Early Hearing Loss upon Disruption of Slc4a10 in C57BL/6 Mice

Abstract

The unique composition of the endolymph with a high extracellular K⁺ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis.

Keywords: NCBE; Slc4a10; Slc4a7; bicarbonate transport; deafness; fibrocyte; pH.

Fig. 1

Slc4a10 expression in spiral ligament fibrocytes starts shortly before hearing onset in mice. a Section of the lower part of the cochlear duct of an adult (16-week-old) wild-type mouse stained for Slc4a10 (magenta) and Pendrin (green). Slc4a10 is expressed in the spiral ligament. It is not expressed in the stria vascularis or the organ of Corti. b Magnification from (a). Slc4a10 expression is restricted to type I, II, IV, and V fibrocytes and does not include root cells, outer sulcus cells, or spiral prominence epithelial cells, which express Pendrin. c The specificity of the Slc4a10 antibody was confirmed by the absence of labeling in corresponding Slc4a10 KO sections. d–i Expression of Slc4a10 in the developing inner ear. At embryonal day 16.5 (E16.5) (d) and postnatal day 7 (P7) (e), Slc4a10 is not yet expressed in the inner ear. At P9 (f), the Slc410 signal is weak and restricted to type II and V fibrocytes. At P12 (g) and P14 (h), Slc4a10 is clearly expressed in spiral ligament fibrocyte types II and V. At P21 (i), the labeling also includes type I and IV fibrocytes. OC, Organ of Corti; os, outer sulcus epithelial cells; rc, root cells; RM, Reissner‘s membrane; SL, spiral limbus; SM, scala media; sp, spiral prominence epithelial cells; ST, scala tympani; StV, stria vascularis; SV, scala vestibuli. The fibrocyte subtypes I–V are indicated. Nuclei are visualized by DAPI staining (blue). Scale bars, a, c, and e–i 120 μm; b 30 μm; d 100 μm

Fig. 2

Acid extrusion is not severely disturbed in explanted spiral ligament fibrocytes of P12 Slc4a10 knockout mice. a pH_i recordings from WT and KO spiral ligament fibrocytes (mean from recordings of 24 KO and 15 WT fibrocytes from ≥ 5 mice per genotype). The intracellular acid load was induced by bath application of 20 mM propionate (horizontal bar). Upward deflections indicate an increase in the pH_i. b Columns represent the maximal peak acidosis (delta pH_i) induced by propionate and amplitudes of the alkaline overshoot during propionate washout, respectively (mean ± standard error of means). No difference in the maximal peak acidosis in response to propionate (ΔpH WT, 0.137 ± 0.0342; ΔpH KO, 0.131 ± 0.0528; T₃₈, t = 0.406, p = 0.69, two-sided Student’s t test) and the alkaline overshoot after propionate removal (ΔpH WT, 0.149 ± 0.0372; KO, 0.145 ± 0.0652; T₃₈, t = 0.541, p = 0.85, two-sided Student’s t test) suggests that Slc4a10 does not play a major role for acid extrusion at the time window analyzed

Fig. 3

Early onset auditory impairment and reduction of the endocochlear potential in Slc4a10 knockout mice. a Hearing thresholds were determined by auditory brain stem recordings in response to click stimuli. Compared with wild-type littermates (dotted gray line), hearing thresholds of Slc4a10 knockout mice (dotted black line) are significantly decreased at 2, 6, 26, and 52 weeks of age. Repeated-measures two-way ANOVA with Bonferroni post-test, *** p < 0.0005. Boxes depict 25th, median, and 75th percentiles and whiskers the 5th/95th percentile range. b In knockout mice, the endocochlear potential is decreased to 45.2 ± 7.9 mV compared with 102.7 ± 6.9 mV in wild-type mice (KO n = 6, WT n = 5) at 2 weeks of age. It remains significantly below wild-type levels at 3 weeks (KO 54.3 ± 8.7 mV vs 110.6 ± 3.3 mV; KO n = 10, WT n = 5) and 6 weeks of age (KO 68.0 ± 7.1 mV vs 112.2 ± 9.9 mV; KO n = 4, WT n = 3). Repeated-measures two-way ANOVA with Bonferroni post-test, ***p < 0.0005. Vertical bars indicate standard deviations

