Critical Signaling Events During the Aminoglycoside-induced Death of Sensory Hair Cells in Vitro

Sensory hair cells undergo apoptosis following exposure to aminoglycoside antibiotics. In neurons, apoptosis is associated with a transient increase in intracellular Ca2+, phosphorylation of the transcription factor c-Jun, and the release of cytochrome c from mitochondria into the cytosol, which along with other cofactors results in the activation of caspases. To examine the possible role of these events in the survival and death of the sensory receptors of the inner ear, we examined the effects of neomycin treatment on cytoplasmic calcium, activation of c-Jun-N-Terminal kinases (JNKs), cytochrome c release, and caspase-3 activation in cultured vestibular hair cells. Increased numbers of phospho-c-Jun-labeled hair cells (a downstream indicator of JNK activation) were observed at 3-12 h after neomycin treatment, whereas increased numbers of cells with cytoplasmic cytochrome c were observed at 12-18 h following the onset of neomycin treatment. This was followed by an increase in the number of cells that contained activated caspase-3 and displayed pyknotic nuclei. Treatment with the general caspase inhibitor BAF did not affect the release of cytochrome c and the number of p-c-Jun-labeled cells, but reduced the number of cells with activated caspase-3 and pyknotic nuclei. In contrast, treatment with CEP-11004, an indirect inhibitor of the JNK signaling pathway, promoted hair cell survival following neomycin treatment and reduced the number of cells with phosphorylated JNK and c-Jun, cytoplasmic cytochrome c, and activated caspase 3. These results suggest that JNK activation occurs upstream of the release of cytochrome c and that cytochrome c release precedes caspase activation. Cytochrome c release and JNK activation were also preceded by large changes in cytoplasmic calcium. Cytoplasmic calcium increases may be causally related to the release of cytochrome c, and may also be a potential pathway for activation of JNK in hair cells.