Modifier variant of METTL13 suppresses human GAB1-associated profound deafness

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Abstract

A modifier variant can abrogate risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin-homology-domain of GAB1, an essential scaffold in the MET/HGF pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescues the GAB1 associated morphant phenotype. In mouse, GAB1 and METTL13 co-localize in auditory sensory neurons, and METTL13 co-immunoprecipitates with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET-signaling provides potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET-signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET-signaling is likely to have broad clinical implications.

Authors

Rizwan Yousaf, Zubair M. Ahmed, Arnaud P.J. Giese, Robert J. Morell, Ayala Lagziel, Alain Dabdoub, Edward R. Wilcox, Sheikh Riazuddin, Thomas B. Friedman, Saima Riazuddin

Mitochondrial calcium uptake underlies ROS generation during aminoglycoside-induced hair cell death

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Abstract

Exposure to aminoglycoside antibiotics can lead to the generation of toxic levels of reactive oxygen species (ROS) within mechanosensory hair cells of the inner ear that have been implicated in hearing and balance disorders. Better understanding of the origin of aminoglycoside-induced ROS could focus the development of therapies aimed at preventing this event. In this work, we used the zebrafish lateral line system to monitor the dynamic behavior of mitochondrial and cytoplasmic oxidation occurring within the same dying hair cell following exposure to aminoglycosides. The increased oxidation observed in both mitochondria and cytoplasm of dying hair cells was highly correlated with mitochondrial calcium uptake. Application of the mitochondrial uniporter inhibitor Ru360 reduced mitochondrial and cytoplasmic oxidation, suggesting that mitochondrial calcium drives ROS generation during aminoglycoside-induced hair cell death. Furthermore, targeting mitochondria with free radical scavengers conferred superior protection against aminoglycoside exposure compared with identical, untargeted scavengers. Our findings suggest that targeted therapies aimed at preventing mitochondrial oxidation have therapeutic potential to ameliorate the toxic effects of aminoglycoside exposure.

Authors

Robert Esterberg, Tor Linbo, Sarah B. Pickett, Patricia Wu, Henry C. Ou, Edwin W. Rubel, David W. Raible

Designer aminoglycosides prevent cochlear hair cell loss and hearing loss

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Abstract

Bacterial infections represent a rapidly growing challenge to human health. Aminoglycosides are widely used broad-spectrum antibiotics, but they inflict permanent hearing loss in up to ~50% of patients by causing selective sensory hair cell loss. Here, we hypothesized that reducing aminoglycoside entry into hair cells via mechanotransducer channels would reduce ototoxicity, and therefore we synthesized 9 aminoglycosides with modifications based on biophysical properties of the hair cell mechanotransducer channel and interactions between aminoglycosides and the bacterial ribosome. Compared with the parent aminoglycoside sisomicin, all 9 derivatives displayed no or reduced ototoxicity, with the lead compound N1MS 17 times less ototoxic and with reduced penetration of hair cell mechanotransducer channels in rat cochlear cultures. Both N1MS and sisomicin suppressed growth of E. coli and K. pneumoniae, with N1MS exhibiting superior activity against extended spectrum β lactamase producers, despite diminished activity against P. aeruginosa and S. aureus. Moreover, systemic sisomicin treatment of mice resulted in 75% to 85% hair cell loss and profound hearing loss, whereas N1MS treatment preserved both hair cells and hearing. Finally, in mice with E. coli–infected bladders, systemic N1MS treatment eliminated bacteria from urinary tract tissues and serially collected urine samples, without compromising auditory and kidney functions. Together, our findings establish N1MS as a nonototoxic aminoglycoside and support targeted modification as a promising approach to generating nonototoxic antibiotics.

Authors

Markus E. Huth, Kyu-Hee Han, Kayvon Sotoudeh, Yi-Ju Hsieh, Thomas Effertz, Andrew A. Vu, Sarah Verhoeven, Michael H. Hsieh, Robert Greenhouse, Alan G. Cheng, Anthony J. Ricci

Optogenetic stimulation of the auditory pathway

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Abstract

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.

