Spiral ganglion neurons (SGNs) die gradually following the loss of hair cells by the process of apoptosis. Electrical stimulation promotes the survival of such deafferented SGNs in vivo, raising the possibility of using electrical stimulation to maintain survival of SGNs in deaf individuals - in effect allowing cochlear implants to replace the trophic as well as the sensory function of hair cells. Our first goal is to provide a detailed timecourse of the key molecular events characteristic of apoptosis during the death of SGNs following deafening and describe how these events are affected by electrical stimulation. Rats will be deafened using ototoxin to destroy the hair cells and, during the approximately 100 day period over which SGNs die, the levels, the phosphorylation state and the subcellular localization of key representative apoptosis regulators and mediators will be determined immunohistochemically and biochemically. This will reveal at what stage(s) in the apoptotic process the SGNs exist following loss of hair cells, thereby identifying the time during which potential therapies for preventing SGN death would be most successfully applied. The second goal is to test the hypothesis implied by our previous studies: depolarization recruits three distinct kinase systems that independently and additively promote SGN survival, acting in distinct cellular compartments and on distinct substrates. Cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase (CaMK) II, appear to function in the cytoplasm and phosphorylate mitochondrial apoptotic regulators, with CaMKII doing so by an indirect route involving tyrosine kinases. CaMKIV functions in the nucleus, phosphorylating the transcription factor CREB. To test this hypothesis, a lentiviral vector will be used to introduce genes encoding activated protein kinase mutants or kinase inhibitor proteins into SGNs, with these proteins restricted to specific subcellular locations, e.g., mitochondria or nucleus, by means of physiological targeting sequences. For these studies, we use the system we have developed, in which SGNs are electrically stimulated or depolarized in vitro to maintain their survival. In parallel, we will infuse pharmacological inhibitors or activators of these kinase systems into the cochleae of hearing or deafened rats, or deafened rats with implanted stimulating electrodes. These experiments will determine the extent to which these intracellular signals promote survival in vivo and verify that they mediate the survival-promoting effect of electrical stimulation in vivo as they do in vitro.
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