Alkaline shock and excitotoxicity in epilepsy: RNAi in vivo - tools for investigation and prospects for therapy

Epilepsy is one of the most common forms of brain disorders, affecting about 1 % of the population. Alkaline shock, the rise of intracellular pH, is one of the few basic explanations of seizures that are now gaining a clear mechanistic description. Very r ecently a direct link between seizures, CO2-metabolism, pH and carbonic anhydrase inhibitors in a rat model, was demonstrated (Schuchmann, 2006). However, it is unclear which of the 15 carbonic anhydrases are involved in alkaline shock. RNA interference (RNAi), combined with lentiviral delivery and microRNA derived hairpin constructs, provide an opportunity to silence each of the CA genes and thus investigate their function in single cells in vivo, using electrophysiology and pH transient measurements i n single GFP positive cells in acute slices of rat hippocampus. A general benefit of our approach is the development of new technology to analyze the function of any gene in single cells in the intact brain, and thereby establish targets for therapy in neuronal diseases. We will target AQP4, GS and Gls in vivo for ultrastructure analysis of excitotoxicity and glia cell swelling. We aim to obtain single-cell ultrastructure resolution for RNAi in the brain using immunogold EM antibodies for the GFP lentiv iral marker. As ischemic and epileptic excitotoxicity are very complex phenomena, only an in vivo approach will provide true validation of a neuroprotective knockdown of a gene. For this reason our RNAi in vivo technology will provide a vital new tool. This project has the potential to identify new drug targets in epilepsy and ischemia, where few or no effective treatments exist in the clinic today.