Endocochlear potential contributes to hair cell death in TMPRSS3 hearing loss

Abstract

Pathogenic variants in the gene TMPRSS3 are a common cause of hearing loss in humans, although the causal mechanisms remain unknown. Previous work has shown that Tmprss3Y260X/Y260X mice exhibit normal hair cell development, mechanosensory transduction, and spiral ganglion patterning, but experience rapid hair cell death from P12 to P14 at the onset of hearing. Here, we demonstrate that Tmprss3Y260X/Y260X mice display an early and temporary spike in endocochlear potential (EP) prior to the onset of hair cell death. In vitro experiments with cochlear explants from Tmprss3Y260X/Y260X mice and in vivo studies with Tmprss3Y260X/Y260X mice crossed with 2 different mutant models that lacked EP generation promoted hair cell survival. Furthermore, systemic administration of furosemide, a drug that reduces EP in vivo, reduced hair cell death in Tmprss3Y260X/Y260X mice. These findings suggest that extracellular factors, including EP, play a role in TMPRSS3-related hair cell survival and hearing loss, and suggest that modulating EP could be a therapeutic strategy.

Keywords: Cell biology; Drug therapy; Genetic diseases; Otology; Proteases.

Figure 1. Tmprss3^Y260X/Y260X mice demonstrate normal HC and stereocilia morphology and physiology prior to rapid-onset degeneration.

(A) Phalloidin immunostaining and scanning electron microscopy (SEM) show normal architecture of IHCs and OHCs at P6 and P14 in wild-type mice and complete HC degeneration at P14 in Tmprss3^Y260X/Y260X mice. Scale bars: 10 μm (P7), 20 μm (P14). (B) FM1-43 uptake by HCs in Tmprss3^Y260X/Y260X mice is equivalent to that of wild-type mice at P7. Scale bars: 20 μm.

Figure 2. Spiral ganglion subtype composition is normal in Tmprss3^Y260X/Y260X mice, aside from type II SGN increase.

(A) Representative sections of spiral ganglion taken at P11 stained for neuron-specific class III β-tubulin (TuJ1) and antibodies specific for type IA SGNs (calretinin), type IB SGNs (calbindin), type IC SGNs (POU4F1, BRN3A), and type II SGNs (NGFR). Top row shows TuJ1 channel, middle row shows SGN subtype-specific channel, and the bottom row shows merged channels. Arrows indicate costaining; arrowheads indicate negative costaining. Scale bars: 20 μm. (B) Counted subtype-specific neurons at P11 and P21 for Tmprss3^Y260X/Y260X and wild-type mice show no difference aside from increased type II SGNs in wild-type mice at P21. Each point represents cell count from 1 cochlea (n = 3 cochleae per genotype and day).

Figure 3. Cochlear explant cultures implicate intracochlear environment in HC death in Tmprss3^Y260X/Y260X mice.

Representative images of cochlear explant cultures from control (Tmprss3^Y260X/+) (A and B) and Tmprss3^Y260X/Y260X (E and F) mice. Tmprss3^Y260X/Y260X explant at P14 equivalent (P7 explant + 7 days in vitro [DIV]) demonstrates no HC degeneration of either IHCs or OHCs, which is maintained to P30 explant (C and G). Tmprss3^Y260X/Y260X mice display near complete HC degeneration at P14 in vivo (D and H). Scale bar: 20 μm. (I and J) Quantification of IHCs and OHCs in explant culture for control and Tmprss3^Y260X/Y260X mice demonstrating slowly progressive OHC loss in both. There was no significant difference in cell counts up to 30 DIV (D30) (P > 0.11 in all cases). Each point represents the cell count from 1 culture. Exact Wilcoxon rank-sum test (Mann-Whitney U test) was used to compare 2 groups (Tmprss3^Y260X/+ and Tmprss3^Y260X/Y260X) in each culture age. Up to 5 cultures were quantified for each day, but only 2 cultures were available for D30 control (see Supporting Data Values for full details).

Figure 4. Direct EP measurements show supraphysiologic rise in EP in Tmprss3^Y260X/Y260X mice.

