Lisa L. Cunningham - US grants

Vertebrate sensory hair cells are sensitive to damage from a variety of genetic and environmental factors, including exposure to aminoglycoside antibiotics. Exposure to these antibiotics results in death of sensory hair cells. In mammals the loss of hair cells can result in permanent hearing loss and/or balance disturbances. Recent evidence indicates that exposure to aminoglycosides activates an intrinsic cell death pathway in hair cells. This cascade involves activation of a family of cysteine proteases called caspases. Caspase activation is the hallmark of programmed cell death in other systems, and different caspases are activated in response to different cell death-promoting stimuli. The overall goal of the proposed research is to utilize an in vitro model of mammalian sensory hair cell ototoxicity to determine the involvement of specific caspases in aminoglycoside-induced hair cell death. In the first set of experiments, I will determine which caspases are activated in sensory hair cells in response to aminoglycoside exposure and evaluate whether the cells activating caspases are subsequently dying. The second set of experiments will focus on determining which upstream caspases mediate activation of specific downstream caspases in hair cells exposed to aminoglycosides. Such an understanding of the order of caspase activation is an essential step toward understanding the signaling cascade that takes place in a damaged hair cell. The third set of experiments is designed to determine which individual caspases are necessary for aminoglycoside-induced programmed cell death in hair cells. Taken together, the experiments are designed to provide the first examination of the cascade of caspase activation in hair cells beginning with the cell death trigger and proceeding through the actual execution of the cell.

DESCRIPTION (provided by applicant): Sensory hair cells are hypersensitive to death induced by noise, aging, and some therapeutic drugs. Two major classes of ototoxic drugs are the aminoglycoside antibiotics and the antineoplastic agent cisplatin. We plan to take advantage of a unique in vitro preparation of the adult mouse utricle that allows us to examine the rrechanisms underlying ototoxic hair cell death and survival at the molecular level. Understanding these cellular mechanisms will be critical for the design of therapies aimed at preventing or reversing hearing loss. The induction of heat shock proteins (HSPs) in response to cellular stress is a ubiquitous and highlyconserved response that can significantly inhibit apoptosis in some systems. Induction of HSPs occurs in hair cells in response to a variety of stimuli. However, there are no studies on the effects of HSPs on ototoxic drug-induced hair cell death. More importantly, there are no data available regarding the cellular interactions between HSPs and apoptotic proteins in hair cells in response to any stimulus. Our preliminary data demonstrate that in vitro heat shock treatment inhibits hair cell death in response to both neomycin and cisplatin exposure. The experiments in this proposal are designed to test the hypothesis that the protective effect of heat shock against ototoxic drug-induced hair cell death is the result of HSP-mediated inhibition of specific apoptotic signaling pathways. Four groups of experiments are proposed: 1). To examine the similarities and differences between the molecular mechanisms mediating cisplatin- vs. aminoglycoside-induced hair cell death. 2). To determine whether heat shock treatment is sufficient to induce a shift in the dose-response relationship between ototcxic drug concentration and hair cell survival. 3). To determine whether induction of HSP-70 is necessary and sufficient for the protective effect of heat shock against ototoxic hair cell apoptosis. 4). To investigate the interactions between HSPs and apoptotic proteins in hair cells exposed to ototoxic drugs.