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.

项目总结/摘要 慢性酒精暴露产生神经适应的一种方式是改变基因表达。 这些变化会影响饮酒行为，并可能导致酒精使用的发展 疾病（AUD）。此外，在戒酒期间，基因表达变化可能会导致 消极情感状态的发展，如焦虑和抑郁，这使得它具有挑战性， 个人停止饮酒。来自许多物种的越来越多的证据表明，慢性酒精 暴露还导致不同脑区域中的选择性剪接转录物。然而， 酒精改变RNA剪接的机制仍然未知。此K99/R 00奖项包括 根据Luana Carvalho博士的初步数据制定的全面职业发展和研究计划 表明雄性大鼠从长期酒精暴露中戒断会增加基因的表达， RNA剪接机器的编码组分，并导致RNA剪接的变化。我的初步 数据显示海马中剪接因子Poly r（C）结合蛋白（PCBP 1）的表达增加 (HPC)酒精戒断大鼠表现出焦虑和抑郁样行为， 诊断为AUD的受试者的尸检HPC。我还发现PCBP 1参与了 Hapln 2基因是神经传导性的重要调节因子，功能丧失可能会对神经传导性产生负面影响。 在酒精使用的情况下影响神经传递。K99/R 00的科学目标是研究 RNA剪接，重点是PCBP 1，作为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PCBP 1表达以测试其在酒精期间的行为相关性 戒断我还将用PCBP 1抗体进行RNA免疫沉淀，然后用下一代 测序以鉴定PCBP 1靶。最后，我将进行全长转录组测序， 在慢性酒精暴露和戒断过程中HPC中的选择性剪接转录物组合。 在我的K99阶段，我将获得额外的技术培训，在尖端分子，生物信息学， 统计方法。我也将提高我的领导能力，并在格兰特接受相当多的培训 写作，口头报告，和道德，这将是至关重要的，我作为一个独立的研究人员的成功。 在R 00阶段，我将应用所有收到的培训，继续这个项目，并进一步探索PCBP 1 被诊断患有AUD的人的HPC中的靶点。总的来说，这项工作将提供深入了解 酒精戒断引起的RNA剪接和负性变化的分子机制 情感状态，为未来的功能研究揭示了新的目标和可检验的假设，并促进 转化研究，寻找治疗AUD的新靶点。