@article{Redfield2023.10.08.23296081,

title = {PKHD1L1, A Gene Involved in the Stereociliary Coat, Causes Autosomal Recessive Nonsyndromic Hearing Loss},

author = {Shelby E Redfield and Pedro De-la-Torre and Mina Zamani and Hina Khan and Tyler Morris and Gholamreza Shariati and Majid Karimi and Margaret A Kenna and Go Hun Seo and Sadaf Naz and Hamid Galehdari and Artur A Indzhykulian and A. Eliot Shearer and Barbara Vona},

url = {https://www.medrxiv.org/content/early/2023/10/09/2023.10.08.23296081},

doi = {10.1101/2023.10.08.23296081},

year  = {2023},

date = {2023-10-08},

urldate = {2023-10-08},

journal = {medRxiv [Preprint]},

publisher = {Cold Spring Harbor Laboratory Press},

abstract = {Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor, given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of three families segregating autosomal recessive moderate to severe nonsyndromic sensorineural hearing loss. Compound heterozygous missense p.[(Gly129Ser)];p.[(Gly1314Val)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families. In vitro functional assessment indicated that both engineered PKHD1L1 mutant p.(Gly129Ser) and p.(Gly1314Val) constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. In silico molecular modelling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on protein folding and stability and exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild-moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild-moderate hearing loss may identify further affected families.Competing Interest StatementGo Hun Seo is an employee of 3Billion, Inc.Funding StatementThis work was supported by NIDCD K08 DC019716 to AES, the De Garay Family Fund to MAK with support from the Boston Childrens Rare Disease Cohort Initiative, and the German Research Foundation (DFG) VO 2138/7-1 grant 469177153 to BV, and by NIH R01DC017166 (NIDCD) and R01DC020190 (NIDCD) to AAI. Funding for work in Pakistan was provided by the University of the Punjab (SN).Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Institutional review boards of Boston Childrens Hospital (IRB P-00031494), University Medical Center Goettingen (No. 3/2/16), and the School of Biological Sciences, University of Punjab, Lahore, Pakistan (IRB No. 00005281) gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37693382,

title = {Large-scale annotated dataset for cochlear hair cell detection and classification},

author = {Christopher J Buswinka and David B Rosenberg and Rubina G Simikyan and Richard T Osgood and Katharine Fernandez and Hidetomi Nitta and Yushi Hayashi and Leslie W Liberman and Emily Nguyen and Erdem Yildiz and Jinkyung Kim and Amandine Jarysta and Justine Renauld and Ella Wesson and Punam Thapa and Pierrick Bordiga and Noah McMurtry and Juan Llamas and Siân R Kitcher and Ana I López-Porras and Runjia Cui and Ghazaleh Behnammanesh and Jonathan E Bird and Angela Ballesteros and A Catalina Vélez-Ortega and Albert Sb Edge and Michael R Deans and Ksenia Gnedeva and Brikha R Shrestha and Uri Manor and Bo Zhao and Anthony J Ricci and Basile Tarchini and Martin Basch and Ruben S Stepanyan and Lukas D Landegger and Mark Rutherford and M Charles Liberman and Bradley J Walters and Corné J Kros and Guy P Richardson and Lisa L Cunningham and Artur A Indzhykulian},

doi = {10.1101/2023.08.30.553559},

year  = {2023},

date = {2023-09-01},

urldate = {2023-09-01},

journal = {bioRxiv [Preprint]},

abstract = {Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following  and  ototoxic drug application. The dataset includes over 90,000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37312453,

title = {Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F},

author = {Cole W Peters and Killian S Hanlon and Maryna V Ivanchenko and Eric Zinn and Elizabeth F Linarte and Yaqiao Li and Jonathan M Levy and David R Liu and Benjamin P Kleinstiver and Artur A Indzhykulian and David P Corey},

doi = {10.1016/j.ymthe.2023.06.007},

issn = {1525-0024},

year  = {2023},

date = {2023-08-01},

journal = {Mol Ther},

volume = {31},

number = {8},

pages = {2439--2453},

abstract = {Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15 mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37214838,

