Bo Zhao

Aminoglycosides are widely used to treat severe infections. However, systemically administered AGs preferentially kill cochlear hair cells, resulting in irreversible hearing loss. Recently, we found that AGs induce a rapid translocation of RIPOR2 in hair cells, a process that relies on functional mechanotransduction, subsequently dysregulates the autophagy/mitophagy pathway, and ultimately leads to irreversible hair cell death. Recent studies found that AGs also trigger rapid phosphatidylserine (PS) externalization in hair cells, probably by activating the scramblase activity of TMC1/2, which are the pore-forming subunits of the mechanotransduction channel. To determine whether AG-triggered rapid RIPOR2 translocation and PS externalization are independent, RIPOR2 translocation and PS externalization were extensively investigated in wild-type hair cells treated with AG for different amounts of time. Next, the potential effect of PS externalization on RIPOR2 translocation in hair cells was studied. Finally, we investigated the extent to which cisplatin, a chemotherapy drug that shares several pathological features of ototoxicity with AGs, affects PS externalization and RIPOR2 localization in hair cells. Our results suggest that AG triggers RIPOR2 translocation and PS externalization by independent mechanisms, and that cisplatin and AGs induce hair cell death via distinct molecular pathways.

Pathogenic variants in the gene TMPRSS3 are a common cause of hearing loss in humans, although the causal mechanisms remain unknown. Previous work has shown that Tmprss3 ^Y260X/Y260X mice exhibit normal hair cell development, mechanosensory transduction, and spiral ganglion patterning, but experience rapid hair cell death from P12 to P14 at the onset of hearing. Here, we demonstrate that Tmprss3 ^Y260X/Y260X mice display an early and temporary spike in endocochlear potential (EP) prior to the onset of hair cell death. In vitro experiments with cochlear explants from Tmprss3 ^Y260X/Y260X mice and in vivo studies with Tmprss3 ^Y260X/Y260X mice crossed with 2 different mutant models that lacked EP generation promoted hair cell survival. Furthermore, systemic administration of furosemide, a drug that reduces EP in vivo, reduced hair cell death in Tmprss3 ^Y260X/Y260X mice. These findings suggest that extracellular factors, including EP, play a role in TMPRSS3-related hair cell survival and hearing loss, and suggest that modulating EP could be a therapeutic strategy.

A functional skin barrier is essential for restricting water losses for terrestrial animals. The outermost layer of this barrier, the stratum corneum, consists of corneocytes (derived from terminally differentiated keratinocytes) coated with a protein‐rich envelope and a conjugated lipid‐rich envelope embedded in an organised hydrophobic intercellular lipid matrix. Gene mutations and environmental insults that affect corneocyte formation or the intercellular lipid matrix organisation cause skin barrier defects in mice and humans. Here we demonstrate mice homozygous for a loss‐of‐function mutation in the Lipm gene exhibit a diminished and discontinuous intercellular lipid matrix and a profound skin barrier defect associated with neonatal lethality. These findings suggest the protein encoded by the Lipm gene, lipase M (LIPM), is essential for skin barrier function by impacting intercellular lipid matrix organisation in the stratum corneum.

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin–bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.

Aminoglycosides (AGs) are highly potent, broad-spectrum antibiotics frequently used as first-line treatments for multiple life-threatening infections. Despite their severe ototoxicity, causing irreversible hearing loss in millions of people annually, no preventive therapy has been approved. We previously reported that GABARAP and several other central autophagy proteins are essential for AG-induced hearing loss. This finding opens avenues for the rational design and development of inhibitors that selectively target proteins in this pathway, thereby mitigating AG ototoxicity. In this study, we generated a mouse model with a targeted deletion of GABARAPL1, a homolog of GABARAP, and another model deficient in both GABARAP and GABARAPL1. We found that normal hearing is unaffected by the depletion of these proteins. Remarkably, both proteins are essential for AG-induced hearing loss, with GABARAP playing a more significant role. To further explore the therapeutic potential, we designed and validated short hairpin RNAs targeting the mouse and human GABARAP gene. By inhibiting GABARAP expression in inner ear hair cells using adeno-associated virus-mediated RNA interference, we successfully prevented AG-induced hair cell death and subsequent hearing loss. Our findings underscore the critical role of GABARAP in AG ototoxicity and highlight its potential as a therapeutic target for preventing AG-induced hearing loss.

Aminoglycosides (AGs) are widely used to treat severe infections. However, systemically administered AGs preferentially kill cochlear hair cells, resulting in irreversible hearing loss. Recently, we found that AGs bind to RIPOR2 and trigger its rapid translocation in cochlear hair cells. Reducing RIPOR2 expression entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Next using yeast two-hybrid screening, we found that RIPOR2 interacts with GABARAP, a key macroautophagy/autophagy pathway protein. Following AG treatment, RIPOR2 colocalizes with GABARAP and regulates the activation of autophagy. Remarkably, reducing the expression of GABARAP, or another key autophagy protein MAP1LC3B/LC3B, entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Furthermore, we found that AGs activate the autophagy pathway specific to mitochondria. Reducing the expression of PINK1 or PRKN/parkin, two key mitophagy proteins, protects hair cells against AG toxicity. Thus, our findings demonstrated that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss and provided potential therapeutic strategies for preventing AG toxicity.

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.