Authors

Victor H. Hernandez, Anna Gehrt, Kirsten Reuter, Zhizi Jing, Marcus Jeschke, Alejandro Mendoza Schulz, Gerhard Hoch, Matthias Bartels, Gerhard Vogt, Carolyn W. Garnham, Hiromu Yawo, Yugo Fukazawa, George J. Augustine, Ernst Bamberg, Sebastian Kügler, Tim Salditt, Livia de Hoz, Nicola Strenzke, Tobias Moser

Sound preconditioning therapy inhibits ototoxic hearing loss in mice

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Abstract

Therapeutic drugs with ototoxic side effects cause significant hearing loss for thousands of patients annually. Two major classes of ototoxic drugs are cisplatin and the aminoglycoside antibiotics, both of which are toxic to mechanosensory hair cells, the receptor cells of the inner ear. A critical need exists for therapies that protect the inner ear without inhibiting the therapeutic efficacy of these drugs. The induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death and hearing loss. We hypothesized that exposure to sound that is titrated to stress the inner ear without causing permanent damage would induce HSPs in the cochlea and inhibit ototoxic drug–induced hearing loss. We developed a sound exposure protocol that induces HSPs without causing permanent hearing loss. We used this protocol in conjunction with a newly developed mouse model of cisplatin ototoxicity and found that preconditioning mouse inner ears with sound has a robust protective effect against cisplatin-induced hearing loss and hair cell death. Sound therapy also provided protection against aminoglycoside-induced hearing loss. These data indicate that sound preconditioning protects against both classes of ototoxic drugs, and they suggest that sound therapy holds promise for preventing hearing loss in patients receiving these drugs.

Authors

Soumen Roy, Matthew M. Ryals, Astrid Botty Van den Bruele, Tracy S. Fitzgerald, Lisa L. Cunningham

Tricellulin deficiency affects tight junction architecture and cochlear hair cells

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Abstract

The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.

Authors

Gowri Nayak, Sue I. Lee, Rizwan Yousaf, Stephanie E. Edelmann, Claire Trincot, Christina M. Van Itallie, Ghanshyam P. Sinha, Maria Rafeeq, Sherri M. Jones, Inna A. Belyantseva, James M. Anderson, Andrew Forge, Gregory I. Frolenkov, Saima Riazuddin

The LINC complex is essential for hearing

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Abstract

Hereditary hearing loss is the most common sensory deficit. We determined that progressive high-frequency hearing loss in 2 families of Iraqi Jewish ancestry was due to homozygosity for the protein truncating mutation SYNE4 c.228delAT. SYNE4, a gene not previously associated with hearing loss, encodes nesprin-4 (NESP4), an outer nuclear membrane (ONM) protein expressed in the hair cells of the inner ear. The truncated NESP4 encoded by the families’ mutation did not localize to the ONM. NESP4 and SUN domain–containing protein 1 (SUN1), which localizes to the inner nuclear membrane (INM), are part of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the nuclear envelope. Mice lacking either Nesp4 or Sun1 were evaluated for hair cell defects and hearing loss. In both Nesp4^–/– and Sun1^–/– mice, OHCs formed normally, but degenerated as hearing matured, leading to progressive hearing loss. The nuclei of OHCs from mutant mice failed to maintain their basal localization, potentially affecting cell motility and hence the response to sound. These results demonstrate that the LINC complex is essential for viability and normal morphology of OHCs and suggest that the position of the nucleus in sensory epithelial cells is critical for maintenance of normal hearing.

Authors

Henning F. Horn, Zippora Brownstein, Danielle R. Lenz, Shaked Shivatzki, Amiel A. Dror, Orit Dagan-Rosenfeld, Lilach M. Friedman, Kyle J. Roux, Serguei Kozlov, Kuan-Teh Jeang, Moshe Frydman, Brian Burke, Colin L. Stewart, Karen B. Avraham

Mechanotransduction in mouse inner ear hair cells requires transmembrane channel–like genes

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Abstract

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel–like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1Δ mice) were deaf and those with a deletion of Tmc2 (Tmc2Δ mice) were phenotypically normal, Tmc1ΔTmc2Δ mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1ΔTmc2Δ mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

Authors

Yoshiyuki Kawashima, Gwenaëlle S.G. Géléoc, Kiyoto Kurima, Valentina Labay, Andrea Lelli, Yukako Asai, Tomoko Makishima, Doris K. Wu, Charles C. Della Santina, Jeffrey R. Holt, Andrew J. Griffith

Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition

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Abstract

Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3– into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.

Authors

Byung Yoon Choi, Hyoung-Mi Kim, Taku Ito, Kyu-Yup Lee, Xiangming Li, Kelly Monahan, Yaqing Wen, Elizabeth Wilson, Kiyoto Kurima, Thomas L. Saunders, Ronald S. Petralia, Philine Wangemann, Thomas B. Friedman, Andrew J. Griffith