Brain region- and cell-specific roles of CRF-binding protein in alcohol use disorders

酒精消费是世界上第三大残疾和疾病风险因素。酗酒有 与许多不利的社会和健康后果有关，包括过渡到 酒精依赖压力是酒精使用的一个关键环境因素，并与酒精使用有关。 在临床和临床前模型中，中枢神经系统应激调节的关键是促肾上腺皮质激素释放 CRF在调节狂饮（BD）和依赖中起重要作用。通用报告格式 系统包括两种不同的GPCR，即CRF受体1（CRF-R1）和CRF受体2（CRF-R2），以及一种分泌的 CRF结合蛋白（CRF-BP），以等于或大于受体的亲和力结合CRF。而 CRF和CRF-R1与AUDs明显相关，CRF-R1拮抗剂在临床上无效 审判引人注目的是，CRHBP单核苷酸多态性与BD和依赖性相关，CRF-BP表达与BD和依赖性相关。 在BD中调节，啮齿动物临床前研究表明，阻断VTA中的CRF-BP活性可能会降低 BD.然而，CRF-BP在应激/奖赏通路中的功能作用及其对CRF受体的调节 在体内的活性还没有很好地理解。CRF-BP在不同脑区的具体作用 暴饮暴食和大量饮酒可能有助于发现新的治疗方法来减少BD， 防止升级为依赖。分泌的CRF-BP抑制CRH在CRF-R1的活性，而CRF-BP出现 增强VTA中的CRF-R2信号传导;其他研究表明，细胞内CRF-BP充当护送蛋白， 对于CRF-R2。我们认为CRF-BP可能以脑区域或细胞类型特异性的方式发挥多种作用， CRF受体亚型作为潜在的决定因素。在本R21提案中，我们将重点关注两个关键的交互 压力/奖励通路中CRF-BP/CRF受体共表达的位点，前额叶皮层（mPFC） 腹侧被盖区（VTA）。使用药理学（CRF 6 -33和受体拮抗剂）的组合， 病毒和遗传工具，我们将比较CRF-BP在细胞类型中的细胞内和细胞外作用 在mPFC和VTA中正常表达CRF-BP。我们将利用在黑暗中饮酒（DID）和慢性 间歇性接触酒精（IAA）模型，以评估CRF-BP在从BD向BD过渡过程中的作用。 依赖性饮酒将研究雄性和雌性小鼠的CRF性别特异性变化， CRF-R2的表达最近在BD中有报道。我们将研究CRF-BP在mPFC中的作用（目标1） 和VTA（Aim 2），使用细胞外CRF-BP拮抗剂（CRF 6 -33）、病毒CRF-BP敲低和cre- 在mPFC sst表达细胞（Aim 1C）和VTA TH表达细胞中CRF-BP依赖性过表达 神经元（Aim 2C）。我们假设CRF-BP在PFC中对CRF-R1具有很大的抑制作用，而CRF-BP 在VTA中可能分别通过CRF-R1和CRF-R2发挥抑制和增强作用。结果从这个 该项目将促进我们对CRF-BP的脑区域和细胞特异性作用的理解，以及 表征CRF-BP拮抗剂作为治疗AUD的新型治疗靶点的潜力。

Alcohol consumption is the world’s third largest risk factor for disability and disease. Binge alcohol drinking has been linked to many adverse social and health consequences, including an increased risk of transitioning to alcohol dependence. Stress is a key environmental factor in alcohol use and has been linked to alcohol use disorders (AUDs) in clinical and preclinical models. The key CNS regulator of stress is corticotropin-releasing factor (CRF), and CRF plays an important role in modulating binge drinking (BD) and dependence. The CRF system includes two distinct GPCRs, CRF receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2), and a secreted CRF-binding protein (CRF-BP) that binds CRF with an affinity equal to or greater than the receptors. While CRF and CRF-R1 have been clearly implicated in AUDs, CRF-R1 antagonists have been ineffective in clinical trials. Strikingly, CRHBP SNPs have been associated with BD and dependence, CRF-BP expression is regulated in BD, and rodent pre-clinical studies suggest that blocking CRF-BP activity in VTA may decrease BD. Yet, the functional roles of CRF-BP within the stress/reward pathways and its modulation of CRF receptor activity in vivo are not well understood. Dissecting the specific roles of CRF-BP in different brain regions in binge and heavy alcohol drinking may enable the discovery of new therapeutic treatments to reduce BD and prevent escalation to dependence. Secreted CRF-BP inhibits CRH activity at CRF-R1, while CRF-BP appears to enhance CRF-R2 signaling in VTA; other studies suggest that intracellular CRF-BP acts as an escort protein for CRF-R2. We propose that CRF-BP plays multiple roles, likely in a brain region or cell-type specific manner, with CRF receptor subtype as a potential determinant. In this R21 proposal, we will focus on 2 key interacting sites of CRF-BP/CRF receptor co-expression within the stress/reward pathways, the prefrontal cortex (mPFC) and ventral tegmental area (VTA). Using a combination of pharmacological (CRF6-33 and receptor antagonists), viral, and genetic tools, we will compare the intracellular and extracellular roles of the CRF-BP in the cell types normally expressing CRF-BP in the mPFC and VTA. We will utilize the drinking in the dark (DID) and chronic intermittent access to alcohol (IAA) models to assess the role of CRF-BP across the transition from BD to dependence-like drinking. Both male and female mice will be studied as sex-specific changes in CRF and CRF-R2 expression have recently been reported in BD. We will examine the role of CRF-BP in mPFC (Aim 1) and VTA (Aim 2) using an extracellular CRF-BP antagonist (CRF6-33), viral CRF-BP knockdown, and cre- dependent overexpression of CRF-BP in mPFC sst-expressing cells (Aim 1C) and VTA TH-expressing neurons (Aim 2C). We hypothesize that CRF-BP has largely inhibitory roles at CRF-R1 in PFC, while CRF-BP in VTA may have both inhibitory and enhancing roles via CRF-R1 and CRF-R2 respectively. Results from this project will advance our understanding of brain region and cell-specific roles of CRF-BP, as well as characterize the potential for CRF-BP antagonists as a novel therapeutic target for treatment of AUDs.