Browsing by Author "Liu, Chang"

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    ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points
    (JCB, 2022-02-17) Krey, Jocelyn F.; Liu, Chang; Belyantseva, Inna A.; Bateschell, Michael; Dumont, Rachel A.; Goldsmith, Jennifer; Chatterjee, Paroma; Morrill, Rachel S.; Fedorov, Lev M.; Foster, Sarah; Kim, Jinkyung; Nuttall, Alfred L.; Jones, Sherri M.; Choi, Dongseok; Friedman, Thomas B.; Ricci, Anthony J.; Zhao, Bo; Barr-Gillespie, Peter G.; Otolaryngology -- Head and Neck Surgery, School of Medicine
    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.
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    Autophagy proteins are essential for aminoglycoside-induced hearing loss
    (Taylor & Francis, 2023) Li, Jinan; Liu, Chang; Müller, Ulrich; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    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.
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    Collapsin Response Mediator Protein 1 (CRMP1) Is Required for High-Frequency Hearing
    (Elsevier, 2022) Li, Jinan; Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Collapsin response mediator protein 1 (CRMP1), also known as dihydropyrimidinase-related protein 1, participates in cytoskeleton remodeling during axonal guidance and neuronal migration. In cochlear hair cells, the assembly and maintenance of the cytoskeleton is of great interest because it is crucial for the morphogenesis and maintenance of hair cells. Previous RNA sequencing analysis found that Crmp1 is highly expressed in cochlear hair cells. However, the expression profile and functions of CRMP1 in the inner ear remain unknown. In this study, the expression and localization of CRMP1 in hair cells was investigated using immunostaining, and was shown to be highly expressed in both outer and inner hair cells. Next, the stereocilia morphology of Crmp1-deficient mice was characterized. Abolishing CRMP1 did not affect the morphogenesis of hair cells. Interestingly, scanning electron microscopy detected hair cell loss at the basal cochlear region, an area responsible for high-frequency auditory perception, in Crmp1-deficient mice. Correspondingly, an auditory brainstem response test showed that mice lacking CRMP1 had progressive hearing loss at high frequencies. In summary, these data suggest that CRMP1 is required for high-frequency auditory perception.
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    GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing
    (Elsevier, 2018-10-30) Liu, Chang; Luo, Na; Tung, Chun-Yu; Perrin, Benjamin J.; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Mutations in human GRXCR2, which encodes a protein of undetermined function, cause hearing loss by unknown mechanisms. We found that mouse GRXCR2 localizes to the base of the stereocilia, which are actin-based mechanosensing organelles in cochlear hair cells that convert sound-induced vibrations into electrical signals. The stereocilia base also contains taperin, another protein of unknown function required for human hearing. We show that taperin and GRXCR2 form a complex and that taperin is diffused throughout the stereocilia length in Grxcr2-deficient hair cells. Stereocilia lacking GRXCR2 are longer than normal and disorganized due to the mislocalization of taperin, which could modulate the actin cytoskeleton in stereocilia. Remarkably, reducing taperin expression levels could rescue the morphological defects of stereocilia and restore the hearing of Grxcr2-deficient mice. Thus, our findings suggest that GRXCR2 is critical for the morphogenesis of stereocilia and auditory perception by restricting taperin to the stereocilia base.
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    Murine GRXCR1 Has a Different Function Than GRXCR2 in the Morphogenesis of Stereocilia
    (Frontiers Media, 2021-07-21) Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Mutations in human glutaredoxin domain-containing cysteine-rich protein 1 (GRXCR1) and its paralog GRXCR2 have been linked to hearing loss in humans. Although both GRXCR1 and GRXCR2 are required for the morphogenesis of stereocilia in cochlear hair cells, a fundamental question that remains unclear is whether GRXCR1 and GRXCR2 have similar functions in hair cells. Previously, we found that GRXCR2 is critical for the stereocilia morphogenesis by regulating taperin localization at the base of stereocilia. Reducing taperin expression level rescues the morphological defects of stereocilia and hearing loss in Grxcr2-deficient mice. So far, functions of GRXCR1 in mammalian hair cells are still unclear. Grxcr1-deficient hair cells have very thin stereocilia with less F-actin content inside, which is different from Grxcr2-deficient hair cells. In contrast to GRXCR2, which is concentrated at the base of stereocilia, GRXCR1 is diffusely distributed throughout the stereocilia. Notably, GRXCR1 interacts with GRXCR2. In Grxcr1-deficient hair cells, the expression level of GRXCR2 and taperin is reduced. Remarkably, different from that in Grxcr2-deficient mice, reducing taperin expression level does not rescue the morphological defects of stereocilia or hearing loss in Grxcr1-deficient mice. Thus, our findings suggest that GRXCR1 has different functions than GRXCR2 during the morphogenesis of stereocilia.
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    N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception
    (Frontiers Media, 2021-05-05) Li, Jinan; Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Stereocilia of cochlear hair cells are specialized mechanosensing organelles that convert sound-induced vibration to electrical signals. Glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2) is localized at the base of stereocilia and is necessary for stereocilia morphogenesis and auditory perception. However, the detailed functions of GRXCR2 in hair cells are still largely unknown. Here, we report that GRXCR2 interacts with chloride intracellular channel protein 5 (CLIC5) which is also localized at the base of stereocilia and required for normal hearing in human and mouse. Immunolocalization analyses suggest that GRXCR2 is not required for the localization of CLIC5 to the stereociliary base during development, or vice versa. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we deleted 60 amino acids near the N-terminus of GRXCR2 essential for its interaction with CLIC5. Interestingly, mice harboring this in-frame deletion in Grxcr2 exhibit moderate hearing loss at lower frequencies and severe hearing loss at higher frequencies although the morphogenesis of stereocilia is minimally affected. Thus, our findings reveal that the interaction between GRXCR2 and CLIC5 is crucial for normal hearing.
