Research Project
9331613
? DESCRIPTION (provided by applicant): Due to hair cells' unique sensitivity to numerous environmental insults, including certain therapeutic medications, hair cell death is a leading cause of hearing loss. While progress has been made in uncovering the cellular mechanisms underlying the ototoxicity of therapeutic medications, to date there is no proven treatment to prevent hair cell death and subsequent hearing loss. The objective of this proposal is to determine how retrograde intraflagellar transport (IFT) genes affect aminoglycoside toxicity. The central hypothesis is that retrograde IFT genes are required for aminoglycoside-induced hair cell death, due to their involvement in aminoglycoside uptake, as well as their role in mitochondria cell death pathways. This hypothesis is based on preliminary data showing mutants in two retrograde IFT genes are resistant to aminoglycoside-induced hair cell death. This hypothesis will be tested by pursuing three specific aims: (1) identify the retrograde IFT genes important for aminoglycoside-induced hair cell death, (2) determine whether retrograde IFT genes affect hair cell susceptibility by influencing aminoglycoside uptake and trafficking, and (3) determine the role of retrograde IFT genes in mitochondria cell death pathways during aminoglycoside-induced hair cell death. In the first aim DNA editing technology will be used to generate zebrafish with mutations in known IFT genes. Using both newly generated and existing mutants we will assess the extent to which hair cells are protected against neomycin-induced death. In the second aim neomycin conjugated to a fluorescent dye will be used to determine the amount of neomycin entering mutant hair cells as well as its localization. Additionally, FM1-43 dye uptake and hair cell stimulation will be used to determine if decreases in aminoglycoside uptake are due to defects in hair cell mechanotransduction activity. In the third aim mutants will be treated with drugs known to affect mitochondria cell death pathways to look for additive interactions. Additionally, mitochondria Ca2+ will be tracked during neomycin-induced hair cell death using in vivo imaging experiments in both wild type and mutant animals. The experiments in this proposal will be carried out using the zebrafish lateral line system. This is an innovative approach because the zebrafish system allows for the rapid generation of mutants. Additionally, due to the maternal contribution of wild-type RNA to mutant embryos in zebrafish, retrograde IFT mutants develop normally in this system. This allows us to distinguish between the effects of these genes on hair cell development from those in mature hair cells. The proposed research is significant because it will provide novel insight into the genes involved in aminoglycoside ototoxicity as well as their mechanisms of action. This knowledge has the potential to be used to develop therapeutics to prevent hair cell death.
