Deletion of Tricellulin Causes Progressive Hearing Loss Associated with Degeneration of Cochlear Hair Cells

Abstract

Tricellulin (also known as MARVELD2) is considered as a central component of tricellular tight junctions and is distributed among various epithelial tissues. Although mutations in the gene encoding tricellulin are known to cause deafness in humans (DFNB49) and mice, the influence of its systemic deletion in vivo remains unknown. When we generated tricellulin-knockout mice (Tric(-/-)), we found an early-onset rapidly progressive hearing loss associated with the degeneration of hair cells (HCs); however, their body size and overall appearance were normal. Tric(-/-) mice did not show any morphological change pertaining to other organs such as the gastrointestinal tract, liver, kidney, thyroid gland and heart. The endocochlear potential (EP) was normal in Tric(-/-) mice, suggesting that the tight junction barrier is maintained in the stria vascularis, where EP is generated. The degeneration of HCs, which occurred after the maturation of EP, was prevented in the culture medium with an ion concentration similar to that of the perilymph. These data demonstrate the specific requirement of tricellulin for maintaining ion homeostasis around cochlear HCs to ensure their survival. The Tric(-/-) mouse provides a new model for understanding the distinct roles of tricellulin in different epithelial systems as well as in the pathogenesis of DFNB49.

Figure 1. Localisation of tricellulin in the organ of Corti.

Organ of Corti harvested from P8-Tric^+/+ (a–c) and Tric^−/− mice (d–f) were immunostained using antibodies against occludin (a,c,d,f) and tricellulin (b,c,e,f). The overall geometries of apical junctions are comparable between Tric^+/+ (a) and Tric^−/− mice (c). Tricellulin is localised mainly to tricellular TJs in Tric^+/+ mice (b) but is undetectable in Tric^−/− mice (e). Scale bar = 10 μm.

Figure 2. Progressive hearing loss and normal EP of Tric^−/− mice.

(a) Representative ABR waveforms to click stimuli for P21-Tric^+/+ and Tric^−/− mice. (b) ABR thresholds of Tric^+/+, Tric^+/− and Tric^−/− mice in response to a broadband click and to tone-burst stimuli of 8, 16, 24 or 32 kHz; n = 15 (Tric^+/+), 6 (Tric^+/−) and 12 (Tric^−/−). Tric^−/− mouse did not generate detectable waves in response to any stimulus, while Tric^+/+ and Tric^+/− mice did not show a significant difference; P = 0.639 (click), 0.135 (8 kHz), 0.468 (16 kHz), 0.797 (24 kHz) and 0.648 (32 kHz). (c) DPOAE for 12, 16 and 20 kHz on P14 (blue) and P21 (red) of Tric^+/+ (solid lines) and Tric^−/− (dotted lines) mice. Note the reduced response on P14 and no response on P21 of Tric^−/− mice; n = 10 (P14) and 16 (P21), P < 0.01 at all frequencies. (d) Rotarod test of Tric^+/+ and Tric^−/− mice on P60-P90 mice. Tric^−/− mice showed normal motor and balance function. (e) EP of Tric^+/+ and Tric^−/− mice on P17–24.

Figure 3. Progressive degeneration of HCs in the organ of Corti of Tric^−/− mice.

(a–f) Middle turn of the organ of Corti of Tric^+/+ (a–c) and Tric^−/− (d–f) mice on P12 (a,d), P14 (b,e) and P21 (c,f) stained with a HC marker myosin VIIa (green) and rhodamin phalloidin (red). IHCs (arrows) and OHCs (arrowheads) are regularly aligned in Tric^+/+ cochlea until P21 (a–c). Hair cells in Tric^−/− cochlea are normal on P12 (d). OHCs progressively degenerate at P14 (e) to P21 (f), and some IHCs degenerate at P21 (f). Scale bar = 10 μm. Progressive loss of OHCs (g) and IHCs (h) from the cochlea of Tric^−/− mice. The loss of HCs is not detected in Tric^+/+ cochlea (blue lines). The degeneration of HCs of Tric^−/− mice (red lines) progresses during P12–P21 in OHCs (g) and P16–P60 in IHCs (h).

Figure 4. Ultrastructure of degenerated HCs of Tric^−/− mice.

(a–d) SEM images of the organ of Corti of Tric^+/+ (a) and Tric^−/− (b–d) mice. (a) IHCs (arrow) and OHCs (arrowheads) with normally organised arrays of stereocilia are observed in Tric^+/+ mice on P21. (b) Morphology of HCs is normal in Tric^−/− cochlea on P10. Note the V-shaped array of stereocilia in OHCs (inset). (c) Degeneration occurs frequently in OHCs but not in IHCs of Tric^−/− cochlea on P14. (d) Most HCs are lost except for some IHCs with severely deformed stereocilia (arrows) in Tric^−/− cochlea on P21. Scale bars = 6 μm (a–d) and 1 μm (inset in a).

Figure 5. Apoptotic hair cell death of Tric^−/− cochlea.

TUNEL assay of Tric+/+. (a) and Tric^−/− (b) cochleae on P15. Nuclei were counterstained with Hoechst 33258 (blue). Note the TUNEL-positive (green) condensed nuclei in Tric^−/− cochlea. Scale bar = 20 μm.

Figure 6. Normal bTJs in Tric^−/− mice.

TEM images of bTJs of Tric^+/+ (a,b) and Tric^−/− (c,d) mice. Note the normally-formed membrane contact sites (arrowheads) and perijunctional densities (arrows) in Tric^−/− mice. SC: supporting cell. Scale bar = 200 nm.

Figure 7. Tric^−/− HCs survive in explant culture.

Tric^+/+ (a) and Tric^−/− (b,c) cochleae in explant culture (a,b) and in vivo (c) stained using an antibody against myosin VIIa (green) and rhodamine phalloidin (red). (a,b) Most OHCs survive in Tric^+/+ and Tric^−/− cochleae cultured until P16. (c) Most OHCs are lost on P16 Tric^−/− cochlea in vivo. (d) The numbers of remaining OHCs in culture does not differ between Tric^+/+ and Tric^−/− cochleae, and they are significantly larger compared with those in vivo. Scale bars: 50 μm.