Defects in hair cells disrupt the development of auditory peripheral circuitry

Abstract

Deafness is the most common form of sensory impairment in humans and frequently caused by defects in hair cells of the inner ear. Here we demonstrate that in male mice which model recessive non-syndromic deafness (DFNB6), inactivation of Tmie in hair cells disrupts gene expression in the neurons that innervate them. This includes genes regulating axonal pathfinding and synaptogenesis, two processes that are disrupted in the inner ear of the mutant mice. Similar defects are observed in mouse models for deafness caused by mutations in other genes with primary functions in hair cells. Gene therapy targeting hair cells restores hearing and inner ear circuitry in DFNB6 model mice. We conclude that hair cell function is crucial for the establishment of peripheral auditory circuitry. Treatment modalities for deafness thus need to consider restoration of the function of both hair cells and neurons, even when the primary defect occurs in hair cells.

Fig. 1. Altered SGN transcriptome in Tmie^-/- mice.

a Diagram of the known mammalian mechanotransduction molecular machinery, located within the stereocilia of hair cells. b Diagram of the mammalian inner ear, highlighting inner hair cells (IHCs), outer hair cells (OHCs) and spiral ganglion neurons (SGNs). c Experimental strategy for single cell RNA-sequencing in Tmie^+/- and Tmie^-/- mice at P21. d Uniform manifold approximation and projection (UMAP) plots representing sequenced neurons combined from wild-type and mutants. Top: annotation by neuronal cell types. Bottom, annotation by genotype. e Violin plots of normalized median expression for top differentially expressed genes in Tmie^-/- versus Tmie^+/- type I SGNs. f Enriched gene ontology (GO) terms plotted by odds ratio related to neuronal development of differentially expressed genes. g Left, fluorescent in situ hybridization (FISH) using probes for three differentially expressed genes (Kcnip3, Marcksl1, Rxrg) in sections of P21 Tmie^+/- and Tmie^-/- SGNs; scale bar, 5 μm. Right, quantification of FISH for Kcnip3 (+/-: 9.247 ± 0.2897, n = 757 neurons/3 mice; -/-: 2.86 ± 0.08, n = 837 neurons/4 mice; p < 0.0001, two-sided Mann-Whitney U test, U = 11451), Marcksl1 (+/-: 19.60 ± 0.34, n = 757 neurons/3 mice; -/-: 40.15 ± 0.71, n = 837 neurons/4 mice; p < 0.0001, two-sided Mann-Whitney U test, U = 107558), and Rxrg puncta/SGN ( + /-: 12.01 ± 0.39, n = 742 neurons/4 mice; -/-: 3.79 ± 0.099, n = 837 neurons/4 mice; p < 0.0001, two-sided Mann-Whitney U test, U = 149603). Values are mean ± SEM.

Fig. 2. Type I SGN subtype maturation is impaired in Tmie^-/- mutants.

a, b UMAP plots for type I spiral ganglion neurons (SGNs) from Tmie^+/- (a, left) and Tmie^-/- (b, right) mice at P21, colored by type I subtype (blue, type IA; orange, type IB; green, type IC) predicted by semi-supervised annotation via single cell Annotation using Variational Inference (scANVI). Bottom, feature plots of SGN subtype specific genes (IA- Calb2; IB- Ttn; IC- Lypd1) in Tmie^+/- and Tmie^-/- mice. Fractional composition of type IA, IB, and IC SGNs in Tmie^+/- and Tmie^-/- mice. c Dot plots of normalized mean expression of differentially expressed genes. d Neuron development-related GO terms enriched in type I SGN dataset, as analyzed separately for each type I SGN subtype.

Fig. 3. Disrupted peripheral SGN projection patterns in Tmie^-/- mice.

a Diagram of spiral ganglion neuron (SGN) innervation patterns of the cochlear sensory epithelium in adult mice. b Experimental strategy for viral labeling with an adeno-associated virus (AAV) encoding enhanced green fluorescent protein (EGFP) under the control of the human synapsin (hSyn) promoter (AAV9.hSyn.EGFP). c Mid-cochlear whole-mounts from P21 Tmie^+/- and Tmie^-/- mice after high titer viral injection. Tissue was immunolabeled for MYO7A (magenta) to visualize inner hair cells (IHCs) and outer hair cells (OHCs) and EGFP (green) to visualize projections of infected neurons; scale bar: 10 μm. d P21 mid-cochlear whole mounts after low titer viral injection. Yellow arrow: SGN projecting past IHC; white arrow: SGN with abnormal branching; arrowhead: SGN with no projection to IHC; scale bar: 10 μm. e Representative images of P11 mid-cochlear whole mounts after low titer viral injection; arrow: single SGN projecting to several IHCs; arrowheads: single SGN projecting both towards the apex and base in OHC region; Scale bar: 10 μm. f Quantified results at P11 ( +/-: IHC contact only- 82%; abnormal projection- 18%; n = 111 neurons/3 mice; -/-: IHC contact only- 34%; abnormal projection- 66%; n = 168 neurons/3 mice). g Quantified results at P21 ( +/-: IHC contact only- 97%; abnormal projection- 3% n = 92 neurons/5 mice; -/-: IHC contact only- 48%; abnormal projection- 52%; n = 22 neurons/5 mice).

