6/11 Astrocyte-specific changes and interventions in alcohol dependence

6/11 星形胶质细胞特异性变化和酒精依赖干预

• 批准号: 10409263
• 负责人: Marina Guizzetti
• 金额: $ 37.78万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10409263
• 关键词: Affect; Affinity Chromatography; Alcohol consumption; Alcohol dependence; Alcohols; Alteplase; Amygdaloid structure; Animal Model; Area; Astrocytes; Attenuated; Automobile Driving; Biological Process; Brain; Brain region; Cell Nucleus; Cell Separation; Chronic; Chronic Disease; Clinical; Data; Development; Disease; Down-Regulation; Ethanol; Extracellular Matrix; Female; Fluorescence; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic Engineering; Genetic Transcription; Heterogeneity; Immune response; Immunohistochemistry; Inflammation; Intervention; Lead; LoxP-flanked allele; Methods; Mission; Modeling; Molecular; Molecular Target; Morphology; Mus; National Institute on Alcohol Abuse and Alcoholism; Neuroimmune; Nuclear; Nuclear RNA; PLAT gene; Pathway Analysis; Phenotype; Plasminogen Activator; Prefrontal Cortex; Procedures; Protein Biosynthesis; Proteins; Proxy; Public Health; RNA; Research; Ribosomal Proteins; Ribosomes; Sorting - Cell Movement; Specificity; Synapses; Technology; Therapeutic; Therapeutic Intervention; Translating; Translations; Western Blotting; alcohol exposure; alcohol use disorder; brain cell; brain remodeling; cell type; cellular targeting; drinking; immune activation; insight; knock-down; male; mouse model; neuroinflammation; novel strategies; promoter; protein expression; response; sex; transcriptome; transcriptome sequencing; translatome

PROJECT SUMMARY Astrocytes respond to CNS damage and disease with changes in gene expression and morphology and with immune activation to become reactive astrocytes. Alcohol Use Disorder is characterized in part by neuroimmune responses that increase with the progression of the disorder. Reactive astrocytes upregulate the expression of Tissue-type Plasminogen Activator (tPA) encoded by the gene Plat, which is involved in brain plasticity, the remodeling of the brain extracellular matrix, and neuroimmune responses including microglial activation and neuroinflammation. tPA is upregulated by ethanol in several brain areas and in astrocytes in several models of ethanol exposure. The main scientific questions driving the proposed studies are: 1) What are the changes in translating RNA occurring in astrocytes after Chronic Intermittent Ethanol-2 Bottle Choice (CIE-2BC)? 2) What are the changes in nuclear gene expression occurring in astrocytes after CIE-2BC? 3) Does Plat/tPA knock-down in astrocytes reduce escalation in drinking, neuroimmune responses, and synaptic changes induced by CIE-2BC? We hypothesize that: CIE-2BC induces changes in the translation of neuroimmune genes in astrocytes and that some, but not all, changes in translation are driven by changes in transcription. We also hypothesize that knocking down Plat selectively in astrocytes will attenuate escalation in drinking, induction of neuroimmune genes, and synaptic changes induced by CIE-2BC. We propose to use the Aldh1l1-EGFP-Rpl10a mouse model that allows the selective pull down of actively translating RNA from astrocytes by the translating ribosome affinity purification (TRAP) method. Moreover, this mouse line has GFP fluorescence in the nucleus that allows for the isolation of astrocyte-specific nuclei by Fluorescent-Activated Cell Sorting (FACS). In Specific Aim 1 we will study the translatome in amygdala and PFC of female and male Aldh1l1-EGFP-Rpl10a mice after CIE-2BC by TRAP-RNA-seq. Pathway analysis will be performed to determine enrichment in the biological processes affected by CIE-2BC. We will employ TRAP-qPCR, Western blot, and immunohistochemistry (IHC) to validate changes in neuroimmune gene translation and protein expression. In Specific Aim 2 we will study the nuclear transcriptome in the amygdala and PFC of female and male Aldh1l1-EGFP-Rpl10a mice after CIE-2BC by FACS sorting of astrocyte nuclei followed by RNA-seq. We will integrate transcriptome and translatome data to identify RNAs regulated by alcohol through transcription- dependent and transcription-independent mechanisms. We will employ FACS-qPCR and Fluorescence In Situ Hybridization (FISH)-RNAScope to validate changes in neuroimmune gene expression. In Specific Aim 3 we will investigate the hypothesis that tPA knock-down in astrocytes attenuates CIE-2BC-induced escalation of drinking, immune response, and synaptic changes. Inducible Aldh1l1-Cre/ERT2 mice will be crossed to floxed Plat mice for the selective knock down of Plat in astrocytes. Mice lacking tPA in astrocytes will undergo CIE- 2BC; drinking escalation, immune response, and synaptic changes will be assessed.

