The Unfolding Role of Microglia in Alcohol Withdrawal

摘要 戒酒是一种医疗紧急情况，也是减少酗酒的行为障碍。不像 戒断到其他药物，有充分的证据表明，戒断成酒后，多次 反复饮酒和戒酒，增加了严重不良并发症的风险，包括 癫痫发作、颤颤性精神错乱和死亡。此外，酒精循环固有的负面强化 戒酒和酗酒使任何减少饮酒的行为干预变得复杂 虐待。许多线索表明，神经炎症与酒精耐受和戒断都有关， 但这一认识并没有带来酒精使用障碍的新疗法。这项建议的目的是 发展我们的假设，即慢性间歇性酒精暴露改变了小胶质细胞的方式，即先天免疫 驻留在大脑中的细胞，调节神经免疫和神经元突触信号。在试行中 多次饮酒后小胶质细胞核糖体相关RNA的RNA测序实验 暴露和戒酒，我们发现酒精戒断会引起小胶质细胞的氧化应激并激活 “未折叠蛋白反应”(UPR)，这是一种参与细胞应激反应的机制，导致 神经炎性信号和小胶质细胞功能紊乱，可导致细胞死亡。 我们建议通过敲除关键转录因子CHOP来阻断小胶质细胞UPR的完全激活 参与这一在戒断过程中小胶质细胞显著上调的途径，使用最近的一种 开发的选择性cre驱动小鼠品系Tem119-2A-CRE-ERT2/TdTomato与商业杂交 可用带绒毛的小鼠来研究这种生物学。我们将研究钝化的UPR信号如何改变 通过检测Chop基因敲除是否改变了几种行为表现并减少了戒酒的迹象 戒断后小胶质细胞形态和基因表达的变化。利用活体双光子 时间推移显微镜，我们将检查小胶质细胞的切割敲除是否改变了形态和运动性 在戒酒期间有关联。我们还将测试小胶质细胞印记是否会影响 在多次酒精蒸气暴露后自愿饮酒的模型中饮酒 戒烟。这些实验共同检验了神经炎症在酒精作用机制中的作用。 从一个以前没有考虑过并与之很好匹配的新角度看依赖和戒断 R21机制具有很高的新颖性和开发新疗法的潜力。

Abstract Alcohol withdrawal is a medical emergency and a behavioral barrier to reducing alcohol abuse. Unlike withdrawal to other drugs, there is ample evidence that withdrawal to alcohol becomes worse after multiple cycles of alcohol consumption and abstinence, increasing the risks for serious adverse complications including seizures, delirium tremens, and death. Furthermore, the negative reinforcement inherent to cycles of alcohol abstinence and relapse to alcohol consumption complicates any behavioral interventions to reduce alcohol abuse. Many clues suggest that neuroinflammation is involved in both alcohol tolerance as well as withdrawal, but this realization has not led to new treatments for alcohol use disorder. The purpose of this proposal is to develop our hypothesis that chronic, intermittent alcohol exposure alters the way microglia, the innate immune cells that reside in the brain, modulate neuroimmune as well as neuronal synaptic signaling. In pilot experiments using RNA sequencing of ribosome-associated RNA from microglia after multiple cycles of alcohol exposure and abstinence, we found that alcohol withdrawal causes oxidative stress in microglia and activates the “unfolded protein response” (UPR), a mechanism that is involved in cellular stress responses leading to neuroinflammatory signaling and derangements in microglia function that can lead to cell death. We propose to block the full activation of the microglial UPR by knocking out CHOP, a key transcription factor involved in this pathway that was dramatically upregulated in microglia during withdrawal, using a recently developed, selective cre-driver mouse line, Tmem119-2A-Cre-ERT2/TdTomato, crossed with commercially available floxed CHOP mice to investigate this biology. We will examine how blunting UPR signaling alters the signs of alcohol withdrawal by testing if CHOP knockout alters several behavioral manifestations and reduces microglia morphological and gene expression changes associated with withdrawal. Using in vivo two-photon time lapse microscopy, we will examine if CHOP knockout from microglia changes morphology and motility associated during alcohol withdrawal. We will also test whether microglial CHOP impacts the motivation to drink alcohol in a model of voluntary alcohol drinking after multiple cycles of ethanol vapor exposure and withdrawal. Together, these experiments examine the role of neuroinflammation in the mechanisms of alcohol dependence and withdrawal from a new angle that has not been previously considered and is well matched to the R21 mechanism due to its high novelty and potential to develop new treatments.