Fig. 4. Aberrant NGFR⁺ SGN projections in Tmie^-/- mice.

a Mid-cochlear whole mounts from P22 Tmie ^+/- (top) and Tmie^-/- mice (bottom); tissue immunolabeled for NGFR (cyan), CALB2 (yellow), and DAPI (red); scale bar: 50 μm. b Mid-cochlear whole mounts at P11 and P23 in Tmie ^+/- and Tmie^-/- mice immunolabeled for NGFR (cyan), CALB2 (yellow), and DAPI (red); arrows: abnormal bulbous NGFR⁺ projections terminating near inner hair cells (IHCs) in Tmie^-/- mice; scale bars: 20 μm. c NGFR⁺ projections terminating near IHCs per 100 μm in mid-cochlear whole mounts at P11 ( +/-: 0.69 ± 0.20, n = 13 whole mounts/3 mice, -/-: 8.61 ± 0.70, n = 18 whole mounts/4 mice, two-sided Welch’s t test: p = 1.435*10^-9, t = 10.83, df = 19.92) and P20 ( +/-: 1.83 ± 0.34, n = 12 whole mounts/3 mice, -/-: 6.14 ± 0.67, n = 14 whole mounts/3 mice, two-sided Welch’s t test: p = 1.881*10^-5, t = 5.66, df = 19.07). d High magnification view of NGFR⁺ side branches to outer hair cells (OHCs) at P11, P20, and P23; left and middle: fibers at innermost row of OHCs; scale bar: 5 μm; right: fibers at all three OHC rows; arrows: NGFR+ branches to OHCs; scale bar: 20 μm. e NGFR⁺ branches to OHCs per 100 μm across mid-cochlea at P11 ( +/-: 74.85 ± 1.48, n = 13 whole mounts/3 mice, -/-: 40.61 ± 1.54, n = 18 whole mounts/4 mice, two-sided Welch’s t test: p = 1.306*10^-15, t = 15.99, df = 28.52) and P20 ( +/-: 75.42 ± 2.40, n = 12 whole mounts/3 mice, -/-: 37.93 ± 2.52, n = 14 whole mounts/3 mice, two-sided Welch’s t test: p = 1.885*10^-10, t = 10.76, df = 23.97). Values are mean ± SEM.

Fig. 5. Defects in synaptic connections between SGNs and hair cells in Tmie^-/- mice.

a Mid-cochlear whole mounts of P14 Tmie^+/- and Tmie^-/- mice immunolabeled for CTBP2 (magenta) and GLUR2 (green). b CTBP2 puncta per 10 inner hair cells (IHCs) at P14 ( +/-: 222.39 ± 3.48, n = 18 whole mounts/4 mice, -/-: 224.35 ± 2.29, n = 20 whole mounts/4 mice; two-sided Welch’s t test: p = 0.6417, t = 0.47, df = 29.88). c GLUR2 puncta per 10 IHCs at P14 ( +/-: 248.53 ± 9.17, n = 17 whole mounts/4 mice; -/-: 161.4 ± 8.25, n = 20 whole mounts/4 mice; two-sided Welch’s t test: p = 4.351*10^-8, t = 7.06, df = 33.78). d % of CTBP2 puncta co-localized with GLUR2 in P14 IHCs (+/-: 80.39 ± 3.45, n = 17 whole mounts/4 mice; -/-: 32.05 ± 1.49, n = 20 whole mounts/4 mice; two-sided Welch’s t test: p = 2.040*10^-11, t = 12.85, df = 21.91). e CTBP2 puncta per 10 outer hair cells (OHCs) at P14 ( +/-: 19.92 ± 0.43, n = 18 whole mounts/4 mice; -/-: 19.72 ± 0.46, n = 20 whole mounts/4 mice; two-sided Welch’s t test: p = 0.7580, t = 0.31, df = 36.00). f Mid-cochlear whole mounts of P21 Tmie^+/- and Tmie^-/- mice immunolabeled for CTBP2 (magenta) and GLUR2 (green). g CTBP2 puncta per 10 IHCs at P20-23 ( +/-: 204.95 ± 4.16, n = 20 whole mounts/4 mice; -/-: 172.95 ± 6.27, n = 21 whole mounts/4 mice; two-sided Welch’s t test: p = 0.0002, t = 4.24, df = 34.47). h GLUR2 puncta per 10 IHCs at P20-23 ( +/-: 220.64 ± 5.60, n = 14 whole mounts/4 mice; -/-: 148.33 ± 5.72, n = 15 whole mounts/4 mice; two-sided Welch’s t test: p = 1.719*10^-9, t = 9.02, df = 26.99). i % of CTBP2 puncta co-localized with GLUR2 in P21 IHCs (+/-: 95.34 ± 0.83, n = 20 whole mounts/4 mice; -/-: 36.76 ± 3.88, n = 21 whole mounts/4 mice; two-sided Welch’s t test: p = 1.526*10^-12, t = 14.73, df = 21.86). j CTBP2 puncta per 10 OHCs at P21 ( +/-: 24.13 ± 0.69, n = 20 whole mounts/4 mice; -/-: 16.36 ± 0.81, n = 21 whole mounts/4 mice; two-sided Welch’s t test: p = 1.264*10^-8, t = 7.21, df = 38.34) per area of 10 IHCs. Scale bars: 10μm. Values are mean ± SEM.

