Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic-and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of l-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycosideinduced hearing loss.

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway.

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