Neurobiological Signatures of Alcohol Dependence Revealed by Protein Profiling

Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction.

酒精滥用会引发大脑出现显著的神经适应性改变，进而导致酒精耐受、依赖以及行为模式改变。既往针对人类酒精成瘾者及动物模型的蛋白质组学研究，已筛选出与酒精成瘾相关的候选蛋白。然而，近期研究证据表明，酒精依赖的本质源于共调控网络的改变，这类改变无法通过单蛋白水平的分析被检测到。本研究针对全局蛋白质组学数据展开分析，整合差异表达、共表达网络以及基因注释信息，以揭示由慢性间歇性乙醇（Chronic Intermittent Ethanol, CIE）双瓶选择（two-bottle choice, 2BC）范式构建的酒精依赖模型中，与酒精依赖转化相关的关键神经生物学重排事件。本研究对接受CIE-2BC范式处理的雄性C57BL/6J小鼠的大脑皮层（cerebral cortices, CTX）及中脑（midbrains, MB）进行了分析；该范式可诱导小鼠大量饮酒，是目前最优秀的酒精依赖及复饮动物模型之一。相较于非依赖对照组小鼠，CIE处理的依赖小鼠体内的蛋白质水平发生了显著改变。多种蛋白质亚型因翻译后修饰，呈现出脑区特异性的差异调控模式。本研究的整合分析筛选出了与CIE处理高度相关的共表达蛋白模块。研究发现，与CIE处理效应最为相关的模块，协同调控了内吞通路及能量代谢通路中的分子失衡，涉及动力蛋白1（dynamin-1）等特定蛋白。实时定量反转录聚合酶链反应（qRT-PCR）实验验证了差异表达与共表达分析的结果；本研究数据与既往针对人类及啮齿类动物的基因组学、蛋白质组学研究结果的一致性，进一步证实了本研究发现的可靠性。上述鉴定出的改变，可能在与酒精依赖相关的乙醇摄食量升级过程中发挥关键作用。本研究针对酒精成瘾的研究策略，将增进我们对药物滥用引发的大脑重塑机制及适应性改变的认知，助力解析大脑皮层与中脑蛋白质组的组织原则，并为酒精成瘾的治疗确定潜在的新型分子靶点。本研究采用的整合分析方法，凸显了系统生物学策略在研究酒精成瘾神经生物学机制中的优势。