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

项目总结 星形胶质细胞对中枢神经系统损伤和疾病的反应是通过基因表达和形态的变化以及与 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 脑相关基因Plat编码的组织型纤溶酶原激活物的表达 可塑性、脑细胞外基质的重塑和包括小胶质细胞在内的神经免疫反应 激活和神经炎症。乙醇使TPA在几个脑区和星形胶质细胞中表达上调 酒精暴露的几种模型。推动这项研究的主要科学问题是：1)什么 慢性间歇性选择酒精2瓶后星形胶质细胞翻译RNA的变化 (CIE-2BC)？2)CIE-2BC后星形胶质细胞核基因表达发生了什么变化？ 星形胶质细胞中Plat/tPA的下调是否可以减少饮酒、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导致了翻译的变化 星形胶质细胞中的神经免疫基因以及翻译中的一些(但不是全部)变化是由 抄写。我们还假设，选择性地敲除星形胶质细胞中的Plat将减缓星形胶质细胞的升级 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，允许选择性地从 翻译核糖体亲和纯化(TRAP)方法获得星形胶质细胞。此外，该鼠标系具有绿色荧光蛋白。 核中的荧光，允许通过荧光激活来分离星形胶质细胞特异的核 细胞分选(FACS)。在特定的目标1中，我们将研究杏仁核和雌性和雄性的PFC的翻译组。 ALDH1L1-EGFP-Rpl10a小鼠接种CIE-2BC后，用TRAP-RNA-seq.将执行路径分析以 确定受CIE-2BC影响的生物过程中的浓缩。我们将采用TRAP-QPCR，WESTERN 印迹和免疫组织化学(IHC)验证神经免疫基因翻译和蛋白的变化 表情。在特定目标2中，我们将研究雌性和雌性杏仁核和PFC中的核转录组。 雄性ALDH1L1-EGFP-Rpl10a小鼠CIE-2BC后，用流式细胞仪分选星形胶质细胞核，然后进行RNA-seq。我们 将整合转录组和翻译组数据，通过转录识别受酒精调控的RNA- 依赖和转录不依赖的机制。我们将采用FACS-qPCR和荧光原位检测技术 杂交(FISH)-RNAScope验证神经免疫基因表达的变化。在具体目标3中，我们 将探讨星形胶质细胞下调tPA可减轻CIE-2BC诱导的血管紧张素转换酶升高的假说。 饮酒、免疫反应和突触变化。可诱导的ALDH1L1-Cre/ERT2小鼠将被杂交到牙线 Plat小鼠对星形胶质细胞Plat的选择性敲除。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。

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.