title = {SKOOTS: Skeleton oriented object segmentation for mitochondria},

author = {Christopher J Buswinka and Hidetomi Nitta and Richard T Osgood and Artur A Indzhykulian},

doi = {10.1101/2023.05.05.539611},

year  = {2023},

date = {2023-05-01},

urldate = {2023-05-01},

journal = {bioRxiv [Preprint]},

abstract = {The segmentation of individual instances of mitochondria from imaging datasets is informative, yet time-consuming to do by hand, sparking interest in developing automated algorithms using deep neural networks. Existing solutions for various segmentation tasks are largely optimized for one of two types of biomedical imaging: high resolution three-dimensional (whole neuron segmentation in volumetric electron microscopy datasets) or two-dimensional low resolution (whole cell segmentation of light microscopy images). The former requires consistently predictable boundaries to segment large structures, while the latter is boundary invariant but struggles with segmentation of large 3D objects without downscaling. Mitochondria in whole cell 3D EM datasets often occupy the challenging middle ground: large with ambiguous borders, limiting accuracy with existing tools. To rectify this, we have developed  eleton  riented  bjec  egmentation (SKOOTS); a new segmentation approach which efficiently handles large, densely packed mitochondria. We show that SKOOTS can accurately, and efficiently, segment 3D mitochondria in previously difficult situations. Furthermore, we will release a new, manually annotated, 3D mitochondria segmentation dataset. Finally, we show this approach can be extended to segment objects in 3D light microscopy datasets. These results bridge the gap between existing segmentation approaches and increases the accessibility for three-dimensional biomedical image analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37068229,

title = {Reprogramming by drug-like molecules leads to regeneration of cochlear hair cell-like cells in adult mice},

author = {Yi-Zhou Quan and Wei Wei and Volkan Ergin and Arun Prabhu Rameshbabu and Mingqian Huang and Chunjie Tian and Srinivas Vinod Saladi and Artur A Indzhykulian and Zheng-Yi Chen},

doi = {10.1073/pnas.2215253120},

issn = {1091-6490},

year  = {2023},

date = {2023-04-01},

journal = {Proc Natl Acad Sci U S A},

volume = {120},

number = {17},

pages = {e2215253120},

abstract = {Strategies to overcome irreversible cochlear hair cell (HC) damage and loss in mammals are of vital importance to hearing recovery in patients with permanent hearing loss. In mature mammalian cochlea, co-activation of  and  reprograms supporting cells (SC) and promotes HC regeneration. Understanding of the underlying mechanisms may aid the development of a clinically relevant approach to achieve HC regeneration in the nontransgenic mature cochlea. By single-cell RNAseq, we show that MYC/NICD "rejuvenates" the adult mouse cochlea by activating multiple pathways including Wnt and cyclase activator of cyclic AMP (cAMP), whose blockade suppresses HC-like cell regeneration despite / activation. We screened and identified a combination (the cocktail) of drug-like molecules composing of small molecules and small interfering RNAs to activate the pathways of  and  We show that the cocktail effectively replaces  and  transgenes and reprograms fully mature wild-type (WT) SCs for HC-like cells regeneration in vitro. Finally, we demonstrate the cocktail is capable of reprogramming adult cochlea for HC-like cells regeneration in WT mice with HC loss in vivo. Our study identifies a strategy by a clinically relevant approach to reprogram mature inner ear for HC-like cells regeneration, laying the foundation for hearing restoration by HC regeneration.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37100771,

title = {Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F},

author = {Maryna V Ivanchenko and Daniel M Hathaway and Alex J Klein and Bifeng Pan and Olga Strelkova and Pedro De-la-Torre and Xudong Wu and Cole W Peters and Eric M Mulhall and Kevin T Booth and Corey Goldstein and Joseph Brower and Marcos Sotomayor and Artur A Indzhykulian and David P Corey},

doi = {10.1038/s41467-023-38038-y},

issn = {2041-1723},

year  = {2023},

date = {2023-04-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {2400},

abstract = {Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36947567,

title = {The hair cell analysis toolbox is a precise and fully automated pipeline for whole cochlea hair cell quantification},

