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500,000人遭受心脏骤停，这是一种与不良神经预后相关的事件。尽管在过去的50年里进行了密集的研究，但没有任何药物干预被证明在改善存活率和预后方面是成功的。缺血诱导神经元死亡的一个特征是谷氨酸过度释放，导致兴奋性毒性。不幸的是，谷氨酸拮抗剂已被证明在人类身上不成功，主要是由于副作用。合理的替代方法是应用激活GABA-A受体的化合物(GABA-AR)，以抵消过度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失望的不同结果。最近的数据表明，缺血导致GABA-AR蛋白的迅速丢失，这表明缺血诱导的GABA-AR蛋白的减少可能导致GABA增强剂疗效的降低。因此，在缺血事件期间稳定GABA-A受体蛋白和功能的治疗是一种有吸引力的和令人兴奋的神经保护新方法。为了获得缺血后神经元GABA-A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，最著名的是突触的全细胞电压钳记录(MIPSCs)和总的GABA-A受体活性(对外源性饱和GABA的响应电流)，以确认和扩展我们的初步观察，即缺血导致功能性GABA-A受体的减少，重要的是，ALLO可以防止这种缺血诱导的功能丧失。这项RO1应用将测试四个特定的假设：1)ALLO可以防止缺血引起的功能性GABA-A受体减少，从而保护PC免受缺血的影响。2)ALLO通过维持缺血时PKC活性和GABA-A受体的磷酸化，防止缺血所致的GABA-A受体功能下降。3)Allo通过防止缺血后GABA-A受体蛋白的蛋白体依赖的降解来稳定缺血期间的GABA-A R蛋白，最后4)Allo诱导的保护GABA-A R功能的作用发生在暴露于全脑缺血(心脏骤停)的完整动物中。我们的发现将开始阐明PC的等位基因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.