Small GTPases mediate transmembrane signaling and regulate the actin cytoskeleton in eukaryotic cells. Here, we characterize the auditory pathology of adult male CBA/J mice exposed to traumatic noise (2–20 kHz; 106 dB; 2 h). Loss of outer hair cells was evident 1 h after noise exposure in the basal region of the cochlea and spread apically with time, leading to permanent threshold shifts of 35, 60, and 65 dB at 8, 16, and 32 kHz. Several biochemical and molecular changes correlated temporally with the loss of cells. Immediately after exposure, the concentration of ATP decreased in cochlear tissue and reached a minimum after 1 h while the immunofluorescent signal for p-AMPKα significantly increased in sensory hair cells at that time. Levels of active Rac1 increased, whereas those of active RhoA decreased significantly 1 h after noise attaining a plateau at 1 to 3 h; the formation of a RhoA-p140mDia complex was consistent with an activation of Rho GTPase pathways. Also at 1 to 3 h after exposure, the caspase-independent cell death marker, endonuclease G, translocated to the nuclei of outer hair cells. Finally, experiments with the inner ear HEI-OC1 cell line demonstrated that the energy-depleting agent oligomycin enhanced both Rac1 activity and cell death. The sum of the results suggests that traumatic noise induces transient cellular ATP depletion and activates Rho GTPase pathways, leading to death of outer hair cells in the cochlea.

Post-translational modification of histones is an important form of chromatin regulation impacting transcriptional activation. Histone acetyltransferases (HATs), for example, acetylate lysine residues on histone tails enhancing gene transcription, while histone deacetylases (HDACs) remove those acetyl groups and repress gene transcription. Deficient histone acetylation is associated with pathologies, and histone deacetylase inhibitors have been studied in the treatment of cancer and neurodegenerative diseases. Here we explore histone acetylation in cochlear sensory cells following a challenge with gentamicin, an aminoglycoside antibiotic known to cause loss of auditory hair cells and hearing. The addition of the drug to organotypic cultures of the mouse organ of Corti decreased the acetylation of histone core proteins (H2A Ack5, H2B Ack12, H3 Ack9, and H4 Ack8) followed by a loss of sensory cells. Protein levels of HDAC1, HDAC3 and HDAC4 were increased while the HATs CBP and p300 remained unchanged. We next hypothesized that protecting histone acetylation should prevent cell death and tested the effects of HDAC-inhibitors on the actions of gentamicin. Co-treatment with trichostatin-A maintained near-normal levels of acetylation of histone core proteins in cochlear outer hair cells and attenuated gentamicin-induced cell death. The addition of sodium butyrate also rescued hair cells from damage by gentamicin. The results are consistent with an involvement of deficient histone acetylation in aminoglycoside-induced hair cell death by and point to the potential value of HDAC-inhibitors in protection from the side effects of these drugs.

We have previously demonstrated that oxidative stress increases in the inner ear of aging CBA/J mice and might contribute to the loss of function of the sensory system. We now investigate the activation of cell death pathways in the cochlea of these animals. Middle-aged (12 months) and old (18-26 months) mice with hearing deficits displayed outer hair cell nuclei with apoptotic and, to a lesser extent, necrotic features. Both intrinsic and extrinsic cell death pathways were activated by translocation or post-translational modification of proteins in the aging cochlea as compared to young (3 months) animals. Cytosolic cytochrome C increased, formed a complex with, and activated caspase 9. Endonuclease G translocated to the nuclei of aging outer hair cells suggesting its function as an apoptotic DNase. The cleaved (and hence active) forms of calpain I and calpain II increased while active cathepsin D was transiently elevated in middle-aged but not old animals. Finally, increases in the phosphorylation of p38 MAPK and JNK implicated the additional involvement of the MAPK pathway. The results suggest that multiple cell death pathways, all potentially linked to oxidative stress, are activated in hair cells of the auditory organ in aging mice.