Deafness in TRbeta mutants is caused by malformation of the tectorial membrane

Abstract

Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.

Figure 1.

A, B, ABR measurements in HC-TRβ-Ctr, HC-TRβ^−/−, and TRβ^−/− mice. At 3–4 months, TRβ^−/− mice show a significant elevation of click-evoked ABR thresholds (A). Frequency-specific ABR thresholds of HC-TRβ^−/− mice show a significant threshold elevation at frequencies <8 kHz (B). C–F, BKα expression (red, arrowheads) in HC-TRβ-Ctr and HC-TRβ^−/− mice at indicated ages. Note lack of BKα expression in IHCs and OHCs of HC-TRβ^−/− mice at P15. Nuclei are stained in blue with DAPI. Scale bars, 20 μm. G–J, Typical IHC voltage-activated K⁺ currents in HC-TRβ-Ctr mice at P15 (G) and P18 (I) compared with IHCs of HC-TRβ^−/− mice (H, J).

Figure 2.

Exocytosis in IHCs (A–D) and CAP responses (E–G). A, Ca²⁺ currents of a P20 TRβ-Ctr (black trace) and a P20 TRβ^−/− IHC (gray trace) evoked by stepping from −80 to 0 mV for 100 ms (top) and capacitance increases indicating exocytosis (bottom). B–D, Box-and-whisker plots of capacitance change ΔC_m (B), Ca²⁺ charge (C) and resulting exocytosis efficiency (D). Horizontal lines at the median (bold), 25 and 75% quartiles (box), and extreme values (whiskers) for TRβ-Ctr and TRβ^−/− IHCs aged P17–P20. E, CAP threshold elevation was evident in the whole frequency range, especially in the frequency range of normal best sensitivity. F, CAP input–output functions in TRβ^−/− mice were shifted to higher SPLs and had steeper slopes than those for TRβ-Ctr mice; maximum CAP amplitudes were similar. G, Time course of CAP and SP responses in TRβ^−/− mice; the stimulus is indicated by the line above the time axis.

Figure 3.

A–C, CMs and DPOAEs of TRβ-Ctr and TRβ^−/− mice. CM are present in both TRβ-Ctr and TRβ^−/− mice, although reduced in TRβ^−/− mice (A). DPOAE amplitudes (B) and DPOAE thresholds (C) reveal a loss of active cochlear mechanics in TRβ^−/− mice at frequencies above f1 = 8 kHz. D–I, TM structure of TRβ-Ctr, HC-TRβ^−/−, and TRβ^−/− mice. Cross-sections of epoxy resin-embedded cochleae reveal morphological abnormalities of the TM in TRβ^−/− mice (F, I) compared with TRβ-Ctr (D, G) and HC-TRβ^−/− mice (E, H). Scale bars, 100 μm. J–L, Dimensional measurements (width, thickness, area) of the TM.

Figure 4.

α- and β-tectorin in TRβ-Ctr and TRβ^−/− mice. A–H, Immunohistochemistry against α-tectorin (A–D, red) and β-tectorin (E–H, red) shows a more abundant expression in TRβ^−/− compared with TRβ-Ctr mice. Nuclei are stained in blue with DAPI. Scale bars, 20 μm. I, J, Western blots and densitometric quantification of α-tectorin (I) and β-tectorin expression levels (J) in TRβ^−/− mice compared with TRβ-Ctr animals. The house keeping protein ezrin was used for normalization. Data are expressed as mean percentage of control ± SD.