author = {Christopher J Buswinka and Richard T Osgood and Rubina G Simikyan and David B Rosenberg and Artur A Indzhykulian},

doi = {10.1371/journal.pbio.3002041},

issn = {1545-7885},

year  = {2023},

date = {2023-03-01},

journal = {PLoS Biol},

volume = {21},

number = {3},

pages = {e3002041},

abstract = {Our sense of hearing is mediated by sensory hair cells, precisely arranged and highly specialized cells subdivided into outer hair cells (OHCs) and inner hair cells (IHCs). Light microscopy tools allow for imaging of auditory hair cells along the full length of the cochlea, often yielding more data than feasible to manually analyze. Currently, there are no widely applicable tools for fast, unsupervised, unbiased, and comprehensive image analysis of auditory hair cells that work well either with imaging datasets containing an entire cochlea or smaller sampled regions. Here, we present a highly accurate machine learning-based hair cell analysis toolbox (HCAT) for the comprehensive analysis of whole cochleae (or smaller regions of interest) across light microscopy imaging modalities and species. The HCAT is a software that automates common image analysis tasks such as counting hair cells, classifying them by subtype (IHCs versus OHCs), determining their best frequency based on their location along the cochlea, and generating cochleograms. These automated tools remove a considerable barrier in cochlear image analysis, allowing for faster, unbiased, and more comprehensive data analysis practices. Furthermore, HCAT can serve as a template for deep learning-based detection tasks in other types of biological tissue: With some training data, HCAT's core codebase can be trained to develop a custom deep learning detection model for any object on an image.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shindyapina2021.02.23.432500,

title = {Integrative analysis reveals aged clonal B cells, microenvironment and c-Myc activation in the origin of age-related lymphoma},

author = {Anastasia V. Shindyapina and José P. Castro and Alessandro Barbieri and Olga S. Strelkova and João A. Paulo and Csaba Kerepesi and Anna P. Petrashen and Marco Mariotti and Margarita Meer and Yan Hu and Alexander Karamyshev and Grigoriy Losyev and Artur A. Indzhykulian and Steven P. Gygi and John M. Sedivy and John P. Manis and Vadim N. Gladyshev},

url = {https://www.biorxiv.org/content/early/2021/12/01/2021.02.23.432500},

doi = {10.1101/2021.02.23.432500},

year  = {2021},

date = {2021-12-01},

urldate = {2021-12-01},

journal = {bioRxiv [Preprint]},

publisher = {Cold Spring Harbor Laboratory},

abstract = {While cancer is an age-related disease, most studies focus on genetically engineered younger mouse models. Here, we uncover how cancer develops as a consequence of the naturally aged immune system in mice. B-cell lymphoma frequently occurs in aged mice and is associated with increased cell size, splenomegaly, and a novel clonal B-cell population. Age-emergent B cells clonally expand outside of germinal centers driven by somatic mutations, activated c-Myc and hypermethylated promoters, and both genetically and epigenetically recapitulate human follicular and diffuse-large B-cell lymphomas. Mechanistically, mouse cancerous B cells originate from age-associated B cells, which are atypical memory B cells. Age-associated B cells secrete a spectrum of proinflammatory cytokines and activate paracrinally the expression of c-Myc in surrounding B cells. Although clonal B cells are a product of an aging microenvironment, they evolve being self-sufficient and support malignancy when transferred into young mice. Inhibition of mTOR and c-Myc attenuates premalignant changes in B cells during aging and emerges as a therapeutic strategy to delay the onset of age-related lymphoma. Together, we uncover how aging generates cancerous B cells, characterize a model that captures the origin of spontaneous cancer during aging and identify interventions that may postpone age-associated lymphoma.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34665127,

title = {Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish},

author = {Melanie Holmgren and Michael E Ravicz and Kenneth E Hancock and Olga Strelkova and Dorina Kallogjeri and Artur A Indzhykulian and Mark E Warchol and Lavinia Sheets},

