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Astrocyte gene expression and translation in an in vivo FASD mouse model

体内 FASD 小鼠模型中的星形胶质细胞基因表达和翻译

基本信息

批准号：
10285484
负责人：
Marina Guizzetti
金额：
$ 32.6万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10285484
关键词：
Address Adult Affect Affinity Chromatography Alcohols Animal Model Astrocytes Behavior Behavioral Biological Process Brain Brain region Cell Nucleus Cell Separation Cells Clinical Data Development Disease Disease Outcome Ethanol Extracellular Matrix Extracellular Matrix Proteins Female Fetal Alcohol Exposure Fetal Alcohol Spectrum Disorder Fluorescence Fluorescent in Situ Hybridization Functional disorder Gene Expression Genes Genetic Transcription Heterogeneity Hippocampus (Brain)Immunohistochemistry Inflammatory Response Knowledge Lead Mediating Mental disorders Messenger RNA Methods Mission Molecular Molecular Target Mus NIH Program Announcements National Institute on Alcohol Abuse and Alcoholism Neonatal Alcohol Exposure Nuclear Nuclear RNA Pathway Analysis Play Prefrontal Cortex Process Proteins Public Health Publishing RNA Reporting Research Ribosomal Proteins Ribosomes Role Sorting - Cell Movement Specificity Synapses System Therapeutic Intervention Translating Translations United States National Institutes of Health Western Blotting alcohol effect alcohol exposure base brain cell cell type cellular targeting disability fetal in vivo insight interest male mouse model neuron development new technology novel strategies promoter response sex synaptogenesis transcriptome transcriptome sequencing translatome

项目摘要

PROJECT SUMMARY The effects of developmental alcohol exposure on astrocytes remains largely unknown, despite the extensive and growing evidence of the roles played by these cells both in the developing and adult brain. This gap in knowledge is due in part to the challenges of studying these cells in vivo. We propose to employ new technologies allowing the study of astrocytes in vivo to gain mechanistic insights into astrocytic functions altered by developmental alcohol exposure to advance the pace of our discoveries of the roles played by astrocytes in Fetal Alcohol Spectrum Disorders (FASD). We propose to use the Aldh1l1-EGFP-Rpl10a mice that allow for the selective pull down of actively translating RNA from astrocytes by the translating ribosome affinity purification (TRAP) method and for the isolation of astrocyte-specific nuclei by Fluorescent-Activated Cell Sorting (FACS). Hence, this system allows to analyze changes in both astrocyte-specific nuclear RNA expression and astrocyte-specific RNA translation by RNA-seq. We hypothesize that neonatal alcohol exposure induces extensive changes in the translation of genes involved in several astrocyte-mediated processes in vivo and on molecular stuctures mainly contributed by astrocytes, such as the extracellular matrix (ECM) that modulate some of these processes. We also hypothesize that changes in translation are in part driven by changes in transcription and in part independent from transcription. Additionally, the proposed studies will assess astrocyte heterogeneity in their response to developmental alcohol exposure across developmental stages, sexes, and brain regions. The cell type-specificity of the proposed studies will help to disentangle astrocyte function and dysfunction in FASD from contributions of other cell types. We are particularly interested in alterations involving the ECM as we have reported that several proteins of the ECM play important roles in neuronal development and are dysregulated by ethanol. We expect the results of our proposed Aims to be very impactful to the FASD field. We will study the prefrontal cortex (PFC) and hippocampus (HPC) of developing (PD7) and adult (PD90) female and male Aldh1l1-EGFP-Rpl10a mice. Aim 1: To identify changes in the astrocyte nuclear transcriptome induced by neonatal ethanol exposure by FACS sorting of astrocyte nuclei followed by RNA-seq and pathway analysis. Aim 2: To identify changes in the astrocyte translatome induced by neonatal ethanol exposure by TRAP-RNA-seq and integrate these findings with transcriptome data. Aim 3: To explore the dysregulation of the astrocyte ECM network that underlies some of the developmental effects of ethanol by TRAP-qPCR, Fluorescence In Situ Hybridization (FISH)-RNAscope, Western blot, and immunohistochemistry to validate at both mRNA and protein levels ethanol-induced changes in ECM proteins. The proposed studies address NIH/NIAA priorities as they will provide mechanistic insights into astrocyte functions altered by developmental alcohol exposure in the developing and adult brain which are likely involved in behavioral abnormalities and mental illnesses developed by adults with FASD.

项目总结