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

项目摘要 星形胶质细胞对CNS损伤和疾病的反应是基因表达和形态的变化， 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 表达由基因Plat编码的组织型纤溶酶原激活剂（tPA），其参与脑 可塑性，脑细胞外基质的重塑和神经免疫反应，包括小胶质细胞 激活和神经炎症。tPA在几个脑区和星形胶质细胞中被乙醇上调， 几种酒精暴露模型。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发生的RNA翻译变化 （CIE-2BC）？2)CIE-2BC后星形胶质细胞核基因表达发生了什么变化？第三章 星形胶质细胞中的Plat/tPA敲低是否减少了饮酒、神经免疫反应和突触 CIE-2BC引起的变化？我们假设：CIE-2BC诱导翻译的变化， 神经免疫基因在星形胶质细胞和一些，但不是全部，翻译的变化是由变化驱动的， 转录。我们还假设，在星形胶质细胞中选择性地敲低Plat将减弱星形胶质细胞中Plat的升高。 饮酒、神经免疫基因的诱导和CIE-2BC诱导的突触变化。我们建议使用 Aldh 111-EGFP-Rpl 10a小鼠模型，其允许选择性下拉来自 星形胶质细胞的翻译核糖体亲和纯化（TRAP）方法。此外，这种小鼠品系具有GFP 在核中的荧光，允许分离星形胶质细胞特异性核的荧光激活 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中的翻译体 通过TRAP-RNA-seq.将进行路径分析， 确定受CIE-2BC影响的生物过程中的富集。我们将采用TRAP-qPCR、Western blot和Western blot。 免疫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表达的变化。 表情在具体目标2中，我们将研究女性杏仁核和PFC的核转录组， 雄性Aldh 111-EGFP-Rpl 10a小鼠在CIE-2BC后通过星形胶质细胞核的FACS分选，随后进行RNA-seq.我们 将整合转录组和翻译组数据，以识别通过转录由酒精调节的RNA- 依赖性和非转录依赖性机制。我们将采用FACS-qPCR和荧光原位 杂交（FISH）-RNAScope验证神经免疫基因表达的变化。具体目标3 将研究星形胶质细胞中tPA敲低减弱CIE-2BC诱导的 饮酒免疫反应和突触变化将诱导型Aldh 1 l1-Cre/ERT 2小鼠与floxed 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- 2BC;将评估饮酒升级、免疫应答和突触变化。

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.