doi = {10.7554/eLife.69264},

issn = {2050-084X},

year  = {2021},

date = {2021-10-01},

journal = {Elife},

volume = {10},

abstract = {Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34544869,

title = {Norrie disease protein is essential for cochlear hair cell maturation},

author = {Yushi Hayashi and Hao Chiang and ChunJie Tian and Artur A Indzhykulian and Albert S B Edge},

doi = {10.1073/pnas.2106369118},

issn = {1091-6490},

year  = {2021},

date = {2021-09-01},

journal = {Proc Natl Acad Sci U S A},

volume = {118},

number = {39},

abstract = {Mutations in the gene for Norrie disease protein () cause syndromic deafness and blindness. We show here that cochlear function in an  knockout mouse deteriorated with age: At P3-P4, hair cells (HCs) showed progressive loss of Pou4f3 and Gfi1, key transcription factors for HC maturation, and Myo7a, a specialized myosin required for normal function of HC stereocilia. Loss of expression of these genes correlated to increasing HC loss and profound hearing loss by 2 mo. We show that overexpression of the  gene in neonatal supporting cells or, remarkably, up-regulation of canonical Wnt signaling in HCs rescued HCs and cochlear function. We conclude that Ndp secreted from supporting cells orchestrates a transcriptional network for the maintenance and survival of HCs and that increasing the level of β-catenin, the intracellular effector of Wnt signaling, is sufficient to replace the functional requirement for  in the cochlea.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34746139,

title = {Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles},

author = {Maryna V Ivanchenko and Artur A Indzhykulian and David P Corey},

doi = {10.3389/fcell.2021.744248},

issn = {2296-634X},

year  = {2021},

date = {2021-01-01},

journal = {Front Cell Dev Biol},

volume = {9},

pages = {744248},

abstract = {Hair cells-the sensory cells of the vertebrate inner ear-bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells' mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the absence or mislocalization within stereocilia of specific proteins. Thus, stereocilia bundles are closely examined to understand most animal models of hereditary hearing loss. Because stereocilia have a diameter less than a wavelength of light, light microscopy is not adequate to reveal subtle changes in morphology or protein localization. Instead, electron microscopy (EM) has proven essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Here we review a set of EM imaging techniques commonly used to study stereocilia, including optimal sample preparation and best imaging practices. These include conventional and immunogold transmission electron microscopy (TEM) and scanning electron microscopy (SEM), as well as focused-ion-beam scanning electron microscopy (FIB-SEM), which enables 3-D serial reconstruction of resin-embedded biological structures at a resolution of a few nanometers. Parameters for optimal sample preparation, fixation, immunogold labeling, metal coating and imaging are discussed. Special attention is given to protein localization in stereocilia using immunogold labeling. Finally, we describe the advantages and limitations of these EM techniques and their suitability for different types of studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32555200,

title = {Serial scanning electron microscopy of anti-PKHD1L1 immuno-gold labeled mouse hair cell stereocilia bundles},

author = {Maryna V Ivanchenko and Marcelo Cicconet and Hoor Al Jandal and Xudong Wu and David P Corey and Artur A Indzhykulian},

doi = {10.1038/s41597-020-0509-4},

issn = {2052-4463},

year  = {2020},

date = {2020-06-01},

journal = {Sci Data},

volume = {7},

number = {1},

pages = {182},

abstract = {Serial electron microscopy techniques have proven to be a powerful tool in biology. Unfortunately, the data sets they generate lack robust and accurate automated segmentation algorithms. In this data descriptor publication, we introduce a serial focused ion beam scanning electron microscopy (FIB-SEM) dataset consisting of six outer hair cell (OHC) stereocilia bundles, and the supranuclear part of the hair cell bodies. Also presented are the manual segmentations of stereocilia bundles and the gold bead labeling of PKHD1L1, a coat protein of hair cell stereocilia important for hearing in mice. This depository includes all original data and several intermediate steps of the manual analysis, as well as the MATLAB algorithm used to generate a three-dimensional distribution map of gold labels. They serve as a reference dataset, and they enable reproduction of our analysis, evaluation and improvement of current methods of protein localization, and training of algorithms for accurate automated segmentation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31797926,