(A) EP was recorded through the basal turn of left cochleae in live mice ranging from P7 to P24. Representative tracing of EP recorded from the scala media of a live control mouse. (B) Direct EP recordings from P28 mice demonstrating no difference between control (n = 5) (Tmprss3^+/–) and Tmprss3^Y260X/Y260X (n = 6) mice. Open circles and squares represent female mice. Filled circles and squares represent male mice. nd, no statistical difference determined. (C) Direct EP recordings from mouse models. Each data point represents 1 reading from 1 animal and is plotted as the mean with SD. Mean EP at P12–P15 is significantly different (P < 0.0001) between Tmprss3^Y260X/Y260X and control (Tmprss3^Y260X/+) mice. Wilcoxon rank-sum test (Mann-Whitney U test) was used to compare 2 groups (control Tmprss3^Y260X/+ and experimental Tmprss3^Y260X/Y260X) in each postnatal age group (P7–11: n = 12, n = 11; P12–15: n = 15, n = 11; P18–P24: n = 10, n = 9, for control and experimental mice). (D) Schematic demonstrating time frame of HC death in Tmprss3^Y260X/Y260X mice in the context of physiologic rapid phase of EP development. Neither Pou3f4^delJ nor Mitf^Mi-wh/+ knockout mice generated EP and neither had HC degeneration despite profound HL.

Figure 5. Rescue of cochlear HCs in Tmprss3^Y260X/Y260X mice through reduction of EP via crossing Mitf^Mi-wh/+ mice and Pou3f4^delJ mice.

(A) Representative cochlear sections of Tmprss3^Y260X/Y260X (T^–/–) mice crossed with Mitf^Mi-wh/+ (M^Mi-wh/+) mice. Sections shown are cochlear apex at P16, labelled with Myo7a. (B) IHC and OHC counts per 100 μm at P16 and P21 for T^–/– and M^Mi-wh/+ crosses. Each dot represents the average of both ears for the same mouse; n = 3 mice per condition. Note loss of OHCs at P16 and P21 in M^Mi-wh/+ mouse lines. (C) Representative cochlear sections of Pou3f4^delJ (P^–/–) crosses at P48. Cross of P^–/– with T^–/– mice rescues HCs. Scale bar: 30 μm. Staining is with Myo7a (red) and phalloidin (blue). (D) IHC and OHC counts for per 100 μm at P48 for Pou3f4^delJ (P^–/–) crosses. There is no loss of OHCs noted over time in this model. n = 2 or 3 mice per condition.

Figure 6. Reduced EP does not rescue KCNMA1 expression.

(A) Representative sections from the base of P15 cochleae from control (Tmprss3^Y260X+-;Mitf^+/+) and double-mutant mice (Tmprss3^Y260X/Y260X; Mitf^Mi-wh/+) demonstrate lack of expression of the outward-rectifying K⁺-channel KCNMA1 surviving HCs that lack Tmprss3 expression. (B) Counts of KCNMA1 puncta show a base to apex gradient of expression in control mice at P12 (n = 9), P13 (n = 9), P14 (n = 6), and P15 (n = 7) and lack of KCNMA1 expression in Tmprss3^Y260X/Y260X Mitf^Mi-wh/+ mice at P12 (n = 9), P13 (n = 6), P14 (n = 7), and P15 (n = 6). Note there were no punctae for Tmprss3^Y260X/Y260X Mitf^Mi-wh/+ mice (all values were 0).

Figure 7. Pharmacologic reduction of EP using furosemide rescues HCs.

(A) Administration of 200 mg/kg furosemide once daily from P10 to P14 leads to HC survival in Tmprss3^Y260X/Y260X mice. Representative confocal images of base, middle, and apex cochlear turns of P14 Tmprss3^Y260X/Y260X-treated mice immunostained against Myo7a (green) and phalloidin (blue). (B) Mean ± SD counts of IHCs and OHCs per 100-μm section from untreated (n = 4 ears) and treated P14 Tmprss3^Y260X/Y260X (n = 7) mice measured in the base, middle, and apex cochlear turns. Each data point represents an individual cochlea. Nonparametric ANOVA with Tukey’s post hoc test for comparison of untreated versus treated IHC: P = 0.0003 for apex, P = 0.0008 for mid, P = 0.006 for base, and OHC: P = 0.009 for apex, P = 0.79 for mid, P = 0.78 for base.