Background The frequent use of systematic aminoglycoside (AG) treatment has resulted in a widespread trend of permanent hearing loss globally. Previously, we found that AGs induce a rapid translocation of RIPOR2 in hair cells, which is a process that relies on functional mechanotransduction and subsequently dysregulates the autophagy/mitophagy pathway, leading to irreversible hair cell death. Recent studies found that AGs also trigger rapid phosphatidylserine (PS) externalization in hair cells, possibly by activating the scramblase activity of TMC1/2, which are the pore-forming subunits of the mechanotransduction channel. Since both processes are AG-triggered and mechanotransduction-dependent, we investigated if RIPOR2 translocation and PS externalization occurred through dependent pathways.

Methods To determine whether AG-triggered rapid RIPOR2 translocation and PS externalization are dependent, RIPOR2 translocation and PS externalization were observed with fluorescent microscopy in wild-type hair cells treated with gentamycin. Tmie -/- cochlea were also treated with gentamycin to investigate the role of core mechanotransduction channel proteins TMC1/2 in the aforementioned processes. To study the effect of PS externalization on RIPOR2 translocation in hair cells, we treated wild type cells with Benzamil, which induces PS externalization through MET channel blockage. Finally, we treated wild type hair cells with cisplatin, a drug that shares several pathological features of ototoxicity with AGs, to observe its effects on PS externalization and RIPOR2 localization in hair cells.

Results Gentamycin can trigger both RIPOR2 translocation and PS externalization in wild type mice, but not in Tmie -/- mice. Throughout the 2-hour gentamycin treatment, PS externalization and RIPOR2 translocation displayed distinct spatiotemporal distributions. We found that Benzamil, which is sufficient to trigger PS externalization, was unable to trigger RIPOR2 translocation under our experimental conditions. In addition, we observed that cisplatin treatment, unlike gentamycin treatment, was unable to trigger either RIPOR2 translocation or PS externalization.

Conclusion Aminoglycoside treatment likely triggers RIPOR2 translocation and PS externalization via independent mechanisms, considering that both phenomena display distinct spatiotemporal patterns with gentamycin treatment, and certain MET-blocking reagents that induce PS externalization (e.g. Benzamil) fail to induce RIPOR2 translocation. However, the failure for gentamycin to induce either phenomenon in Tmie -/- cells suggests that the MET channel plays a significant role in the function of both pathways. Because cisplatin could neither induce PS externalization nor RIPOR2 translocation, it is probable that cisplatin triggers hair cell death through a pathway distinct from that of aminoglycoside-mediated ototoxicity.