title = {Renewed proliferation in adult mouse cochlea and regeneration of hair cells},

author = {Yilai Shu and Wenyan Li and Mingqian Huang and Yi-Zhou Quan and Deborah Scheffer and Chunjie Tian and Yong Tao and Xuezhong Liu and Konrad Hochedlinger and Artur A Indzhykulian and Zhengmin Wang and Huawei Li and Zheng-Yi Chen},

doi = {10.1038/s41467-019-13157-7},

issn = {2041-1723},

year  = {2019},

date = {2019-12-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {5530},

abstract = {The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31444330,

title = {PKHD1L1 is a coat protein of hair-cell stereocilia and is required for normal hearing},

author = {Xudong Wu and Maryna V Ivanchenko and Hoor Al Jandal and Marcelo Cicconet and Artur A Indzhykulian and David P Corey},

doi = {10.1038/s41467-019-11712-w},

issn = {2041-1723},

year  = {2019},

date = {2019-08-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {3801},

abstract = {The bundle of stereocilia on inner ear hair cells responds to subnanometer deflections produced by sound or head movement. Stereocilia are interconnected by a variety of links and also carry an electron-dense surface coat. The coat may contribute to stereocilia adhesion or protect from stereocilia fusion, but its molecular identity remains unknown. From a database of hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a single transmembrane domain. Using serial immunogold scanning electron microscopy, we show that PKHD1L1 is expressed at the tips of stereocilia, especially in the high-frequency regions of the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1 is a component of the surface coat and is required for normal hearing in mice.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31444330b,

title = {PKHD1L1 is a coat protein of hair-cell stereocilia and is required for normal hearing},

author = {Xudong Wu and Maryna V Ivanchenko and Hoor Al Jandal and Marcelo Cicconet and Artur A Indzhykulian and David P Corey},

doi = {10.1038/s41467-019-11712-w},

issn = {2041-1723},

year  = {2019},

date = {2019-08-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {3801},

abstract = {The bundle of stereocilia on inner ear hair cells responds to subnanometer deflections produced by sound or head movement. Stereocilia are interconnected by a variety of links and also carry an electron-dense surface coat. The coat may contribute to stereocilia adhesion or protect from stereocilia fusion, but its molecular identity remains unknown. From a database of hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a single transmembrane domain. Using serial immunogold scanning electron microscopy, we show that PKHD1L1 is expressed at the tips of stereocilia, especially in the high-frequency regions of the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1 is a component of the surface coat and is required for normal hearing in mice.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30581889,

title = {Gene Transfer with AAV9-PHP.B Rescues Hearing in a Mouse Model of Usher Syndrome 3A and Transduces Hair Cells in a Non-human Primate},

author = {Bence György and Elise J Meijer and Maryna V Ivanchenko and Kelly Tenneson and Frederick Emond and Killian S Hanlon and Artur A Indzhykulian and Adrienn Volak and K Domenica Karavitaki and Panos I Tamvakologos and Mark Vezina and Vladimir K Berezovskii and Richard T Born and Maureen O'Brien and Jean-François Lafond and Yvan Arsenijevic and Margaret A Kenna and Casey A Maguire and David P Corey},

doi = {10.1016/j.omtm.2018.11.003},

issn = {2329-0501},

year  = {2019},

date = {2019-06-01},

journal = {Mol Ther Methods Clin Dev},

volume = {13},

pages = {1--13},

abstract = {Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene ), we use AAV9-PHP.B encoding  to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28165476,

title = {Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c},

author = {Bifeng Pan and Charles Askew and Alice Galvin and Selena Heman-Ackah and Yukako Asai and Artur A Indzhykulian and Francine M Jodelka and Michelle L Hastings and Jennifer J Lentz and Luk H Vandenberghe and Jeffrey R Holt and Gwenaëlle S Géléoc},

