Establishing the Molecular Mechanisms of Reduced Ethanol Drinking in Calcineurin-Mediated Immunosuppression Treated Rodents

建立钙调神经磷酸酶介导的免疫抑制治疗的啮齿动物减少乙醇饮酒的分子机制

• 批准号: 10477241
• 负责人: THOMAS P BERESFORD
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477241
• 关键词: Abstinence; Address; Admission activity; Affect; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; American; Americas; Anatomy; Brain; Calcineurin; Calcineurin inhibitor; Caring; Chemicals; Consumption; Cyclosporine; DSM-V; Data; Diagnosis; Enzyme-Linked Immunosorbent Assay; Ethanol; FK506; Family; Flow Cytometry; Frequencies; Functional Magnetic Resonance Imaging; Future; General Population; Genetic; Glutamates; Goals; Health; Health care facility; Human; Immune; Immune system; Immunohistochemistry; Immunosuppression; Immunosuppressive Agents; Injections; Inpatients; International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10); Intervention; Knock-out; Knockout Mice; Knowledge; Lead; LoxP-flanked allele; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Mediating; Medical; Medicine; Mental Depression; Metabolic; Metabolism; Microglia; Microinjections; Mitochondria; Modeling; Molecular; Morbidity - disease rate; Mus; Neuroglia; Neuroimmune; Neurons; Neurotransmitters; Organ Transplantation; Pathway interactions; Patients; Peripheral; Persons; Play; Prevention; Procedures; Productivity; Protein Inhibition; Protein phosphatase; Protocols documentation; Quantitative Reverse Transcriptase PCR; Rattus; Recovery; Regulation; Research; Research Personnel; Resolution; Rewards; Rodent; Rodent Model; Role; Self Administration; Signal Pathway; Signal Transduction Pathway; Signaling Molecule; Sirolimus; T-Lymphocyte; Tacrolimus; Testing; Time; Transplantation; Ventricular; addiction; alcohol use disorder; analog; base; brain metabolism; clinical application; consumption measures; cost; design; drinking; experimental study; gamma-Aminobutyric Acid; innovation; inpatient service; liver transplantation; military veteran; mortality; mouse model; neurochemistry; neuroinflammation; neuromechanism; neurotransmission; novel strategies; pandemic disease; preference; preservation; prevent; problem drinker; psychosocial; translational study

Veteran populations present alcoholism rates that are far higher than general population frequencies. Despite this, the treatment options for alcoholism are very few and much in need of new approaches for medical interventions. This project grows from the observation that alcoholic persons receiving liver transplant evidence very high rates of sustained abstinence after the liver grafting procedure that we could not explain by psychosocial or selection factors. We asked whether this might be due to a common factor shared by all of these patients: immunosuppressant medicines given for very long periods after transplant to keep the grafted organ in a state of health. In two separate rodent experiments we found that 1) cyclosporine (CSA) had an antidipsic effect in an open choice model of ethanol (alternatively alcohol, or ethyl alcohol) drinking, and 2) that CSA and tacrolimus (TRL, alternately FK506) had similar effects in a drinking-in-the-dark (DID) ethanol drinking model while sirolimus (SRL, alternately rapamycin) had no effect but had lower concentrations in the brain. These findings implicate the immunosuppressants blocking brain calcineurin (CLN) activity that may enhance the rodent's choice against drinking ethanol through 1) direct CLN modulation in the brain or 2) through the systemic immunosuppressive and neuroimmune pathways. This study asks whether further evidence supports one or the other of these possible mechanisms of action. Following the overall goal of this project--to elucidate the mechanism(s) of reduction of alcohol preference by CLN directly or by CLN-mediated immunosuppression, the study's primary hypothesis asserts that inhibition limiting CLN activity in the brain itself will decrease rodent ethanol choice. An alternative hypothesis states that the peripheral effects of CLN inhibition in the immune system will result in a decrease in ethanol preference. The overall aim of the project is therefore to establish which of these two possible mechanisms mediates the effect of immunosuppressant agents on rodent's choice not to drink ethanol. Our experimental approaches address the primary hypothesis using a genetic knockout approach. In Specific Aim 1, we will compare immunosuppressants in brain-specific CLN knock-out mice that include a) pan-brain knockout (CamKIIα Cre x floxed CLN) rodents, b) CRF neuron specific calcineurin knockout (CRHCrex floxed CLN), and c) focal CLN knockouts in extended reward regions (VTA, NAc, CeA) utilizing AAV-Cre microinjections in floxed CLN mice. In Specific Aim 2, we will characterize the possible mechanisms of central effects of CLN inhibition on alcohol consumption by assessing 1) the brain's metabolic protection by CLN inhibitors, and 2) addiction related downstream signal molecules in extended brain reward networks. In Specific Aim 3, we test whether drinking causes neuroinflammation that drives subsequent drinking. We will use a combination of approaches (flow cytometry, immunohistochemistry, qRT-PCR, ELISA) to determine the effects of CLN inhibition on ethanol induced neuroinflammation. And in Specific Aim 4 we will non-invasively assess anatomical, metabolic and functional changes in the mouse brain using multi-parametric magnetic resonance imaging (MRI). We will use non-invasive 9.4 Tesla MRI protocols longitudinally on our mouse models of drinking preference treated with CsA, TRL and SRL. We expect the data from these procedures to answer the study questions in a definitive manner and to point the path to new answers in the neurochemistry of alcohol choice. This in turn can lead to better understanding of the mechanisms involved in stopping or limiting drinking and alcohol addiction in veteran populations. The overall goal is to find new medicinal agents that can treat alcoholism a condition that affects up to one in every two patients admitted to VA inpatient services with an estimated half of those actively drinking.

