Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.
Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.
Lgr5+ cochlear supporting cells (SCs) have been reported to be hair cell (HC) progenitor cells that have the ability to regenerate HCs in the neonatal mouse cochlea, and these cells are regulated by Wnt signaling. Frizzled-9 (Fzd9), one of the Wnt receptors, has been reported to be used to mark neuronal stem cells in the brain together with other markers and mesenchymal stem cells from human placenta and bone marrow. Here we used Fzd9-CreER mice to lineage label and trace Fzd9+ cells in the postnatal cochlea in order to investigate the progenitor characteristic of Fzd9+ cells. Lineage labeling showed that inner phalangeal cells (IPhCs), inner border cells (IBCs), and third-row Deiters’ cells (DCs) were Fzd9+ cells, but not inner pillar cells (IPCs) or greater epithelial ridge (GER) cells at postnatal day (P)3, which suggests that Fzd9+ cells are a much smaller cell population than Lgr5+ progenitors. The expression of Fzd9 progressively decreased and was too low to allow lineage tracing after P14. Lineage tracing for 6 days
in vivo
showed that Fzd9+ cells could also generate similar numbers of new HCs compared to Lgr5+ progenitors. A sphere-forming assay showed that Fzd9+ cells could form spheres after sorting by flow cytometry, and when we compared the isolated Fzd9+ cells and Lgr5+ progenitors there were no significant differences in sphere number or sphere diameter. In a differentiation assay, the same number of Fzd9+ cells could produce similar amounts of Myo7a+ cells compared to Lgr5+ progenitors after 10 days of differentiation. All these data suggest that the Fzd9+ cells have a similar capacity for proliferation, differentiation, and HC generation as Lgr5+ progenitors and that Fzd9 can be used as a more restricted marker of HC progenitors.
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