Autophagy proteins are essential for aminoglycoside-induced hearing loss

ABSTRACT

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.

Aminoglycosides (AGs) are highly potent antibiotics with a broad spectrum of activity against Gram-negative and Gram-positive bacteria. AGs are frequently used as first-line treatments for multiple life-threatening infections, particularly in developing countries, due to their low cost and low incidence of antibiotic resistance. However, AGs preferentially damage cochlear hair cells and cause permanent hearing loss in 20%-47% of AG-treated patients. Despite the fact that hundreds of millions of AG doses are consumed annually on a global scale, no effective treatment has been approved by the FDA to prevent hearing loss caused by AGs.

Tremendous progress has been made throughout the years in elucidating the mechanisms by which AGs cause irreversible hearing loss. Systemically administered AGs are transported across the blood-endolymph barrier and enter hair cells through their mechanotransducer (MET) channels. Previous studies have demonstrated that AG treatment activates the autophagy pathway in hair cells. However, the extent to which the autophagy pathway contributes to AG ototoxicity is controversial, based on pharmacological studies.

In our recent work [1], we found that AG binds directly to RIPOR2 (RHO family interacting cell polarization regulator 2), a protein abundantly expressed in cochlear hair cells and required for auditory perception. Immunostaining results show a rapid translocation of RIPOR2 in hair cells from their stereocilia to the apical part of the cell body within minutes, which requires the entry of AGs via MET channels (Figure 1). As AGs specifically trigger a rapid localization change of RIPOR2 in hair cells, we hypothesized that RIPOR2 is essential for AG-induced hair cell death. To test this hypothesis, we injected AGs subcutaneously into wild-type control mice and heterozygous Ripor2^+/- mice, which have normal hearing but significantly reduced RIPOR2 expression. After 14 days of AG treatment, wild-type mice lose more than 75% of their hair cells and their hearing threshold is elevated by over 60 dB. Remarkably, neither significant hair cell death nor hearing loss is detected in Ripor2^+/- mice, suggesting that Ripor2^+/- mice are completely insusceptible to AG-induced hair cell death and subsequent hearing loss.

Figure 1.

Model of AG-induced hair cell death. AGs enter hair cells via the mechanotransduction (MET) channels localized on the top of the stereocilia, which are specialized mechanosensory subcellular organelles in hair cells. AGs bind to RIPOR2 and trigger its rapid translocation. Then, RIPOR2 binds to GABARAP, activates the autophagy/mitophagy pathway, and ultimately causes hair cell death. The diagram is redrawn from Li et al. (2022).

We hypothesized that RIPOR2 initiates a specific molecular pathway that eventually results in hair cell death. Full-length RIPOR2 was then used as the bait to screen a previously constructed inner ear-specific yeast two-hybrid library. Interactions between RIPOR2 and GABARAP (gamma-aminobutyric acid receptor associated protein) and GABARAPL1, orthologs of yeast Atg8 and key autophagy proteins, were identified. GABARAP is abundantly expressed in cochlear hair cells. Following AG treatment, RIPOR2 colocalizes with and activates GABARAP in hair cells. Monitoring LC3B lipidation by western blotting, and fluorescence intensity of an autophagy dye by imaging of living cells revealed the activation of autophagy in wild-type hair cells, but not in Ripor2 or Gabarap mutant hair cells, suggesting that RIPOR2 and GABARAP are essential for AG-induced autophagy activation. Next, mice lacking GABARAP were characterized. The gabarap null mutant mice have normal hearing and their hair cells take up AGs normally. In a remarkable similarity to the Ripor2 heterozygous mice, gabarap^−/− mutant mice do not exhibit significant hair cell loss or hearing threshold elevation, whereas robust hair cell death and profound hearing loss are observed in control wild-type mice. To further investigate whether the autophagy pathway is essential for AG-induced hearing loss, mice lacking LC3B, which also have normal hearing, were characterized. Similar to the Ripor2^+/- and gabarap^−/− mutant mice, lc3b^−/− mutant mice are also entirely resistant to AG ototoxicity, suggesting that the autophagy pathway is indeed essential for AG-induced hair cell death and subsequent hearing loss.

Given the fact that mutations in mt-Rnr1 (mitochondrially encoded 12S rRNA) are associated with a higher vulnerability to AG ototoxicity in humans, mitochondria probably play a substantial role in AG-induced hearing loss. It is important to note that cells with those mutations in the mitochondrial mt-Rnr1 rRNA exhibit elevated mitophagy, which is a mitochondrial-specific autophagy. Thus, we hypothesized that mitophagy is involved in the AG ototoxicity. Indeed, treatment with AG induces an increase in the fluorescence intensity of a mitophagy dye in hair cells. Furthermore, by phenotyping corresponding knockout mice, we studied the functions of PINK1 and PRKN, two key mitophagy proteins, in AG ototoxicity. Similar to the Ripor2, gabarap and lc3b mutant mice, both Pink1 and Prkn heterozygous or homozygous mice are resistant to AG ototoxicity.

Taken together, our findings suggest that AGs aberrantly activate the autophagy/mitophagy pathway, and ultimately cause hair cell death and permanent hearing loss. Several key components in the autophagy pathway identified in our studies are highly promising therapeutic drug targets for the prevention of hearing loss caused by AGs.

Author contribution

Funding acquisition - B. Z., U. M.; Writing (original draft) - B. Z.; Writing (review & editing) - all authors.

Funding Statement

This work was supported by National Institute on Deafness and Other Communication Disorders (NIDCD) grant DC017147 (B. Z.), DC018785 (B. Z.), DC005965 (U. M.) and Indiana University School of Medicine startup funding (B. Z.).

Disclosure statement

J. L., U. M. and B. Z. are named inventors on patent applications related to this work. U. M. is a co-founder of Decibel Therapeutics.