Fig. 6. Conditional knockout of Tmie in cochlear hair cells.

a Experimental strategy. b Auditory brainstem response thresholds of control (Tmie^FL/FL; yellow, n = 5) and cKO mice (Gfi1-GCE;Tmie^FL/FL; orange, n = 7) at P21. c Mid-cochlear whole mounts from P21 control (Con) and cKO mice immunolabeled for CALB2 (yellow), NGFR (cyan), and DAPI (red), scale bar: 50 μm; right panels, high magnification images; yellow arrowheads: NGFR⁺ branches to outer hair cells (OHCs), white arrowheads: endings of NGFR⁺ neurons near inner hair cells (IHCs); scale bar: 10 μm. d Terminals near IHCs/100 μm (Con: 1.81 ± 0.46, n = 6 mice; cKO: 10.48 ± 1.62, n = 6 mice; two-sided Welch’s t test: p = 0.0024, t = 5.13, df = 5.80) and branches to OHCs/100μm (Con: 79.28 ± 2.13, n = 6 mice; cKO: 44.43 ± 2.54, n = 6 mice; two-sided Welch’s t test: p = 2.342*10^-6, t = 10.52, df = 9.70). e Mid-cochlear whole mounts of control and cKO mice at P21 immunolabeled for CTBP2 (magenta) and GLUR2 (green); scale bar: 10 μm. f CTBP2 puncta/10 IHCs (Con: 179.49 ± 2.95, n = 12 whole mounts/6 mice; cKO:135.20 ± 5.61, n = 14 whole mounts/7 mice, two-sided Welch’s t test: p = 1.189*10^-6, t = 6.98, df = 19.45); GLUR2 puncta/10 IHCs (Con: 183.63 ± 3.33, n = 12 whole mounts/6 mice; cKO: 140.43 ± 8.01, n = 14 whole mounts/7 mice, two-sided Welch’s t test: p = 0.0001, t = 4.98, df = 17.28); %GLUR2 + CTBP2 IHC puncta (Con: 97.93 ± 0.35, n = 12 whole mounts/6 mice; cKO: 72.16 ± 2.32, n = 14 whole mounts/7 mice, two-sided Welch’s t test: p = 6.284*10^-8, t = 10.94, df = 13.62). g Left, mid-cochlear whole mounts of control and cKO mice at P21 immunolabeled for MYO7A; scale bar: 20 μm. Right, P21 IHCs/100 μm (Con: 12.96 ± 0.42, n = 3 mice; cKO: 12.89 ± 0.28, n = 4 mice, two-sided Welch’s t test: p = 0.9013, t = 0.13, df = 3.70) and OHCs/100 μm (Con: 40.66 ± 0.75, n = 3 mice; cKO: 40.02 ± 0.51, n = 4 mice, two-sided Welch’s t test: p = 0.5260, t = 0.70, df = 3.74). Values are mean ± SEM.

