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).

项目概要 酒精使用障碍 (AUD) 是一种慢性、复发性脑部疾病，会造成巨大的损失 在美国，虽然已经制定了 AUD 的诊断标准，但其基本的分子水平仍然存在。 之前的 AUD 研究已经证明，AUD 的发病机制在很大程度上是未知的。 糖皮质激素信号传导遍及多个大脑区域，包括内侧前额皮质 (mPFC)。 糖皮质激素受体（GR）活性的特定改变对于酒精的进展很重要 依赖性，澳元的一个关键方面，导致酒精消费、戒断和酗酒的升级 绝大多数用于理解 AUD 的 GR 研究都集中在焦虑样表型上。 尽管只有不到 2% 的基因组是蛋白质编码基因，但蛋白质编码基因却发挥着至关重要的作用。 该提案将检验一个新的假设，即长期的作用。 非编码 RNA (lncRNA) 生长停滞特异性 5 (Gas5) 是一种性别依赖性和[神经元]特异性调节剂 Gas5 是一种已知的 GR 活性抑制因子。 我们之前的研究表明，它与小鼠和人类的 AUD 表型有关。 Gas5 表达的变化，特别是 mPFC 神经元中的变化，与酒精慢性诱导的 雄性小鼠戒断和乙醇消耗增加然而，这些的生物学机制。 此外，雄性动物中与 Gas5 相关的变化尚不清楚，也没有同时进行的研究。 在雌性动物中进行，以确定 Gas5 对慢性酒精诱发的潜在性别依赖性影响 该项目将直接研究 mPFC 神经元 Gas5 在酒精依赖性转录组中的作用。 以及与两性 GR 相关的行为表型 为了实现这一目标，慢性间歇性乙醇。 蒸气暴露（CIEV）模型将与腺相关病毒的立体定位注射同时使用 (AAV) 诱导 mPFC 神经元中 Gas5 的 CRISPR/Cas9 介导的敲低 (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。 本研究的第一个目标是使用 RNA 免疫沉淀，然后进行实时定量 PCR (RIP-qPCR) 定义 mPFC Gas5-GR 结合的变化第二个目标使用[下一代 RNA 测序（3'Tag-）。 Seq）以确定 GR 依赖性转录组中神经元特异性的改变。 用于测定慢性乙醇暴露相关行为表型的综合电池该项目是一个至关重要的项目。 AUD 中 Gas5 功能表征的步骤以及 GR 依赖性表型如何出现。 拟议的研究可能有助于合理开发更有效的 GR 介导的 AUD 药物疗法 和共病（例如慢性压力和焦虑）。