Lysosomal ion homeostasis drives delayed hair cell death after aminoglycoside uptake.
Aminoglycoside ototoxicity has been widely reported and remains an important public health issue. Unfortunately, the molecular mechanisms of ototoxicity are not well understood. Here, we report the lysosome compartment as the main driver of delayed cell death triggered by aminoglycosides. By labeling early and late endosomes we show that endocytosis is not an significant path of aminoglycoside uptake. Instead, we show that aminoglycosides are delivered to lysosomes primarily through autophagy. Hair cells can be protected from damage by activation of the dual function lysosomal Two-Pore-Channel 2 (TPC2), stimulated by NAADP agonist but not by phosphoinositide PI(3,5,)P2 agonist. These treatments neutralize lysosomal pH. Moreover, luminal pH changes are also accompanied by changes in ferrous iron availability, though classical ferroptosis inhibitors do not prevent a delayed hair cell death. These findings reveal that lysosomal-driven delayed hair cell death is ferroptosis independent, suggesting that toxicity relies on a distinct mechanism that based on the internal conditions of the lysosomal compartment.