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    Reducing Taperin Expression Restores Hearing in Grxcr2 Mutant Mice
    (Elsevier, 2022) Liu, Chang; Luo, Na; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Recessive mutations in GRXCR2 cause deafness in both humans and mice. In Grxcr2 null hair cells, the sensory receptors for sound in the inner ear, stereocilia are disorganized. Reducing the expression of taperin, a protein that interacts with GRXCR2 at the base of stereocilia, corrects the morphological defects of stereocilia and restores hearing in Grxcr2 null mice. To further validate this finding, this study generated two novel taperin mutant mouse lines that exhibit progressive hearing loss. Then Grxcr2 null mice were crossed with one of these taperin mutant mice. The following morphological analysis revealed that reducing taperin expression indeed corrected stereocilia morphological abnormalities in Grxcr2 null mice. Functional analysis further confirmed that reducing taperin expression partially restored hearing in Grxcr2 null mice.
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    A RIPOR2 in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss
    (BMJ, 2020) de Bruijn, Suzanne E.; Smits, Jeroen J.; Liu, Chang; Lanting, Cornelis P.; Beynon, Andy J.; Blankevoort, Joëlle; Oostrik, Jaap; Koole, Wouter; de Vrieze, Erik; Cremers, Cor W.R.J.; Cremers, Frans P.M.; Roosing, Susanne; Yntema, Helger G.; Kunst, Henricus P.M.; Zhao, Bo; Pennings, Ronald J.E.; Kremer, Hannie; DOOFNL Consortium; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Background: Hearing loss is one of the most prevalent disabilities worldwide, and has a significant impact on quality of life. The adult-onset type of the condition is highly heritable but the genetic causes are largely unknown, which is in contrast to childhood-onset hearing loss. Methods: Family and cohort studies included exome sequencing and characterisation of the hearing phenotype. Ex vivo protein expression addressed the functional effect of a DNA variant. Results: An in-frame deletion of 12 nucleotides in RIPOR2 was identified as a highly penetrant cause of adult-onset progressive hearing loss that segregated as an autosomal dominant trait in 12 families from the Netherlands. Hearing loss associated with the deletion in 63 subjects displayed variable audiometric characteristics and an average (SD) age of onset of 30.6 (14.9) years (range 0-70 years). A functional effect of the RIPOR2 variant was demonstrated by aberrant localisation of the mutant RIPOR2 in the stereocilia of cochlear hair cells and failure to rescue morphological defects in RIPOR2-deficient hair cells, in contrast to the wild-type protein. Strikingly, the RIPOR2 variant is present in 18 of 22 952 individuals not selected for hearing loss in the Southeast Netherlands. Conclusion: Collectively, the presented data demonstrate that an inherited form of adult-onset hearing loss is relatively common, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an attractive target for developing a (genetic) therapy.
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    RIPOR2-mediated autophagy dysfunction is critical for aminoglycoside-induced hearing loss
    (Elsevier, 2022) Li, Jinan; Liu, Chang; Müller, Ulrich; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Aminoglycosides (AGs) are potent antibiotics capable of treating a wide variety of life-threatening infections, however, they are ototoxic and cause irreversible damage to cochlear hair cells. Despite substantial progress, little is known about the molecular pathways critical for hair cell function and survival that are affected by AG exposure. We demonstrate here that gentamicin, a representative AG antibiotic, binds to and triggers within minutes translocation of RIPOR2 in murine hair cells from stereocilia to the pericuticular area. Then, by interacting with a central autophagy component GABARAP, RIPOR2 affects autophagy activation. Reducing the expression of RIPOR2 or GABARAP completely prevents AG-induced hair cell death and subsequent hearing loss in mice. Additionally, abolishing the expression of PINK1 or Parkin, two key mitochondrial autophagy proteins, prevents hair cell death and subsequent hearing loss caused by AG. In summary, our study demonstrates that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss.
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    The Mechanotransduction Channel and Organic Cation Transporter Are Critical for Cisplatin Ototoxicity in Murine Hair Cells
    (Frontiers, 2022-02-10) Li , Jinan; Liu, Chang; Kaefer , Samuel; Youssef , Mariam; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Cisplatin is one of the most widely used chemotherapeutic drugs across the world. However, the serious ototoxic effects, leading to permanent hair cell death and hearing loss, significantly limit the utility of cisplatin. In zebrafish, the functional mechanotransduction channel is required for cisplatin ototoxicity. However, it is still unclear the extent to which the mechanotransduction channel is involved in cisplatin uptake and ototoxicity in mammalian hair cells. Herein, we show that genetically disrupting mechanotransduction in mouse partially protects hair cells from cisplatin-induced hair cell death. Using a fluorescent-dye conjugated cisplatin, we monitored cisplatin uptake in cochlear explants and found that functional mechanotransduction is required for the uptake of cisplatin in murine hair cells. In addition, cimetidine, an inhibitor of the organic cation transporter, also partially protects hair cells from cisplatin ototoxicity. Notably, the otoprotective effects of cimetidine do not require mechanotransduction. These findings suggest that both the mechanotransduction channel and the organic cation transporter are critical for cisplatin ototoxicity in murine hair cells.