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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：该提案探讨了慢性酒精（乙醇； ETOH）的后果，并在性别 - 特定的星形胶质细胞激活，表达和功能上戒断。慢性ETOH滥用与神经毒性和反应性星形胶质细胞增多有关，但所涉及的机制和细胞类型的特征仍然很差。此外，与酒精中毒相关的脑损伤是性二态性的，但尚未完全探索慢性EtOH暴露的性别特异性作用。该建议扩展了我们先前的分析，该分析使用基因表达转录分析比较了男性与女性，以表征与从慢性ETOH暴露中退出有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，性别是神经适应和慢性暴露后的转录反应更强大的决定因素。对基因表达差异的生物学确认表明，女性更容易受到酗酒引起的脑损伤的影响，这与某些（但不是全部）临床研究一致。在前额叶皮层中慢性EtOH显着调节的基因中，有些主要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有关，这可能是由于星形胶质细胞来源。 GABA受体也是神经适应的靶标，对慢性ETOH滥用，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性和突触神经传递（包括兴奋性和抑制性信号），以影响神经毒性，并且在酒精滥用的情况下观察到星形胶质细胞激活，因此对慢性ETOH对星形胶质细胞功能的影响进行了研究。鉴于有证据表明ETOH戒断差异损害了女性与男性大脑，因此目前的应用研究了以下假设：慢性酒精暴露和戒断以性别方式靶向星形胶质细胞功能，从而导致基因表达和功能变化，从而导致兴奋性毒性信号升高。 AIM 1表征了慢性乙醇离体对男性与女性皮质星形胶质细胞培养物的细胞作用。该目标将开发主要的单性皮质星形胶质细胞培养方法，以直接测试在受控环境中男性与女性星形胶质细胞种群中对慢性ETOH暴露和戒断的反应。这个目的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存储的贡献。 AIM 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差异。方法包括QRT-PCR分析在谷氨酸能和GABA能信号中重要的转录物，并通过西方或免疫细胞化学分析来确认表达差异。利用在这种发育R21应用中开发的原发性皮质星形胶质细胞男性与女性前体培养方法和慢性ETOH治疗范式，这些研究将使人们更好地了解酒精对星形胶质细胞功能的特定影响，从而潜在地鉴定出对脑部和女性遭受慢性EtoH滥用的性别损害的治疗靶标的治疗靶标。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。尽管许多滥用药物（包括酒精）会导致神经毒性和脑损伤，但所涉及的特定细胞类型的机制和贡献的表征很差。此外，关于ETOH行为和性别特异性脑损伤，男性和女性之间存在许多差异，但是对所涉及的途径的分析很少。星形胶质细胞能够调节神经元活性和突触神经传递（包括兴奋性和抑制性信号），以影响神经毒性，并在酗酒中观察到星形胶质细胞激活。但是，尚未确定慢性酒精暴露和戒断对星形胶质细胞表达和功能的影响。本应用利用性别特异性的原发性皮质星形胶质细胞培养作为离体模型，以阐明酒精如何影响星形胶质细胞生存力，男性与女性的基因表达和功能，重点是谷氨酸能和GABA能信号。这些研究将更好地了解男性和女性的慢性酒精暴露和戒断对星形胶质细胞功能的后果，并可能导致针对性特异性治疗或改善与慢性乙醇滥用相关的脑损伤的新目标。