Fig. 7. Peripheral SGN defects in USH1F model mice.

a Upper, Diagram of developing hair cell stereocilia links. Lower, PDCH15 (green) forms the transient kinocilial links and mature tip links with CDH23 (magenta). b Left, mid-cochlear whole mounts of P21 Pcdh15^+/av-3J and Pcdh15^av-3J/av-3J mice immunolabeled for CALB2 (yellow), NGFR (cyan), and DAPI (red); arrowheads: nerve endings of NGFR⁺ neurons near inner hair cells (IHCs); right, mid-cochlear whole mounts of P21 Pcdh15^+/av-3J and Pcdh15^av-3J/av-3J mice immunolabeled for NGFR; arrows indicate NGFR⁺ side branches to outer hair cells (OHCs); scale bar: 20 μm. c NGFR+ terminals near IHCs/100μm (+/av3J: 2.08 ± 0.25, n = 12 whole mounts/3 mice; av3J/av3J: 5.45 ± 0.49, n = 11 whole mounts/3 mice; two-sided Welch’s t test: p = 2.222 × 10^-5, t = 6.05, df = 15.26) and NGFR+ branches to OHCs/100μm (+/av3J: 80.67 ± 2.22, n = 12 whole mounts/3 mice; av3J/av3J: 42.45 ± 3.75, n = 11 whole mounts/3 mice; two-sided Welch’s t test: p = 1.665*10^-7, t = 8.76, df = 16.40). d Mid-cochlear whole mounts in Pcdh15^+/av-3J and Pcdh15^av-3J/av-3J mice at P21 immunolabeled for CTBP2 (magenta) and GLUR2 (green); scale bar: 20 μm. e CTBP2 puncta/10 IHCs (+/av3J: 176.89 ± 2.84, n = 12 whole mounts/6 mice; av3J/av3J: 140.66 ± 5.68, n = 9 whole mounts/4 mice, two-sided Welch’s t test: p = 0.0001, t = 5.70, df = 11.97), GLUR2 puncta/10 IHCs (+/av3J: 179.47 ± 2.75, n = 12 whole mounts/6 mice; av3J/av3J: 136.57 ± 4.10, n = 9 whole mounts/4 mice, two-sided Welch’s t test: p = 5.225*10^-7, t = 8.68, df = 14.65), and %GLUR2 + CTBP2 IHC puncta (+/av3J: 97.10 ± 0.43, n = 12 whole mounts/6 mice; av3J/av3J: 68.21 ± 3.08, n = 9 whole mounts/4 mice, two-sided Welch’s t test: p = 1.490*10^-5, t = 9.27, df = 8.32) at P21. Values are mean ± SEM.

Fig. 8. Peripheral SGN defects in DFNB117 model mice.

a Diagram of the CLRN2 protein; TM, transmembrane domain. b Left, mid-cochlear whole mounts of Clrn2^+/- and Clrn2^-/- mice at P21 immunolabeled for CALB2 (yellow), NGFR (cyan), and DAPI (red). Right, mid-cochlear whole mounts of Clrn2^+/- and Clrn2^-/- mice at P21 immunolabeled for NGFR, scale bars: 10 μm. c NGFR+ terminals near IHCs/100μm (+/-: 0.87 ± 0.16, n = 5 mice; -/-: 1.03 ± 0.45, n = 5 mice; two-sided Welch’s t test: p = 0.7454, t = 0.34, df = 5.06) and branches to OHCs/100μm (+/-: 77.73 ± 2.13, n = 5 mice; -/-: 58.78 ± 2.33, n = 5 mice; two-sided Welch’s t test: p = 0.0003, t = 5.99, df = 7.94) at P21. d Mid cochlear whole mounts of Clrn2^+/- and Clrn2^-/- mice at P21 immunolabeled for CTBP2 (magenta) and GLUR2 (green); scale bar: 20 μm. e CTBP2 puncta/10 IHCs (+/-: 191.02 ± 1.58, n = 5 mice; -/-: 167.59 ± 5.44, n = 5 mice; two-sided Welch’s t test: p = 0.0104, t = 4.13, df = 4.67), GLUR2 puncta/10 IHCs (+/-: 253.56 ± 11.66 n = 5 mice; -/-: 249.37 ± 3.98, n = 5 mice; two-sided Welch’s t test: p = 0.7480, t = 0.33, df = 4.92) and % GLUR2 + CTBP2 IHC puncta (+/-: 94.25 ± 2.24, n = 5 mice; -/-: 93.26 ± 1.16, n = 5 mice; two-sided Welch’s t test: p = 0.8370, t = 0.21, df = 5.936) at P21. Values are mean ± SEM.

