ABR thresholds of Tprn −/− , Tprn N259/N259 , and Tprnin103/in103mice and VsEP measurements of Tprn−/−mice. ANKRD24, TRIOBP-5, GPSM2, and ESPN-1 localization in WT and Tprnin103/in103postnatal hair cell stereocilia and hair bundle morphology of TPRN-deficient mice. (A) Mean ABR thresholds at P18, P30, and P60 of Tprn+/+, Tprn+/−, and Tprn−/− littermates at 8, 16, and 32 kHz. Using linear mixed effects regression, we found that Tprn−/− mice had significantly worse hearing overall (Table S2) and exhibited progressive deafness (Table S3) with greater early hearing loss at high frequencies (Tables S4 and S5). (B) Mean ABR thresholds of TprnN259/N259, Tprn+/N259, and Tprn+/+ littermate controls at P18, P30, and P60, n = number of animals tested. Similar to Tprn−/− mice, TprnN259/N259 mice exhibit worse hearing overall and a significant worsening of hearing over time (Tables S6 and S7). (C) Mean ABR thresholds of Tprnin103/in103 and WT control mice (Tprn+/+) at P21, P42, and 2 mo of age, n = number of animals tested. At all ages and frequencies, Tprnin103/in103 mice exhibited worse hearing than P60 Tprn+/+ (Tables S8 and S9). (D–F) Mean VsEP threshold (D), P1 amplitude (E), and P1 latency (F) for Tprn+/+, Tprn+/−, and Tprn−/− littermates at P30 and P60 time points. Tprn−/− mice did not exhibit significant differences in VsEP thresholds, P1 amplitudes, or P1 latencies except for a significant difference in VsEP threshold at P60 compared with Tprn+/+, P = 0.04 (Tables S10 and S11), which is likely attributable to expected test-retest variation between time points. N indicates the number of animals tested at P30/P60, correspondingly. Graphs A–F display mean ± SD. *, **, ***—significant difference compared with Tprn+/+ animals at P < 0.05, P < 0.01, and P < 0.001, respectively. Color of asterisks indicates the group exhibiting the difference. Brackets indicate differences within a genotype between time points. (G–G″) Whole mounts of cochlea from P7 WT and Tprnin103/in103 mice were stained with antibodies against TRIOBP-5 (G–G″) and phalloidin (magenta) to reveal stereocilia. (G′ and G″) Enlarged images of a representative IHC (G′) and OHC (G″) at stereocilia level. (H) Quantification of TRIOBP-5 puncta at the bases of different rows of OHC stereocilia of P7 Tprn+/+ and Tprnin103/in103 mice. Thirty-four WT and forty-two Tprn-deficient OHCs from at least three mice were analyzed per group. Data are means ± SEM. ***P < 0.001 by unpaired two-sided t test. (I–J′) Whole mounts of cochlea from P5 (I and I′) and P7 (J and J′) WT and Tprnin103/in103 mice were stained with antibodies against ANKRD24, and counterstained by phalloidin (magenta) to show F-actin of stereocilia. (I′ and J′) Enlarged images of a representative IHC at stereocilia level. Number of TRIOBP-5 and ANKRD24 puncta at bases of immature short-row stereocilia were significantly reduced in P5 and P7 Tprn-deficient hair cells. (K–L′) Cochlear whole mounts from P5 WT (K and L) and Tprnin103/in103 mice (K′ and L′) were stained for GPSM2 (K and K′) or ESPN-1 (L and L′), which is localized at tips of stereocilia. No significant change of GPSM2 or ESPN-1 localization was observed in Tprn-deficient mice (K′ and L′). Scale bars are 5 µm.
