The vast majority of hearing loss occurs due to the death of mechanosensory hair cells of the inner ear. Many environmental and genetic factors contribute to hair cell death in the human population, including noise, age, and ototoxic drug exposure. This project focuses on hair cell death caused by exposure to a family of ototoxic antibiotics-the aminoglycosides. Aminoglycosides specifically kill hair cells and cells of the proximal tubule of the kidney, in addition to their desired pathogenic targets. While kidney damage is often temporary, loss of inner ear hair cells results in permanent hearing loss. Because the aminoglycosides are effective antibiotics, blocking their ototoxicity has been a longstanding goal of the hearing research community (Forge and Schacht, 2000). Reducing hair cell death caused by aminoglycoside antibiotics would allow us to better treat tuberculosis, meningitis, cystic fibrosis, and a range of systemic infections (Cheng et al., 2009;Lima et al., 2006;Yeat et al., 1997). Why hair cells are uniquely sensitive to aminoglycoside exposure remains unclear. Activity of the mechanotransduction (MET) channel-the channel that opens in response to sound stimuli-- facilitates entry of aminoglycosides into hair cells. Genetic mutations and small molecules that perturb MET channel activity inhibit entry of aminoglycosides and protect hair cells. However, while entry of aminoglycosides is critical for their toxicity, other intracellular events mediate aminoglycoside-induced hair cell death. In a genetic screen, we identified the sentinel mutant which shows striking protection from aminoglycosides, while having no effect on the entry of aminoglycosides into hair cells. Because this mutant affects intracellular trafficking events in other contexts, it suggests that altered intracellular traffickng of aminoglycosides may underlie protection in sentinel. Additionally, we observe that the dynamin inhibitor dynasore-which perturbs many intracellular trafficking events--also protects hair cells from aminoglycoside- induced toxicity. Both of these findings suggest that perturbing the intracellular trafficking of aminoglycosides can reduce their toxicity. To study how aminoglycosides are trafficked within hair cells, I propose to follow the trafficking of the labele aminoglycoside neomycin-TexasRed in hair cells. I will use zebrafish lateral line hair cells because we can easily image these cells in their native context while exposing them to Neomycin- TexasRed, vital dyes, and bioactive molecules. In preliminary pulse label studies, we have monitored movement of labeled neomycin from the stereocilia into intracellular structures, and see alterations in those structures during neomycin exposure. I propose to characterize the intracellular compartments transited by labeled neomycin, and developing tools to perturb intracellular trafficking pathways to identify key trafficking events that underlie aminoglycoside toxicity.
Aminoglycosides are very effective antibiotics used to treat a variety of systemic infections. Unfortunately, treatment with these antibiotics frequently causes permanent hearing loss by killing cells of the inner ear that convert sound into nerve impulses. I propose to investigate ways we can reduce aminoglycosides toxicity by determining where aminoglycosides accumulate and whether we can decrease their toxicity by altering delivery to those intracellular sites.