退伍军人群体的酗酒率远远高于一般人群的频率。 尽管如此，酒精中毒的治疗选择很少，而且非常需要新的方法 医疗干预。该项目源于对接受肝移植的酗酒者的观察 证据表明，肝移植手术后持续禁欲的比率非常高，但我们无法用以下方法来解释： 社会心理或选择因素。我们询问这是否可能是由于所有人共有的共同因素造成的 这些患者：移植后长期服用免疫抑制剂以保持移植物 器官处于健康状态。在两个单独的啮齿动物实验中，我们发现 1) 环孢菌素 (CSA) 具有 在乙醇（或者酒精或乙醇）饮用的开放选择模型中的解酒作用，以及 2) CSA 和他克莫司（TRL，或者 FK506）在黑暗饮用 (DID) 乙醇中具有相似的效果 饮酒模型，而西罗莫司（SRL，或者雷帕霉素）没有效果，但体内浓度较低 脑。这些发现表明免疫抑制剂会阻断大脑钙调神经磷酸酶 (CLN) 活性，从而可能 通过 1) 直接调节大脑中的 CLN 或 2) 增强啮齿动物对饮用乙醇的选择 通过全身免疫抑制和神经免疫途径。本研究询问是否进一步 证据支持这些可能的作用机制中的一种或另一种。 遵循该项目的总体目标——阐明减少酒精的机制 该研究的主要假设断言，CLN 直接偏好或通过 CLN 介导的免疫抑制偏好 限制大脑本身 CLN 活性的抑制会减少啮齿动物对乙醇的选择。另一种选择 假设指出，免疫系统中 CLN 抑制的外周效应将导致免疫系统中 乙醇偏好。因此，该项目的总体目标是确定这两种可能中的哪一种 机制介导免疫抑制剂对啮齿动物选择不喝乙醇的影响。 我们的实验方法使用基因敲除方法解决了主要假设。在 具体目标 1，我们将在大脑特异性 CLN 敲除小鼠中比较免疫抑制剂，其中包括： 全脑敲除 (CamKIIα Cre x floxed CLN) 啮齿动物，b) CRF 神经元特异性钙调神经磷酸酶敲除 (CRHCrex floxed CLN)，以及 c) 利用扩展奖励区域 (VTA、NAc、CeA) 进行局部 CLN 敲除 floxed CLN 小鼠中 AAV-Cre 显微注射。在具体目标 2 中，我们将描述可能的机制 通过评估 1) 大脑的代谢保护，了解 CLN 抑制对饮酒的中枢影响 CLN 抑制剂，2) 扩展大脑奖励网络中成瘾相关的下游信号分子。在 具体目标 3，我们测试饮酒是否会导致导致随后饮酒的神经炎症。我们将 使用组合方法（流式细胞术、免疫组织化学、qRT-PCR、ELISA）来确定 CLN 抑制对乙醇诱导的神经炎症的影响。在具体目标 4 中，我们将非侵入性地 使用多参数磁力评估小鼠大脑的解剖、代谢和功能变化 磁共振成像（MRI）。我们将在鼠标上纵向使用非侵入性 9.4 Tesla MRI 协议 使用 CsA、TRL 和 SRL 治疗的饮酒偏好模型。 我们期望这些程序中的数据能够以明确的方式回答研究问题并 为酒精选择的神经化学提供新答案。这反过来又可以带来更好的结果 了解退伍军人停止或限制饮酒和酒精成瘾的机制 人口。总体目标是寻找新的药物来治疗酗酒（一种影响酒精中毒的疾病） 每两名患者中就有一名接受 VA 住院服务，其中估计有一半患者积极参与 喝。