Fig. 9. Characterization of SGNs in Vglut3^-/- mice.

a VGLUT3 is expressed in inner hair cells (IHCs) and is required for synaptic glutamate release unto spiral ganglion neurons (SGNs). b Mid-cochlear whole mounts from P21 Vglut3^+/- and Vglut3^-/- mice immunolabeled for CALB2 (yellow), NGFR (cyan) and DAPI (red); scale bar: 20 μm. c NGFR branches to outer hair cells (OHCs) ( +/-: 78.31 ± 2.42, n = 12 whole mounts/6 mice; -/-: 82.27 ± 1.16, n = 11 whole mounts/4 mice, two-sided Welch’s t test: p = 0.1427, t = 1.543, df = 15.73) and NGFR endings near inner hair cells (IHCs) ( +/-: ± , n = 12 whole mounts/6 mice; -/-: ±, n = 11 whole mounts/ 4 mice, two-sided Welch’s t test: p = 0.0103, t = 2.987, df = 13.33) at P21. d Mid-cochlear whole mounts of P21 Vglut3^+/- and Vglut3^-/- mice immunolabeled for CTBP2 (magenta) and GLUR2 (green). Scale bar: 10 μm. e CTBP2 puncta/10 IHCs (+/-: 117.20 ± 2.48, n = 10 whole mounts/3 mice; -/-: 122.80 ± 3.28, n = 10 whole mounts/3 mice; two-sided Welch’s t test: p = 5.168*10^-10, t = 13.19, df = 16.76), %GLUR2 + CTBP2 IHC puncta (+/-: 79.53 ± 2.84, n = 10 whole mounts/3 mice; -/-: 78.48 ± 2.69%, n = 10 whole mounts/3 mice; two-sided Welch’s t test: p = 0.7919, t = 0.26, df = 17.95) and CTBP2 puncta /10 OHCs (+/-: 20.94 ± 0.87, n = 10 whole mounts/3 mice; -/-: 26.68 ± 1.47, n = 10 whole mounts/3 mice; two-sided Welch’s t test: p = 0.0052, t = 3.24, df = 15.61) at P21. Values are mean ± SEM. f Experimental strategy for sparse labeling of SGNs with adeno-assocated virus (AAV) encoding mCherry under the human synapsin (hSyn) promoter (AAV9.hSyn.mCherry). g Mid-cochlear whole mounts in Vglut3^+/- and Vglut3^-/- mice at P21 immunolabeled for mCherry (magenta) and CALB2 (green); scale bar: 10 μm. h Quantified sparse label results at P21 ( +/-: IHC contact only- 97.82%; abnormal projection- 2.17% n = 92 neurons/7 mice; -/-: IHC contact only- 95.15%; abnormal projection- 4.84%; n = 166 neurons/7 mice).

Fig. 10. AAV mediated gene therapy restores the function of both hair cells and SGNs.

a Diagram of experimental strategy. Adeno-associated viruses (AAV) were used to drive enhanced green fluorescent protein (EGFP) or TMIE and EGFP expression in hair cells of Tmie^-/- mice. b Auditory brainstem response thresholds for click and tone stimulation in Tmie^+/- (non-injected, black, n = 11) and Tmie^-/- mice injected with AAV.PHP.eB.EGFP (green, n = 11) or AAV.PHP.eB.TMIE::EGFP (blue, n = 15). c Representative apex, middle, and base regions of whole mounts of cochleae at P21 following AAV.PHP.eB.TMIE::EGFP injection at P1. Tissue was stained for EGFP (green) and DAPI (magenta). Scale bar, 20 μm. d Quantification of AAV.PHP.eB.TMIE::EGFP transduction in cochlear inner hair cells (IHCs, hollow points) and outer hair cells (OHCs, filled points) as a function of distance from apex (n = 5 Tmie^-/- mice). e Representative apical-cochlear whole mounts at P21 immunolabeled for EGFP and NGFR; scale bars, left and middle panels: 10 μm, right panels: 5 μm. f NGFR⁺ terminals near IHCs/100 μm in P21 Tmie^-/- mice infected with AAV.PHP.eB.EGFP (n = 3 mice) or AAV.PHP.eB.TMIE::EGFP (n = 5 mice) (apex: two-sided Welch’s t test: p = 0.0003, t = 8.21, df = 5.30; middle: two-sided Welch’s t test: p = 0.034, t = 8.24, df = 3.07; base: two-sided Welch’s t test: p = 0.0426, t = 3.27, df = 3.19). g NGFR⁺ branches to OHCs/100 μm in P21 Tmie^-/- mice infected with AAV.PHP.eB.EGFP or AAV.PHP.eB.TMIE::EGFP (apex: two-sided Welch’s t test: p = 2.024*10⁻⁴, t = 9.56, df = 5.945; middle: two-sided Welch’s t test: p = 0.0058, t = 6.76, df = 3.12; base: two-sided Welch’s t test: p = 0.0123, t = 7.79, df = 2.19). Values are mean ± SEM.