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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要星形胶质细胞通过基因表达和形态的变化来响应中枢神经系统损伤和疾病，并通过免疫激活成为反应性星形胶质细胞。酒精使用障碍的部分特征是神经免疫反应随着疾病的进展而增加。反应性星形胶质细胞上调 Plat 基因编码的组织型纤溶酶原激活剂 (tPA) 的表达，该基因参与大脑可塑性、大脑细胞外基质的重塑以及包括小胶质细胞在内的神经免疫反应激活和神经炎症。乙醇在多个大脑区域和星形胶质细胞中上调 tPA 乙醇暴露的几种模型。推动拟议研究的主要科学问题是：1）什么慢性间歇性乙醇2瓶选择后星形胶质细胞中翻译RNA发生的变化是什么（CIE-2BC）？ 2）CIE-2BC后星形胶质细胞核基因表达发生了哪些变化？ 3）星形胶质细胞中 Plat/tPA 敲低是否会减少饮酒、神经免疫反应和突触的升级 CIE-2BC引起的变化？我们假设：CIE-2BC 会引起翻译的变化星形胶质细胞中的神经免疫基因，一些但不是全部的翻译变化是由转录。我们还假设选择性地敲低星形胶质细胞中的 Plat 将减弱星形胶质细胞中的升级饮酒、神经免疫基因的诱导以及 CIE-2BC 诱导的突触变化。我们建议使用 Aldh1l1-EGFP-Rpl10a 小鼠模型，允许选择性下拉主动翻译的 RNA 通过翻译核糖体亲和纯化（TRAP）方法分离星形胶质细胞。此外，该小鼠系具有 GFP 细胞核中的荧光，允许通过荧光激活分离星形胶质细胞特异性细胞核细胞分选（FACS）。在具体目标 1 中，我们将研究女性和男性杏仁核和 PFC 中的翻译组通过 TRAP-RNA-seq 检测 CIE-2BC 后的 Aldh1l1-EGFP-Rpl10a 小鼠。将进行路径分析确定受 CIE-2BC 影响的生物过程的富集。我们将采用 TRAP-qPCR、Western 印迹和免疫组织化学 (IHC) 来验证神经免疫基因翻译和蛋白质的变化表达。在具体目标 2 中，我们将研究女性和女性杏仁核和 PFC 中的核转录组。 CIE-2BC 后的雄性 Aldh1l1-EGFP-Rpl10a 小鼠通过星形胶质细胞核的 FACS 分选，然后进行 RNA 测序。我们将整合转录组和翻译组数据，通过转录识别受酒精调节的 RNA 依赖性和转录非依赖性机制。我们将采用 FACS-qPCR 和原位荧光杂交 (FISH)-RNAScope 用于验证神经免疫基因表达的变化。在具体目标 3 中，我们将研究以下假设：星形胶质细胞中的 tPA 敲除会减弱 CIE-2BC 诱导的饮酒、免疫反应和突触变化。诱导型 Aldh1l1-Cre/ERT2 小鼠将与 floxed 杂交 Plat 小鼠用于选择性敲低星形胶质细胞中的 Plat。星形胶质细胞中缺乏 tPA 的小鼠将经历 CIE- 公元前2年；将评估饮酒增加、免疫反应和突触变化。