The role of lncRNA Gas5 in Glucocorticoid-Mediated Ethanol Dependence Phenotypes

lncRNA Gas5在糖皮质激素介导的乙醇依赖表型中的作用

• 批准号: 10824488
• 负责人: Rachel Rice
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10824488
• 关键词: Abstinence; Adult; Alcohol consumption; Alcohol dependence; Alcohol withdrawal syndrome; Alcohols; Anhedonia; Animals; Anxiety; Behavior; Behavioral; Bilateral; Biological; Biological Assay; Brain Diseases; Brain region; CRISPR/Cas technology; Cause of Death; Cell physiology; Chronic; Chronic stress; Code; Cohort Studies; Consumption; Convulsions; Data; Dependence; Dependovirus; Development; Disease; Ethanol; Ethanol dependence; Feedback; Female; Gene Expression; Genes; Genetic Transcription; Genome; Glucocorticoid Receptor; Glucocorticoids; Goals; Growth; Guide RNA; Human; Immunoprecipitation; Impairment; Injections; Laboratories; Link; Literature; Medial; Mediating; Mediator; Mentors; Messenger RNA; Modeling; Molecular; Mus; Neurobiology; Neurons; Outcome; Pathogenesis; Pharmacotherapy; Phenotype; Physiology; Play; Prefrontal Cortex; Proteins; Public Health; RNA; Receptor Signaling; Relapse; Research; Research Training; Response Elements; Rodent; Role; Scientist; Signal Transduction; Sorting; Sucrose; System; Testing; Time; Tissues; United States; Untranslated RNA; Withdrawal; alcohol abuse therapy; alcohol behavior; alcohol exposure; alcohol use disorder; behavioral outcome; behavioral phenotyping; biobehavior; chronic alcohol ingestion; comorbidity; diagnostic criteria; disability; economic cost; glucocorticoid-induced orphan receptor; human disease; hypothalamic-pituitary-adrenal axis; knock-down; male; molecular phenotype; negative affect; neuronal growth; next generation; novel; preference; rational design; receptor binding; receptor function; response; sex; skills; socioeconomics; stressor; therapeutically effective; transcriptome; transcriptome sequencing; transcriptomics; vapor; vector

Project Summary Alcohol use disorder (AUD) is a chronic, relapsing brain disease that imposes a tremendous socioeconomic cost in the United States. Although the diagnostic criteria for AUD have been established, the underlying molecular pathogenesis is largely unknown. Prior AUD studies have demonstrated a critical role for dysregulation of glucocorticoid signaling throughout multiple brain-regions, including the medial prefrontal cortex (mPFC). Specific alterations in glucocorticoid receptor (GR) activity are important for the progression of alcohol dependence, a critical facet of AUD that contributes to escalation of alcohol consumption, withdrawal, and anxiety-like phenotypes. The overwhelming majority of GR studies for understanding AUD have focused on protein-coding genes despite less than 2% of the genome being protein-coding. Non-coding RNAs play vital roles in basic cellular functions in physiology and disease. This proposal will test a novel hypothesis that the long non-coding RNA (lncRNA) growth arrest specific 5 (Gas5) is a sex-dependent and [neuron]-specific modulator of alcohol responsive GR-dependent gene expression and behavior. Gas5 is a known repressor of GR activity and has previously been linked to AUD phenotypes in mice and humans. Our previous studies have shown that changes in the expression of Gas5, particularly in mPFC neurons, are related to chronic alcohol-induced withdrawal and escalated ethanol consumption in male mice. However, the biological mechanism(s) for these Gas5-associated changes in male animals are unknown. Additionally, no concurrent studies have been conducted in female animals to determine potential sex-dependent effects of Gas5 for chronic alcohol-induced behaviors. This project will directly study the role of mPFC neuronal Gas5 in the alcohol dependent transcriptome and behavioral phenotypes related to GR in both sexes. To accomplish this, the chronic intermittent ethanol vapor exposure (CIEV) model will be used in tandem with stereotaxic injections of an adeno-associated virus (AAV) to induce CRISPR/Cas9-mediated knockdown (KD) of Gas5 in mPFC neurons. The three aims of this study will determine the molecular and behavioral outcomes of mPFC neuron-specific Gas5 KD and CIEV. The first aim of this study uses RNA immunoprecipitation followed by real time, quantitative PCR (RIP-qPCR) to define changes in mPFC Gas5-GR binding. The second aim uses [next-generation RNA Sequencing (3’Tag- Seq) to determine neuron-specific] alterations in the GR-dependent transcriptome. The third aim employs a comprehensive battery to assay chronic ethanol exposure-related behavioral phenotypes. This project is a vital step in the functional characterization of Gas5 in AUD and how GR-dependent phenotypes emerge. The proposed studies may aid in rational development of more effective GR-mediated pharmacotherapies for AUD and co-morbid disorders (e.g., chronic stress and anxiety).

项目摘要 酒精使用障碍（AUD）是一种慢性传递的脑部疾病，不可能巨大的社会经济成本 在美国。尽管已经建立了AUD的诊断标准，但基础分子 发病机理在很大程度上未知。先前的AUD研究证明了失调的关键作用 糖皮质激素信号在多个大脑区域，包括介质前额叶皮层（MPFC）。 糖皮质激素受体（GR）活性的特异性改变对于酒精的发展很重要 依赖性，AUD的关键方面有助于升级饮酒，退出和 类似焦虑的表型。理解AUD的绝大多数GR研究都集中在 蛋白质编码基因dospite小于基因组的2％是蛋白质编码。非编码RNA播放至关重要 在生理和疾病中的基本细胞功能中的作用。该提议将检验一个新的假设，即长期 非编码RNA（LNCRNA）生长滞后特异性5（GAS5）是性别依赖性和[神经元]特异性调节剂 酒精反应性GR依赖性基因表达和行为。 Gas5是GR活性的已知复制品 并以前与小鼠和人类的AUD表型相关。我们以前的研究表明 GAS5表达的变化，特别是在MPFC神经元中，与慢性酒精诱导的 雄性小鼠的提取和升级乙醇消耗。但是，这些生物学机制 雄性动物的气5相关变化尚不清楚。另外，没有并发研究 在雌性动物中进行的，以确定慢性酒精诱导的气5的潜在性依赖性作用 行为。该项目将直接研究MPFC神经元气体5在依赖酒精转录组中的作用 和与性别中GR有关的行为表型。为此，慢性间歇性乙醇 蒸气暴露（CIEV）模型将与立体定位注射腺相关病毒同时使用 （AAV）在MPFC神经元中诱导CRISPR/CAS9介导的GAS5的敲低（KD）。这三个目标 研究将确定MPFC神经元特异性GAS5 KD和CIEV的分子和行为结果。这 这项研究的第一个目的使用RNA免疫沉淀，然后是实时，定量PCR（RIP-QPCR）到 定义MPFC GAS5-GR结合的变化。第二个目的使用[下一代RNA测序（3'tag-- Seq）确定GR依赖性转录组中神经元特异性的变化。第三个目标采用 综合电池以主张慢性乙醇暴露相关的行为表型。这个项目至关重要 介入AUD中GAS5的功能表征以及如何出现GR依赖性表型。这 拟议的研究可能有助于合理发展AUD的更有效的GR介导的药物治疗 和合并症（例如，慢性压力和动画）。