Biology subjects

Riazuddin, S.

Publications and source records attributed to Riazuddin, S..

8 recordsLinked to original sources

bioRxiv2025

Methionine sulfoxide reductase B3 antioxidant activity is indispensable for mouse inner ear cuticular plate structure and hair bundle integrity

Methionine sulfoxide reductases (MSR) are enzymes responsible for catalyzing the reduction of methionine-sulfoxides. We previously demonstrated that variants in human MSRB3, a member of the MSR family, are associated with profound autosomal recessive prelingual non-syndromic hearing loss DFNB74. To better understand the role of MSRB3 in the auditory pathway, we generated a complete MsrB3 gene knock-out mouse model of human DFNB74 deafness. The MsrB3 deficient mouse showed profound hearing loss by postnatal day 16 (P16), which was accompanied by hair cell morphological abnormalities including altered stereocilia bundle shape and cuticular plate degeneration followed by hair cell apoptotic death. Although the absence of MSRB3 primarily affected the actin cytoskeleton, rootlets were present, and the expression and localization of major F-actin stereocilia-core proteins were unaltered. Biochemical assays demonstrated that wild-type MSRB3, but not MSRB3 harboring p.Cys89Gly, the same variant reported for human deafness DFNB74, can repolymerize oxidized actin. Consistent with these in vitro data, we observed a decreased ratio of reduced/total actin in the inner ears of Msrb3 knock-out mice. These data suggest a protective role of MSRB3 in the maintenance and maturation of stereocilia and hair cells, a conserved mechanism aimed at maintaining actin redox dynamics in these sensory cells.

genetics↗

bioRxiv2024

CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2KO/+;Whrnwi/+) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2KO/KO;Whrnwi/wi) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrnwi/wi mutants. Furthermore, over-expression of Whrn in Cib2KO/KOmice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.

neuroscience↗

bioRxiv2023

Calcium and Integrin-binding protein 2 (CIB2) controls force sensitivity of the mechanotransducer channels in cochlear outer hair cells

Calcium and Integrin-Binding Protein 2 (CIB2) is an essential subunit of the mechano-electrical transduction (MET) complex in mammalian auditory hair cells. CIB2 binds to pore-forming subunits of the MET channel, TMC1/2 and is required for their transport and/or retention at the tips of mechanosensory stereocilia. Since genetic ablation of CIB2 results in complete loss of MET currents, the exact role of CIB2 in the MET complex remains elusive. Here, we generated a new mouse strain with deafness-causing p.R186W mutation in Cib2 and recorded small but still measurable MET currents in the cochlear outer hair cells. We found that R186W variant causes increase of the resting open probability of MET channels, steeper MET current dependence on hair bundle deflection (I-X curve), loss of fast adaptation, and increased leftward shifts of I-X curves upon hair cell depolarization. Combined with AlphaFold2 prediction that R186W disrupts one of the multiple interacting sites between CIB2 and TMC1/2, our data suggest that CIB2 mechanically constraints TMC1/2 conformations to ensure proper force sensitivity and dynamic range of the MET channels. Using a custom piezo-driven stiff probe deflecting the hair bundles in less than 10 {micro}s, we also found that R186W variant slows down the activation of MET channels. This phenomenon, however, is unlikely to be due to direct effect on MET channels, since we also observed R186W-evoked disruption of the electron-dense material at the tips of mechanotransducing stereocilia and the loss of membrane-shaping BAIAP2L2 protein from the same location. We concluded that R186W variant of CIB2 disrupts force sensitivity of the MET channels and force transmission to these channels.

neuroscience↗

bioRxiv2023

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

neuroscience↗

bioRxiv2021

Potential therapy for progressive vision loss due to PCDH15-associated Usher syndrome developed in an orthologous Usher mouse