Fig. 4

At early stages, the anatomy of the inner ear of Slc4a10 knockout mice is roughly intact. a, b As in wild-type (a), the gross morphology of HE stained sections of the middle part of the cochlear duct appears intact in 20-week-old Slc4a10 knockout mice (b). c, d Toluidine blue stained semi-thin sections of the stria vascularis from the middle part of the cochlear duct of 20-week-old wild-type and knockout mice. e, f At 60 weeks of age, the spiral ganglion, the stria vascularis, and the spiral ligament appear intact in the middle part of the cochlear duct of wild-type (e) and knockout (f) mice. g, h Higher magnifications of the stria vascularis in the middle part of the cochlear duct of wild-type and knockout mice. i Quantification of the thickness of the stria vascularis in 60-week-old WT (37.69 ± 3.52 μm, n = 4) and KO (32.15 ± 5.02 μm, n = 5) mice. j Quantification of the nuclei within the stria vascularis of 60-week-old mice (WT n = 4 and KO n = 5). k Quantification of the nuclei within the spiral ligament of 60-week-old mice (WT n = 4 and KO n = 5). Scale bars, in a, b, e, and f 80 μm; in c and d 10 μm; g and h 30 μm. OC, organ of Corti; RM, Reissner‘s membrane; SG, spiral ganglion; SM, scala media; ST, scala tympani; SV, scala vestibuli

Fig. 5

Outer hair cells are progressively lost in Slc4a10 knockout mice. a, b Magnifications of the organ of Corti in HE stained sections of the middle part of the cochlear duct at 20 weeks of age show outer hair cell loss. c–j Representative images taken from phalloidin stained whole mounts of the basal (b), middle (m), and apical (a) part of the cochlear duct at P7 (c, d), 3 (e, f), 6 (g, h), and 24 weeks of age (i, j). k–n Quantification of outer hair cells in the basal, middle, and apical part of the cochlear duct at the age of P7 (k; n = 2 per genotype), 3 (l; n = 3 per genotype), 6 (m; n = 3 per genotype), and 24 (n; n = 5 per genotype) weeks of age (two-sided Student’s t test; **p < 0.005, ***p < 0.0005). Scale bars, a, b 40 μm; c–j 35 μm

Fig. 6

Gjb2 and Gjb6 expressions are not altered upon disruption of Slc4a10. a The distribution of Gjb2 and Gjb6 is not altered in Slc4a10 knockout mice. Scale bars, 100 μm. b Immunoblot analysis of lysates from spiral ligaments do not suggest an alteration of Gjb2 or Gjb6 protein abundance in Slc4a10 knockout mice (samples were pooled from n = 3 mice for each genotype). Beta-actin served as a loading control

Fig. 7

Expression of Slc4a7 is lost in spiral ligament fibrocytes upon disruption of Slc4a10. a Immunoblot analysis shows lack of Slc4a7 expression in protein lysates of spiral ligaments of Slc4a10 knockout mice and decreased Slc4a7 protein abundance in heterozygous (HET) Slc4a10 knockout mice at the age of 3 and 6 weeks of age (samples were pooled from n = 3 mice for each genotype and age). b Slc4a7 protein abundance in the brain, kidney, or pancreas protein lysates is preserved in 56-week-old Slc4a10 knockout mice but absent in spiral ligament lysates (n = 3 per genotype). c, d The Slc4a7 labeling (magenta) of fibrocytes is lost in Slc4a10 knockout mice (n = 2 mice). e, f The Slc4a10 labeling of spiral ligament fibrocytes (green) is preserved in Slc4a7 knockout mice (n = 2 mice). Nuclei are stained with DAPI (blue). Scale bars, c–f 100 μm

Fig. 8

Mitochondria in fibrocytes of Slc4a10 knockout mice are altered. a–f Morphological analysis of the spiral ligament. Semi-thin sections from the basal part of the cochlea of a 24-week-old wild-type (a) or knockout (d) mouse suggest that the regular structure of the spiral ligament is preserved in Slc4a10 knockout mice. The ultrastructural analysis of spiral ligament fibrocytes of 14-week-old controls (b, c) compared with Slc4a10 knockout mice (e, f) reveals mitochondrial alterations (arrows) in knockout mice (n = 3 mice per genotype). g–i Co-stainings for Slc4a10 (magenta) and the mitochondrial protein cytochrome C (green) of the spiral ligament. g Overview of a stained spiral ligament obtained from a 24-week-old wild-type mouse. A higher magnification (h) reveals that Slc4a10 localizes to the fibrocyte plasma membrane, while the cytochrome C signals localize to intracellular structures. The Slc4a10 signal is absent in knockout fibrocytes (i). j, k At 24 weeks of age, cytochrome C signals (green) do not differ between genotypes (wild-type (j) and knockout (k), n = 3 per genotype). Nuclei are stained with DAPI (blue). Scale bars, a and d 30 μm; b and e 1 μm; g 10 μm; in c and f 0.2 μm; h and i 5 μm; j and k 15 μm. StV, stria vascularis