doi = {10.1038/nbt.3801},

issn = {1546-1696},

year  = {2017},

date = {2017-03-01},

journal = {Nat Biotechnol},

volume = {35},

number = {3},

pages = {264--272},

abstract = {Because there are currently no biological treatments for hearing loss, we sought to advance gene therapy approaches to treat genetic deafness. We focused on Usher syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and studied a knock-in mouse model, Ush1c c.216G>A, for Usher syndrome type IC (USH1C). As restoration of complex auditory and balance function is likely to require gene delivery systems that target auditory and vestibular sensory cells with high efficiency, we delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80-90% of sensory hair cells. We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28350294,

title = {Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells},

author = {A Catalina Vélez-Ortega and Mary J Freeman and Artur A Indzhykulian and Jonathan M Grossheim and Gregory I Frolenkov},

doi = {10.7554/eLife.24661},

issn = {2050-084X},

year  = {2017},

date = {2017-03-01},

journal = {Elife},

volume = {6},

abstract = {Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca into the cell. Here, we show that this constitutive Ca influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca or intracellular Ca buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28082074,

title = {Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV},

author = {Bence György and Cyrille Sage and Artur A Indzhykulian and Deborah I Scheffer and Alain R Brisson and Sisareuth Tan and Xudong Wu and Adrienn Volak and Dakai Mu and Panos I Tamvakologos and Yaqiao Li and Zachary Fitzpatrick and Maria Ericsson and Xandra O Breakefield and David P Corey and Casey A Maguire},

doi = {10.1016/j.ymthe.2016.12.010},

issn = {1525-0024},

year  = {2017},

date = {2017-02-01},

journal = {Mol Ther},

volume = {25},

number = {2},

pages = {379--391},

abstract = {Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27458190,

title = {Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing},

author = {Christian Vogl and Iliana Panou and Gulnara Yamanbaeva and Carolin Wichmann and Sara J Mangosing and Fabio Vilardi and Artur A Indzhykulian and Tina Pangršič and Rosamaria Santarelli and Montserrat Rodriguez-Ballesteros and Thomas Weber and Sangyong Jung and Elena Cardenas and Xudong Wu and Sonja M Wojcik and Kelvin Y Kwan and Ignacio Del Castillo and Blanche Schwappach and Nicola Strenzke and David P Corey and Shuh-Yow Lin and Tobias Moser},

doi = {10.15252/embj.201593565},

issn = {1460-2075},

year  = {2016},

date = {2016-12-01},

journal = {EMBO J},

volume = {35},

number = {23},

pages = {2536--2552},

abstract = {The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27007841,

title = {Primary cilia are not calcium-responsive mechanosensors},

author = {M Delling and A A Indzhykulian and X Liu and Y Li and T Xie and D P Corey and D E Clapham},

doi = {10.1038/nature17426},

issn = {1476-4687},

year  = {2016},

date = {2016-03-01},

journal = {Nature},

volume = {531},

number = {7596},

pages = {656--660},

abstract = {Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium. This Ca(2+)-responsive mechanosensor hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers. Here we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. We developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca(2+) influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signalling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27196058,

title = {Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice},

author = {Xudong Wu and Artur A Indzhykulian and Paul D Niksch and Roxanna M Webber and Miguel Garcia-Gonzalez and Terry Watnick and Jing Zhou and Melissa A Vollrath and David P Corey},

doi = {10.1371/journal.pone.0155577},

issn = {1932-6203},

year  = {2016},

date = {2016-01-01},

journal = {PLoS One},

volume = {11},

number = {5},

pages = {e0155577},

abstract = {Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26352665,

title = {In the Right Place at the Right Time: Is TMC1/2 the Transduction Channel for Hearing?},

author = {Artur A Indzhykulian and David P Corey},

doi = {10.1016/j.celrep.2015.08.064},

issn = {2211-1247},

year  = {2015},

date = {2015-09-01},

journal = {Cell Rep},

volume = {12},

number = {10},

pages = {1531--1532},

abstract = {Recent papers suggest that TMC1 and TMC2 constitute the ion channels mediating hearing and balance. Kurima et al. (2015) now show these proteins are expressed in the right place and at the right time, bringing us closer to definitive identification.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26302205,

