title: Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.
abstract: Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation.
  
  
  CONCLUSIONS
  Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. This approach illuminates the role of clarin-1 and the molecular mechanism linked to the CLRN1(N48K) mutation in sensory hair cells of the inner ear. Additionally, the investigation provided an in vivo model to guide future drug discovery to rescue the hCLRN1(N48K) in hair cells.
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and.
authors.forename: Suhasini R
authors.initials: SR
authors.lastname: Gopal
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and.
authors.forename: Daniel H-C
authors.initials: DH
authors.lastname: Chen
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Shih-Wei
authors.initials: SW
authors.lastname: Chou
authors.affiliation: Institute of Molecular Life Sciences, University of Zurich, CH-8057 Zurich, Switzerland, and.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zang
authors.affiliation: Institute of Molecular Life Sciences, University of Zurich, CH-8057 Zurich, Switzerland, and.
authors.forename: Stephan C F
authors.identifier: http://orcid.org/0000-0002-9615-480X
authors.initials: SC
authors.lastname: Neuhauss
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and.
authors.forename: Ruben
authors.initials: R
authors.lastname: Stepanyan
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106, Departments of Genetics and Genome Sciences and Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Brian M
authors.initials: BM
authors.lastname: McDermott
authors.affiliation: Departments of Otolaryngology-Head and Neck Surgery, University Hospitals Case Medical Center and Departments of Genetics and Genome Sciences and Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106 kna3@case.edu.
authors.forename: Kumar N
authors.initials: KN
authors.lastname: Alagramam
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001306:Auditory Pathways / Q000378:metabolism / Q000473:pathology; D019521:Body Patterning / Q000187:drug effects / Q000235:genetics; D015820:Cadherins / Q000235:genetics; D004195:Disease Models, Animal; D004706:Endodeoxyribonucleases / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics*; D005838:Genotype; D006198:Hair Cells, Auditory / Q000473:pathology*; D034381:Hearing Loss / Q000235:genetics; D006801:Humans; D007814:Larva; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D004856:Postural Balance / Q000235:genetics; D020539:Sequence Analysis, Protein; D013569:Synapses / Q000378:metabolism / Q000473:pathology; D052245:Usher Syndromes / Q000150:complications / Q000235:genetics / Q000473:pathology*; D014786:Vision Disorders / Q000209:etiology; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C000588831:CLRN1 protein, zebrafish; D015820:Cadherins; D008565:Membrane Proteins; D029961:Zebrafish Proteins; C499092:cdh23 protein, zebrafish; D004706:Endodeoxyribonucleases
keywords: clarin-1; hair cells; hearing