项目摘要 星形胶质细胞对中枢神经系统的损伤和疾病反应，基因表达和形态的变化以及 免疫激活成为反应性星形胶质细胞。饮酒障碍的一部分是 随着疾病进展而增加的神经免疫反应。反应性星形胶质细胞上调 由基因PLAP编码的组织型纤溶酶原活化剂（TPA）的表达，该基因参与脑 可塑性，大脑外基质的重塑以及包括小胶质细胞的神经免疫反应 激活和神经炎症。 TPA在几个大脑区域和星形胶质细胞中被乙醇上调 乙醇暴露的几种模型。推动拟议研究的主要科学问题是：1） 慢性间歇性乙醇2瓶选择后，在星形胶质细胞中翻译RNA的变化是否发生 （CIE-2BC）？ 2）在CIE-2BC之后，星形胶质细胞中发生了核基因表达的变化？ 3） 星形胶质细胞中的平台/TPA是否会减少饮酒的升级，神经免疫反应和突触 CIE-2BC引起的变化？我们假设：CIE-2BC引起了翻译的变化 星形胶质细胞中的神经免疫基因，有些但不是全部，翻译的变化是由变化驱动的 转录。我们还假设，在星形胶质细胞中有选择地击倒平台将减轻升级 饮酒，诱导神经免疫基因以及CIE-2BC诱导的突触变化。我们建议使用 AldH1L1-EGFP-RPL10A鼠标模型，可以选择性地向下拉下主动翻译RNA 通过翻译核糖体亲和力纯化（TRAP）方法的星形胶质细胞。此外，该鼠标线具有GFP 核中的荧光，可以通过荧光激活分离星形胶质细胞特异性核 细胞分类（FACS）。在特定目标1中，我们将研究杏仁核和男性的PFC中的翻译组 CIE-2BC之后，AldH1L1-EGFP-RPL10A小鼠由TRAP-RNA-SEQ。途径分析将进行 确定在受CIE-2BC影响的生物过程中的富集。我们将采用西方陷阱-QPCR 印迹和免疫组织化学（IHC）以验证神经免疫基因翻译和蛋白质的变化 表达。在特定目的2中，我们将研究女性杏仁核和PFC的核转录组和 CIE-2BC之后的雄性AldH1L1-EGFP-RPL10A小鼠通过FACS对星形胶质细胞核进行排序，然后是RNA-Seq。我们 将集成转录组和翻译数据，以识别通过转录调节酒精调节的RNA- 依赖和转录无关的机制。我们将使用FACS-QPCR和原位荧光 杂交（FISH） - rnaScope以验证神经免疫基因表达的变化。在特定目标3中我们 将调查以下假设：星形胶质细胞中的TPA敲低可减轻CIE-2BC诱导的升级 饮酒，免疫反应和突触变化。诱导型ALDH1L1-CRE/ERT2小鼠将被杂交 柏拉图小鼠在星形胶质细胞中的选择性敲低。在星形胶质细胞中缺乏TPA的小鼠将经历CIE- 2BC;将评估饮酒升级，免疫反应和突触变化。