RNA splicing regulation during alcohol withdrawal

酒精戒断过程中的 RNA 剪接调节

• 批准号: 10785159
• 负责人: Luana Martins Carvalho
• 金额: $ 14.97万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10785159
• 关键词: Affect; Alcohol consumption; Alcohol withdrawal syndrome; Alcohols; Alternative Splicing; Antibodies; Anxiety; Automobile Driving; Autopsy; Award; Behavior; Behavioral; Big Data; Binding; Binding Proteins; Bioinformatics; Brain; Brain region; Cell physiology; Chronic; Complementary DNA; Complex; Darkness; Data; Data Science; Development; Diagnosis; Dorsal; Environment; Ethanol; Ethics; Exclusion; Exons; Extracellular Matrix Proteins; Future; Gene Delivery; Gene Expression; Genes; Genetic; Goals; Grant; Heavy Drinking; High-Throughput Nucleotide Sequencing; Hippocampus; Human; Immune response; Immunoprecipitation; Individual; Knowledge; Leadership; Learning; Length; Light; Mediating; Mental Depression; Messenger RNA; Molecular; Morphogenesis; Nature; Neural Conduction; Neurons; Oral; Phase; Procedures; Process; Production; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Rattus; Regulation; Research; Research Personnel; Ribonucleoproteins; Role; Signal Transduction; Site; Spliceosomes; Sucrose; Techniques; Testing; Training; Transcript; Translational Research; Untranslated RNA; Variant; Viral; Viral Vector; Withdrawal; Work; Writing; alcohol behavior; alcohol exposure; alcohol use disorder; anxiety-like behavior; behavior test; career development; chronic alcohol ingestion; design; drinking behavior; experience; genetic manipulation; innovation; insight; knock-down; loss of function; mRNA Expression; male; negative affect; neuroadaptation; neurogenesis; neuroinflammation; neurotransmission; next generation sequencing; novel; overexpression; preference; research and development; skills; small hairpin RNA; success; theories; tool; transcriptome sequencing

Project Summary/Abstract One way in which chronic alcohol exposure produces neuronal adaptations is by changing gene expression. These changes can influence alcohol-drinking behavior and may lead to the development of alcohol use disorder (AUD). Additionally, during alcohol withdrawal, gene expression changes can contribute to the development of negative affective states, such as anxiety and depression, which makes it challenging for individuals to stop consuming alcohol. Mounting evidence from many species indicates that chronic a lcohol exposure also leads to alternatively spliced transcripts in different brain regions. Yet the molecular mechanisms by which alcohol alters RNA splicing remains unknown. This K99/R00 award includes a comprehensive career development and research plan based on Dr. Luana Carvalho’s preliminary data showing that withdrawal from chronic alcohol exposure in male rats increases the expression of genes encoding components of the RNA splicing machinery and leads to changes in RNA splicing. My preliminary data shows increased expression of the splicing factor Poly r(C) binding protein (PCBP1) in the hippocampus (HPC) of ethanol withdrawn rats that present with anxiety and depression-like behavior, as well as in the postmortem HPC of subjects diagnosed with AUD. I also found that PCBP1 is implicated in the mis-splicing of the Hapln2 gene, an important regulator of neuronal conductivity in which loss of function could negatively impact neurotransmission in the context of alcohol use. The scientific goal of this K99/R00 is to investigate RNA splicing, with a focus on PCBP1, as a mechanism by which chronic alcohol exposure and withdrawal leads to molecular alterations and contributes to the emergence of negative affective states. I will manipulate PCBP1 expression using viral-mediated gene delivery to test its behavioral relevance during alcohol withdrawal. I will also perform RNA immunoprecipitation with a PCBP1 antibody, followed by next generation sequencing to identify PCBP1-targets. Finally, I will perform full-length transcriptome sequencing to identify the portfolio of alternatively spliced transcripts in the HPC during chronic alcohol exposure and withdrawal. During my K99 phase, I will gain additional technical training in cutting-edge molecular, bioinformatic and statistical approaches. I will also enhance my leadership skills and receive considerable training in grant writing, oral presentations, and ethics that will be crucial to my success as an independent researcher. During the R00 phase, I will apply all training received to continue this project and further explorer PCBP1 targets in the HPC of humans diagnosed with AUD. Collectively, this work will provide insights into the molecular mechanisms underlying alcohol withdrawal-induced changes on RNA splicing and negative affective states, reveal novel targets and testable hypothesis for future functional studies, and facilitate translational research for finding new targets for AUD treatment.

项目摘要/摘要 慢性酒精暴露会产生神经元适应的一种方法是改变基因表达。 这些变化会影响酒精饮用的行为，并可能导致饮酒的发展 障碍（AUD）。此外，在戒酒期间，基因表达变化可能有助于 焦虑和抑郁等负面情感状态的发展，这使其挑战 个人停止饮酒。来自许多物种的越来越多的证据表明慢性lcohol 暴露还导致不同大脑区域的剪接转录本。但是分子 酒精改变RNA剪接的机制仍然未知。该K99/R00奖项包括 基于Luana Carvalho博士的初步数据的全面职业发展和研究计划 表明在雄性大鼠中退出慢性酒精暴露会增加基因的表达 编码RNA剪接机械的组件并导致RNA剪接变化。我的初步 数据显示，海马中的剪接因子poly R（C）结合蛋白（PCBP1）的表达增加 乙醇的（HPC）拔出了具有动画和抑郁行为的大鼠，以及 被诊断为AUD的受试者的后HPC。我还发现在错误的拼写中暗示了PCBP1 hapln2基因是神经元电导率的重要调节因子，其中功能损失可能负面 在饮酒的背景下影响神经传递。该K99/R00的科学目标是调查 RNA剪接，重点是PCBP1，作为一种慢性酒精暴露和戒断的机制 导致分子改变，并导致负面情感状态的出现。我会操纵 使用病毒介导的基因递送的PCBP1表达在酒精期间测试其行为相关性 提取。我还将使用PCBP1抗体进行RNA免疫沉淀，然后是下一代 测序以识别PCBP1目标。最后，我将执行全长转录组测序以识别 在慢性酒精暴露和退出期间，HPC中剪接的转录本的投资组合。 在我的K99阶段，我将在尖端分子，生物信息学和 统计方法。我还将提高我的领导能力，并接受Grant的考虑培训 写作，口头演示和道德规范对我作为独立研究人员的成功至关重要。 在R00阶段，我将应用所有收到的培训来继续此项目，并进一步探索器PCBP1 人类HPC中的目标被诊断为AUD。总的来说，这项工作将提供有关 酒精戒断诱导的RNA剪接变化和阴性的分子机制 情感状态，揭示新的目标和可检验的假设，用于未来的功能研究，并促进 转化研究，用于寻找新的AUD治疗目标。