Usher syndrome type I (USH1) is characterized by congenital deafness, vestibular areflexia, and progressive retinal degeneration with age. The protein-truncating p.Arg245* founder variant of PCDH15 has an ~2% carrier frequency among Ashkenazi Jews, accounting for nearly 60% of their USH1 cases. Here, longitudinal ocular phenotyping in thirteen USH1F individuals harboring the p.Arg245* variant revealed progressive retinal degeneration, leading to severe loss of vision with macular atrophy by the sixth decade. Half of the affected individuals met either the visual acuity or visual field loss definition for legal blindness by the middle of their fifth decade of life. Mice homozygous for p.Arg250* (Pcdh15R250X; equivalent to human p.Arg245*) also have early visual deficits evaluated using electroretinography. Light-dependent translocation of phototransduction cascade proteins, arrestin and transducin, was found to be impaired in Pcdh15R250X mice. Retinal pigment epithelium-(RPE) specific visual retinoid cycle proteins, RPE65 which converts all-trans retinoids to 11-cis retinoids and CRALBP that transports retinoids, and key retinoid levels were also reduced in Pcdh15R250X mice, suggesting a dual role for protocadherin-15 in photoreceptors and RPE. Administration of exogenous 9-cis retinal, an analog of the naturally occurring 11-cis retinal, improved ERG amplitudes in these mutant mice, suggesting a basis for a clinical trial of exogenous FDA approved retinoids to preserve vision in USH1F patients. SummaryIn a preclinical setting studying exogenous retinoids using a novel Usher syndrome mouse model, we describe a potential therapy to treat PCDH15-mediated visual dysfunction.

neuroscience↗

bioRxiv2020

CIB2 regulates autophagy via Rheb-mTORC1 signaling axis

Age-related macular degeneration (AMD), a multifactorial neurodegenerative disorder, is the most common cause of vision loss in the elderly. Deficits in autophagy have been associated with age-related retinal pigment epithelium (RPE) pathology in mice, and dry-AMD in humans. In this study, we establish that the calcium and integrin binding protein 2 (CIB2) regulates autophagy in the RPE via Rheb-mTORC1 signaling axis. Cib2 mutant mice have reduced autophagic clearance in RPE and increased mTORC1 signaling - a negative regulator of autophagy. Concordant molecular deficits were also observed in RPE/choroid tissues from humans affected with dry AMD. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to nucleotide empty or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in aging, Tuberous sclerosis complex (TSC), and lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts and Tsc2 null cell line down-regulates hyperactive mTORC1 signaling. Thus, our findings have significant ramifications for the etiology of AMD and mTORC1 hyperactivity disorders and treatments.

cell biology↗

bioRxiv2020

Loss of CIB2 causes non-canonical autophagy deficits and visual impairment

Non-canonical autophagy or LC3-associated phagocytosis (LAP) is essential for the maintenance and functioning of the retinal pigment epithelium (RPE) and photoreceptors. Although molecular mechanisms still remain elusive, deficits in LAP have been found to be associated with age-related retinal pathology in both mice and humans. In this study, we found that calcium and integrin-binding protein 2 (CIB2) regulates LAP in the RPE. Mice lacking CIB2, both globally and specifically within RPE, have an impaired ability to process the engulfed photoreceptor outer segments due to reduced lysosomal capacity, which leads to marked accumulation of improperly digested remnants, lipid droplets, fused phago-melanosomes in RPE, and impaired visual function. In aged mice, we also found marked accumulation of drusen markers APOE, C3, and A{beta}, along with esterified cholesterol. Intriguingly, we were able to transiently rescue the photoreceptor function in Cib2 mutant mice by exogenous retinoid delivery. Our study links LAP and phagocytic clearance with CIB2, and their relevance to the sense of sight.

cell biology↗

bioRxiv2020

Novel Loss-of-Function Mutations in COCH Cause Autosomal Recessive Nonsyndromic Deafness

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly deafness in mice and humans. Two forms of deafness are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used mini-gene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA-splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA-splicing, and highlights the need to investigate the effect of coding variants on RNA-splicing.

genetics↗