Loss of vacuolar-type H+-ATPase induces caspase-independent necrosis-like death of hair cells in zebrafish neuromasts

Abstract

The vacuolar type H+-ATPase (V-ATPase) is a ubiquitous membrane-bound, multi-subunit proton pump that regulates pH of cellular compartments. V-ATPase activity is known to modulate several cellular processes, but cell type-specific V-ATPase functions remain poorly understood. Patients with mutations in specific V-ATPase subunits can develop sensorineural deafness, but underlying mechanisms are unclear. Here, we show that V-ATPase mutations disrupt formation of zebrafish neuromasts, which serve as a model system to investigate the underpinnings of hearing loss. Neuromasts consist of support cells surrounding mechanosensory hair cells that function similarly to hair cells in the mammalian inner ear. In V-ATPase mutant zebrafish embryos, neuromasts are small, malformed, and contain pyknotic nuclei that denote dying cells. Using molecular markers and live imaging, we find that loss of V-ATPase induces hair cells, but not neighboring support cells, to undergo caspase-independent necrosis-like cell death. This is the first demonstration that loss of V-ATPase can lead to necrosis-like cell death in a specific cell type in vivo. Mechanistically, loss of V-ATPase reduces mitochondrial membrane potential in hair cells, which has previously been associated with necrotic cell death. Modulating the mitochondrial permeability transition pore, which regulates mitochondrial membrane potential, improves hair cell survival. These results have implications for understanding causes of sensorineural deafness, and more broadly, reveal functions for V-ATPase in regulating mitochondrial function and promoting survival of a specific cell type in vivo.

The Role of Cilia in the Development, Survival, and Regeneration of Hair Cells

“…To do this fish were treated with RU360 at 500nM from 3-5 dpf and then fish were fixed and hair cells were labeled for counting. Similar to what had previously been found with extended RU360 treatment (Santra & Amack, 2021) we found a significant decrease in the number of hair cells in wild-type siblings. However, we saw no changes in hair cell number in either IFT gene mutant following RU360 treatment (Figure 4).…”

“…In other hair cell mutants with reduced hair cell number and mitochondrial membrane potential decreases it has been shown that treatment with the mitochondrial calcium uniporter inhibitor RU360 can partially rescue decreases in hair cell number (Santra & Amack, 2021). We wished to test if this was true for IFT gene mutants.…”

Calcium signaling mediates proliferation of the precursor cells that give rise to the ciliated left-right organizer in the zebrafish embryo

“…Fluorescent immunostaining was performed as previously described ( Santra and Amack, 2021 ). Embryos were fixed in 4% paraformaldehyde +1% tween-20 in 1X phosphate-buffered saline (PBS) overnight at 4°C, and then dechorionated and permeabilized in 100% acetone at −20°C for 8 min.…”