GABA-A Receptor Rescue as a Neuroprotective Strategy in Cerebral Ischemia

描述（由申请人提供）：在美国，每年约有50万人患有心脏骤停，这是一种与神经系统预后不良相关的事件。尽管在过去的50年里进行了大量的研究，但没有任何药物干预措施被证明可以成功地提高生存率和结局。缺血诱导的神经元死亡的标志是谷氨酸的过度释放，导致兴奋性毒性。不幸的是，谷氨酸拮抗剂已被证明在人类中不成功，主要是由于副作用。合乎逻辑的替代方法将是应用激活GABA-A受体（GABA-A R）的化合物，以抵消过量的谷氨酸释放和兴奋性毒性。有趣的是，GABA能化合物产生了令人不安的可变结果。最近的数据表明，局部缺血导致GABA-A R蛋白的快速损失，表明局部缺血诱导的GABA-A R蛋白的减少可能导致GABA增强化合物的功效降低。因此，在缺血事件期间稳定GABA-A R蛋白和功能的治疗是一种吸引人和令人兴奋的神经保护新方法。为了获得缺血后神经元GABA受体功能的电生理记录，我们建立了小脑神经元培养模型。我们将使用多种方法的组合，最值得注意的是突触（mIPSC）和总GABA-A R活性（响应于外源施加的饱和GABA的电流）的全细胞电压钳记录，以确认和扩展我们的初步观察，即缺血引起功能性GABA-A R的减少，重要的是ALLO防止这种缺血诱导的功能丧失。该RO 1应用将测试四个特定假设：1）ALLO防止缺血诱导的功能性GABA-A R减少，从而保护PC免受缺血。2)ALLO通过在缺血期间维持PKC活性和GABA-A R的磷酸化来防止缺血诱导的GABA-A R功能降低。3)ALLO通过防止缺血后GABA-A受体蛋白的蛋白体依赖性降解来稳定缺血期间的GABA-A R蛋白，并且最后4）ALLO诱导的GABA-A R功能保护发生在暴露于全脑缺血（心脏骤停）的完整动物中。我们的研究结果将开始阐明ALLO对PC神经保护的细胞机制，并确定可能代表神经保护新靶点的分子途径。

DESCRIPTION (provided by applicant): Each year approximately 500,000 people suffer from cardiac arrest in the United States, an event associated with poor neurological outcome. Despite intense research over the past 50 years, there are no pharmacological interventions that have proven successful in improving survival and outcome. A hallmark of ischemia-induced neuronal death is excessive release of glutamate leading to excitotoxicity. Unfortunately, glutamate antagonists have proven unsuccessful in humans, predominantly due to side effects. The logical alternative approach would be to apply compounds that activate GABA-A receptors (GABA-A R) in order to counteract excessive glutamate release and excitotoxicity. Interestingly, GABAergic compounds have yielded disappointingly variable results. Recent data has demonstrated that ischemia results in a rapid loss of GABA-A R protein, indicating that the ischemia-induced decrease in GABA-A R protein may cause a decrease in efficacy of GABA-potentiating compounds. Therefore, a treatment that stabilizes GABA-A R protein and function during an ischemic event is an appealing and exciting new approach to neuroprotection. In order to obtain electrophysiological recordings of neuronal GABA-A R function following ischemia, we have developed a cerebellar neuronal culture model. We will use a combination of methods, most notably whole-cell voltage- clamp recordings of synaptic (mIPSCs) and total GABA-A R activity (Current in response to exogenously applied saturating GABA) to confirm and extend upon our preliminary observation that ischemia causes a reduction in functional GABA-A Rs and importantly that ALLO prevents this ischemia-induced loss of function. This RO1 application will test four specific hypotheses 1) that ALLO prevents ischemia-induced reduction in functional GABA-A R, thereby protecting PCs from ischemia. 2) ALLO prevents ischemia-induced reduction in GABA-A R function by maintaining PKC activity and phosphorylation of GABA-A Rs during ischemia. 3) ALLO stabilizes GABA-A R protein during ischemia by preventing proteosome-dependent degradation of GABA-A receptor protein following ischemia and finally 4) that the ALLO-induced protection of GABA-A R function occurs in intact animals exposed to global ischemia (cardiac arrest). Our findings will begin to elucidate the cellular mechanisms of ALLO neuroprotection of PCs and determine molecular pathways that may represent novel targets for neuroprotection.