A novel proteomics approach to identify alcohol-induced changes in synapse-specific presynaptic protein interactions.

项目摘要 酗酒和酗酒是一种日益严重、破坏性越来越大的社会、经济、国家和全球危机。 迫切需要新的治疗干预措施，这种干预措施不是控制症状，而是逆转 酒精引起的神经生理学改变。酒精和无数其他辅助药物产生的一种影响 在大脑中，是长期突触抑制(LTD)的中断，特别是LTD的突触前形式 这发生在皮质纹状体突触上。纹状体与强迫和习惯性行为有关， 包括吸毒成瘾。药物引起的背外侧纹状体LTD的破坏与 习惯性行为增加，而LTD恢复则恢复药物破坏的目标导向行为。我们有 研究发现，酒精破坏了纹状体突触前阿片受体(MOR)介导的LTD，LTD在DLS中发生 仅在前岛叶皮质输入(AIC-DLS突触)。丘脑纹状体的MOR-短期抑郁 投入不会受到酒精暴露的影响。识别突触前发生的分子变化 酒精暴露于AIC-DLS突触，而不是丘脑纹状体突触，可能揭示出新的靶点 酒精使用障碍和成瘾的药物干预。然而，具体的问题是 使用药理学和遗传学工具识别蛋白质-蛋白质相互作用的突触前特异性变化 费力、低效，并且依赖于信号通路的先验知识，可能缺少关键分子 可能受到影响的球员。根据我们的初步数据，我们的中心假设是体内酒精 暴露改变MOR介导的LTD所需的PKA依赖的突触前蛋白组织 归纳法。我们将利用研究人员的生理学和蛋白质组学专业知识来 建立一种新的生化方法，能够选择性地测定精确的体内外 恰好发生在AIC突触终末内的蛋白质相互作用体的变化(但不发生在 在MOR介导的LTD诱导和体内暴露后DLS中的丘脑-纹状体终末) 酒精。酒精敏感型AIC-DLS突触与酒精不敏感型突触的比较 丘脑的输入将允许我们确定是什么使一些突触对酒精的有害影响敏感 而其他人则持抵抗态度。这种方法采用了小鼠脑片电生理学，体内酒精暴露， 以及病毒载体驱动的蛋白质表达，允许在突触前终末内进行邻近标记 随后的蛋白质组学分析。使用这个新工具，我们将打开突触前蛋白质组的“黑匣子” 我们预测的变化将揭示以前未知的突触可塑性蛋白质-蛋白质相互作用 会被酒精和其他令人上瘾的药物破坏。此外，我们的目标是确定新的蛋白质相互作用 这可能被证明是治疗酒精使用障碍和成瘾的治疗靶点。

Project Summary Alcohol misuse and addiction is a growing and increasingly destructive socioeconomic national and global crisis. There is a critical need for novel therapeutic interventions that, rather than managing symptoms, reverse alcohol-induced neurophysiological changes. One effect that alcohol, and myriad other addicitive drugs, produce in the brain, is the disruption of long-term synaptic depression (LTD), particularly the presynaptic form of LTD that occurs at corticostriatal synapses. The striatum is associated with compulsive and habitual behaviors, including drug addiction. Drug-induced disruption of LTD in dorsolateral striatum (DLS) is associated with increased habitual behavior whereas LTD restoration restores drug-disrupted goal-directed behavior. We have found that alcohol disrupts striatal presynaptic mu opioid receptor (MOR)-mediated LTD that, in the DLS, occurs exclusively at anterior insular cortex inputs (AIC-DLS synapses). MOR-short-term depression at thalamic striatal inputs are unaffected by alcohol exposure. Identifying the molecular changes that occur presynaptically following alcohol exposure at AIC-DLS synapses, but not at thalamostriatal synapses, may reveal new targets for pharmacological interventions in alcohol use disorder and addiction. The specific problem though is that identifying presynaptic-specific changes in protein-protein interactions using pharmacological and genetic tools is laborious, inefficient, and relies on a priori knowledge of signaling pathways, likely missing key molecular players that could be affected. Based on our preliminary data, our central hypothesis is that in vivo alcohol exposure alters PKA-dependent presynaptic protein organization that is required for MOR-mediated LTD induction. We will capitalize on the combined physiological and proteomic expertise of the investigators to develop a novel biochemical methodology that is able to selectively determine the precise ex vivo and in vivo changes that occur in the protein interactome precisely within AIC synaptic terminals (but do not occur within thalamostriatal terminals) in DLS during the induction of MOR-mediated LTD and following in vivo exposure to alcohol. The comparison between these alcohol-sensitive AIC-DLS synapses and the alcohol-insensitive thalamic inputs will allow us to determine what makes some synapses susceptible to alcohol’s deleterious effects and others resistant. This methodology employs mouse brain slice electrophysiology, in vivo alcohol exposure, and viral vector-driven expression of proteins that allow for proximity labeling within presynaptic terminals with subsequent proteomic analyses. Using this new tool we will open the “black box” of presynaptic proteome changes that we predict will uncover previously unidentified synaptic plasticity protein-protein interactions that are disrupted by alcohol and other addictive drugs. Furthermore, we aim to identify novel protein interactions that may prove to be therapeutic targets for treating alcohol use disorder and addiction.