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

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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.