The Unfolding Role of Microglia in Alcohol Withdrawal

摘要 酒精戒断是一种医疗紧急情况，也是减少酒精滥用的行为障碍。不像 戒断其他药物，有充分的证据表明，戒断酒精变得更糟后，多次 酒精消费和禁欲的周期，增加了严重不良并发症的风险，包括 癫痫谵妄和死亡此外，酒精循环所固有的负面强化 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有关， 但这一认识并没有导致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，先天免疫细胞， 存在于脑中的细胞调节神经免疫以及神经元突触信号传导。在试点 使用多次酒精循环后来自小胶质细胞的核糖体相关RNA的RNA测序的实验 暴露和禁欲，我们发现酒精戒断会导致小胶质细胞的氧化应激，并激活 “未折叠蛋白反应”（UPR），一种参与细胞应激反应的机制， 神经炎性信号传导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 参与这一通路，在戒断期间小胶质细胞中显著上调，使用最近的 开发的选择性Cre-driver小鼠系Tmem 119 -2A-Cre-ERT 2/TdTomato，与商业上的 可用的floxed CHOP小鼠来研究这种生物学。我们将研究钝化UPR信号如何改变 通过测试CHOP敲除是否改变了几种行为表现并减少了酒精戒断的迹象 与戒断相关的小胶质细胞形态学和基因表达变化。使用体内双光子 延时显微镜，我们将检查是否从小胶质细胞敲除CHOP改变形态和运动性 与酒精戒断有关。我们还将测试小胶质细胞CHOP是否会影响 在多次乙醇蒸汽暴露后自愿饮酒模型中饮酒， 戒断总之，这些实验研究了神经炎症在酒精机制中的作用 从一个新的角度，以前没有考虑过，并很好地匹配， R21机制，因为它具有高度的新奇和开发新治疗的潜力。

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.