Tricellulin deficiency affects tight junction architecture and cochlear hair cells
Gowri Nayak, … , Gregory I. Frolenkov, Saima Riazuddin
Gowri Nayak, … , Gregory I. Frolenkov, Saima Riazuddin
Published August 27, 2013
Citation Information: J Clin Invest. 2013;123(9):4036-4049. https://doi.org/10.1172/JCI69031.
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Research Article

Tricellulin deficiency affects tight junction architecture and cochlear hair cells

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

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Figure 1

p.Arg497* mutation results in loss of tricellulin from the organ of Corti.

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p.Arg497* mutation results in loss of tricellulin from the organ of Cort...
(A) Schematic of organ of Corti cross-section, showing the tight junctions that separate endolymph (blue) from perilymph (yellow). At tricellular junctions, tricellulin (green) spans the entire depth of the cuticular plate and intersects ZO-1 (red) at apical and basal ends of these junctions. OP, outer pillar cell; D, Deiters’ cell. (B) Scheme for generating the TricR497X/R497X mice. Exons 5 and 6 were flanked by LoxP (red arrowheads) sites, and the neomycin selection cassette (Neo) that was flanked by FRT sites (gray bars) was removed by crossing TricR497X/R497X mice with mice expressing Flp recombinase gene (59). The blue arrows represent the primers used to genotype the knockin mice. (C) PCR detection of wild-type allele (440 bp) and targeted allele (579 bp). (D) Schematic of the wild-type prematurely truncated tricellulin that is expected from the targeted allele. TM, transmembrane domain. (E) Schematics of transcripts (a, d, and e) that were amplified by RT-PCR from Tric+/+ and TricR497X/R497X inner ear cDNA and the location of the primers used for the reaction (arrows). Gray boxes indicate untranslated regions, and other colored boxes depict coding exons. Tric-a and Tric-d from TricR497X/R497X inner ear cDNA had the knockin mutation (red bars). Brown exons encode the occludin-ELL domain. The magenta bar shows the location of the peptide used to generate the tricellulin antibody, PB705 (24). (F) Tricellulin (green) is absent from tricellular junctions in the organ of Corti of TricR497X/R497X mice. Scale bar: 5 μm.