A systems approach implicates a brain mitochondrial oxidative homeostasis co-expression network in genetic vulnerability to alcohol withdrawal

Differential gene expression between mouse distal chromsome 1 congenic and background and reciprocal congenic and background Genetic factors significantly affect vulnerability to alcohol dependence (alcoholism). We previously identified a quantitative trait locus on chromosome 1 (Adw1) with a large effect on predisposition to alcohol physiological dependence and associated withdrawal following both chronic and acute alcohol exposure in mice. We fine-mapped these loci to a 1.1-1.7 Mb interval syntenic with human 1q23.2-23.3. Adw1 interval genes show remarkable genetic variation among mice derived from the C57BL/6J and DBA/2J strains, the two most widely studied genetic animal models for alcohol-related traits. Here, we report the creation of a novel recombinant Adw1 congenic model (R2) in which the Adw1 interval from a donor C57BL/6J strain is introgressed onto a uniform, inbred DBA/2J genetic background. As expected, R2 mice demonstrate significantly less severe alcohol withdrawal compared to wild-type littermates. Additionally, comparing R2 and background strain animals, as well as reciprocal congenic (R8) and appropriate background strain animals, we assessed Adw1 dependent brain gene expression using microarray and quantitative PCR analyses. To our knowledge this includes the first Weighted Gene Co-expression Network Analysis using reciprocal congenic models. Importantly, this allows detection of co-expression patterns limited to one or common to both genetic backgrounds with high and low predisposition to alcohol withdrawal severity. The gene expression patterns (modules) in common contain genes related to oxidative phosphorylation, building upon human and animal model studies that implicate involvement of oxidative phosphorylation in alcohol use disorders. Finally, we demonstrate that administration of N-acetylcysteine, an FDA-approved antioxidant, significantly reduces symptoms of alcohol withdrawal (convulsions) in mice, thus validating a phenotypic role for this network. Taken together, these studies support the importance of mitochondrial oxidative homeostasis in alcohol withdrawal and identify this network as a valuable therapeutic target in human alcohol use disorders. Whole brain, naïve, males. n=10 inbred C57BL/6J (B6), n=10 R8 small interval Chr 1 congenic (1.1 Mb D2 interval on B6 background), n=6 inbred DBA/2J (D2), n=6 R2 small interval Chr 1 congenic (10.2 Mb B6 interval on D2 background)小鼠1号染色体远端同源重组系（congenic）与背景品系、以及互同类系与背景品系之间的差异基因表达分析。遗传因素可显著影响酒精依赖（酒精成瘾）的易感性。我们此前在小鼠1号染色体上鉴定出一个数量性状位点（quantitative trait locus, QTL）Adw1，该位点对小鼠在慢性和急性酒精暴露后产生酒精生理依赖及相关戒断症状的易感性具有显著调控作用。我们已将该位点精细定位至与人类1q23.2-23.3区域存在同线性关系的1.1~1.7 Mb区间内。Adw1区间内的基因在C57BL/6J与DBA/2J这两种最常用于酒精相关性状研究的近交系小鼠品系间存在显著遗传差异。本研究构建了一种新型重组Adw1同源重组系模型（R2）：将供体C57BL/6J品系的Adw1区间导入至纯合近交DBA/2J遗传背景中。如预期一致，与野生型同窝小鼠相比，R2小鼠的酒精戒断症状显著更轻微。此外，通过比对R2小鼠与背景品系小鼠，以及互同类系（R8）小鼠与对应背景品系小鼠的差异，我们采用基因芯片（microarray）与定量聚合酶链式反应（quantitative PCR, qPCR）分析了Adw1依赖的大脑基因表达模式。据我们所知，本研究首次采用互同类系模型开展加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）。该分析可精准检测仅在某一种遗传背景中存在，或在酒精戒断严重程度高低两种遗传背景中共同存在的共表达模式。两种背景中共有的基因表达模式（模块）富集了与氧化磷酸化相关的基因，这与此前人类及动物模型研究中发现氧化磷酸化参与酒精使用障碍的结论一致。最后，我们证实给予经美国食品药品监督管理局（Food and Drug Administration, FDA）批准的抗氧化剂N-乙酰半胱氨酸（N-acetylcysteine, NAC）可显著减轻小鼠的酒精戒断症状（惊厥），从而验证了该基因共表达网络的表型调控作用。综上，本研究证实线粒体氧化稳态在酒精戒断过程中发挥关键作用，并将该基因共表达网络确定为人类酒精使用障碍的潜在治疗靶点。实验样本为全脑组织、未接触过酒精的雄性小鼠。其中近交C57BL/6J（B6）品系小鼠10只，1号染色体小片段同源重组系R8小鼠（B6背景下导入1.1 Mb D2品系区间）10只，近交DBA/2J（D2）品系小鼠6只，1号染色体小片段同源重组系R2小鼠（D2背景下导入10.2 Mb B6品系区间）6只。