title = {The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing},

author = {Qing Fang and Artur A Indzhykulian and Mirna Mustapha and Gavin P Riordan and David F Dolan and Thomas B Friedman and Inna A Belyantseva and Gregory I Frolenkov and Sally A Camper and Jonathan E Bird},

doi = {10.7554/eLife.08627},

issn = {2050-084X},

year  = {2015},

date = {2015-08-01},

journal = {Elife},

volume = {4},

abstract = {The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25772365,

title = {XIRP2, an actin-binding protein essential for inner ear hair-cell stereocilia},

author = {Déborah I Scheffer and Duan-Sun Zhang and Jun Shen and Artur Indzhykulian and K Domenica Karavitaki and Yichao Joy Xu and Qinchuan Wang and Jim Jung-Ching Lin and Zheng-Yi Chen and David P Corey},

doi = {10.1016/j.celrep.2015.02.042},

issn = {2211-1247},

year  = {2015},

date = {2015-03-01},

journal = {Cell Rep},

volume = {10},

number = {11},

pages = {1811--1818},

abstract = {Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN) are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23776407,

title = {Molecular remodeling of tip links underlies mechanosensory regeneration in auditory hair cells},

author = {Artur A Indzhykulian and Ruben Stepanyan and Anastasiia Nelina and Kateri J Spinelli and Zubair M Ahmed and Inna A Belyantseva and Thomas B Friedman and Peter G Barr-Gillespie and Gregory I Frolenkov},

doi = {10.1371/journal.pbio.1001583},

issn = {1545-7885},

year  = {2013},

date = {2013-01-01},

journal = {PLoS Biol},

volume = {11},

number = {6},

pages = {e1001583},

abstract = {Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23023331,

title = {Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48},

author = {Saima Riazuddin and Inna A Belyantseva and Arnaud P J Giese and Kwanghyuk Lee and Artur A Indzhykulian and Sri Pratima Nandamuri and Rizwan Yousaf and Ghanshyam P Sinha and Sue Lee and David Terrell and Rashmi S Hegde and Rana A Ali and Saima Anwar and Paula B Andrade-Elizondo and Asli Sirmaci and Leslie V Parise and Sulman Basit and Abdul Wali and Muhammad Ayub and Muhammad Ansar and Wasim Ahmad and Shaheen N Khan and Javed Akram and Mustafa Tekin and Sheikh Riazuddin and Tiffany Cook and Elke K Buschbeck and Gregory I Frolenkov and Suzanne M Leal and Thomas B Friedman and Zubair M Ahmed},

doi = {10.1038/ng.2426},

issn = {1546-1718},

year  = {2012},

date = {2012-11-01},

journal = {Nat Genet},

volume = {44},

number = {11},

pages = {1265--1271},

abstract = {Sensorineural hearing loss is genetically heterogeneous. Here, we report that mutations in CIB2, which encodes a calcium- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). One mutation in CIB2 is a prevalent cause of deafness DFNB48 in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In mice, CIB2 is localized to the mechanosensory stereocilia of inner ear hair cells and to retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of calcium binding, CIB2 significantly decreased the ATP-induced calcium responses in heterologous cells, whereas mutations in deafness DFNB48 altered CIB2 effects on calcium responses. Furthermore, in zebrafish and Drosophila melanogaster, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We also show that CIB2 is a new member of the vertebrate Usher interactome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21879401,

title = {TRPA1-mediated accumulation of aminoglycosides in mouse cochlear outer hair cells},

author = {Ruben S Stepanyan and Artur A Indzhykulian and A Catalina Vélez-Ortega and Erich T Boger and Peter S Steyger and Thomas B Friedman and Gregory I Frolenkov},

doi = {10.1007/s10162-011-0288-x},

issn = {1438-7573},

year  = {2011},

date = {2011-12-01},

journal = {J Assoc Res Otolaryngol},

volume = {12},

number = {6},

pages = {729--740},

abstract = {Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}