule; Source; Synapses; Testing; Testosterone; Time; Tissue-Specific Gene Expression; Transaminases; Transcript; Transgenic Organisms; Withdrawal; alcohol effect; alcohol exposure; astrogliosis; brain tissue; cell type; cytotoxicity; drug of abuse; excitotoxicity; gamma-Aminobutyric Acid; in vivo; male; neuroadaptation; neurosteroids; neurotoxicity; neurotransmission; novel; public health relevance; receptor; release of sequestered calcium ion into cytoplasm; response; sex; synthetic enzyme; therapeutic target; uptake

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.

描述（由申请方提供）：本提案探讨了长期酒精（乙醇; EtOH）暴露和戒断对性别特异性星形胶质细胞活化、表达和功能的影响。慢性乙醇滥用与神经毒性和反应性星形胶质细胞增生有关，但所涉及的机制和细胞类型仍然缺乏表征。此外，与酒精中毒相关的脑损伤是性别二态性的，但慢性乙醇暴露的性别特异性影响尚未得到充分探讨。这一建议扩展了我们之前的分析，即使用基因表达转录谱比较男性与女性，以表征与慢性EtOH暴露戒断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的决定因素。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。在前额皮质中受慢性EtOH显著调节的基因中，有一些主要或专门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导致的兴奋性毒性相关，可能来自星形胶质细胞来源。GABA受体也是对慢性EtOH滥用的神经适应的靶点，并且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），以影响神经毒性，并且在酗酒时观察到星形胶质细胞活化，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EtOH戒断对女性与男性大脑的损害不同的证据，本申请检验了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，导致基因表达和功能的变化，导致兴奋性毒性信号传导升高的假设。目的1表征慢性乙醇离体对雄性与雌性皮质星形胶质细胞培养物的细胞效应。该目标将开发原代单一性别皮质星形胶质细胞培养方法，以直接测试受控环境中雄性与雌性星形胶质细胞群体对慢性EtOH暴露和戒断的反应。这一目标还表征了星形胶质细胞增殖、凋亡、谷氨酸摄取和释放、GABA能信号传导以及慢性乙醇暴露和戒断后钙储存的贡献。目的2将定义慢性EtOH损伤后星形胶质细胞中性别特异性基因表达的差异。方法包括qRT-PCR分析在谷氨酸能和GABA能信号传导中重要的转录物，用Western或免疫细胞化学分析确认表达差异。利用原代皮质星形胶质细胞雄性与雌性离体培养方法和在该开发性R21应用中开发的慢性EtOH处理范例，这些研究将导致更好地理解酒精对星形胶质细胞功能的特定影响，以潜在地确定男性和女性中与慢性EtOH滥用相关的脑损伤的性别特异性治疗的治疗靶点。 公共卫生相关性：慢性酒精滥用是一个主要的公共卫生问题。虽然许多滥用药物，包括酒精，会导致神经毒性和脑损伤，但其机制和所涉及的特定细胞类型的贡献还很不清楚。此外，男性和女性之间在乙醇行为方面存在许多差异，并且已经报道了性别特异性脑损伤，但很少分析所涉及的途径。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）以影响神经毒性，并且在酒精滥用时观察到星形胶质细胞活化。然而，长期酒精暴露和戒断对星形胶质细胞表达和功能的影响尚未确定。本申请利用性别特异性原代皮质星形胶质细胞培养物作为离体模型，以阐明酒精如何影响雄性与雌性中的星形胶质细胞活力、基因表达和功能，重点是谷氨酸能和GABA能信号传导。这些研究将提供一个更好的理解的后果，慢性酒精暴露和退出对星形胶质细胞功能的男性和女性，并可能导致新的目标性别特异性治疗或改善与慢性酒精滥用相关的脑损伤。