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. 2021 Dec 15:15:804345.
doi: 10.3389/fncel.2021.804345. eCollection 2021.

Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

Pengcheng Xu  1   2   3 , Longhao Wang  1   2   3 , Hu Peng  4 , Huihui Liu  1   2   3 , Hongchao Liu  1   2   3 , Qingyue Yuan  1   2   3 , Yun Lin  1   2   3 , Jun Xu  1   2   3 , Xiuhong Pang  5 , Hao Wu  1   2   3 , Tao Yang  1   2   3
Affiliations

Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

Pengcheng Xu et al. Front Cell Neurosci. .

Abstract

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo. In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

Keywords: HARS2; apoptosis; hair cells; hearing loss; mitochondrial.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cochlear expression of HARS2 in the wild-type and Hars2 CKO mice. (A) Immunofluorescence staining of HARS2 in cochlea (left) and organ of corti (right) of the wild-type mice at P30 using anti-HARS2 (red) and anti-Myo7a (green, labeling hair cells) antibodies and DAPI (blue). Scale bar = 100 μm. OHC, outer hair cell; IHC, inner hair cell; SGN, spiral ganglion neuron; SV, stria vascularis. (B) Relative protein expression levels of HARS2 at different ages by Western blotting, which was normalized by endogenous β-actin expression. (C) Generation of the hair cell-specific Hars2 CKO mice with the Loxp sites inserted into intron 1 and 8 of mouse Hars2 and crossed with the Gfi1Cre. (D) Verification of targeted genomic deletion of the Hars2-Loxp allele by PCR amplification. (E) Immunofluorescence staining of HARS2 in the cochleae of P30 control and Hars2 CKO mice, showing the successful knockout of Hars2 in hair cells. Scale bars = 10 μm.
FIGURE 2
FIGURE 2
Auditory brainstem response threshold and Wave-I analysis of the Hars2 CKO mice. (A) Indistinguishable ABR thresholds of the Hars2 CKO and control mice at P21. (B) Significantly elevated ABR thresholds at frequencies of 11–32 kHz at P30. (C) Further elevated ABR thresholds at all frequencies at P45. (D) Profound hearing loss at P60. (E) Significantly reduced ABR suprathreshold wave-I amplitudes at frequencies of 8, 16, and 22.6 kHz at P30. (F) Significantly prolonged ABR suprathreshold wave-I latencies at frequencies of 8, 16, and 22.6 kHz at P30. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.
FIGURE 3
FIGURE 3
Hair cell loss in the Hars2 CKO mice. (A,B) No significant hair cell loss at P21 and P30. (C) Partial loss of hair cells at the basal turn at P45. (D) Severe hair cell loss in all turns at P60. Left panels: Whole-mount cochleae were immunostained with anti-Myo7a antibody (green) and DAPI (blue). Right panels: Quantification of the numbers of OHCs (left) and IHCs (right). control: n = 5; Hars2 CKO: n = 5. Scale bars = 20 μm. * represents P < 0.05, *** represents P < 0.001.
FIGURE 4
FIGURE 4
Apoptosis analysis by TUNEL and Western blot in the cochleae of the Hars2 CKO mice at P45. (A) TUNEL (green) and anti-Myo7a (red), DAPI (blue) staining of whole-mount cochlea in the Hars2 CKO and control mice. Scale bars = 20 μm. (B) Quantification of the number of TUNEL positive OHCs and IHCs at the apical, middle, and basal turns of cochlea. (C) Protein expression of cleaved caspase-3, BCL-2, cytochrome C, and cleaved caspase-9 detected by Western blotting. (D) Relative protein expression levels after normalization against endogenous β-actin. All experiments were replicated in triplets. ** represents P < 0.01, *** represents P < 0.001.
FIGURE 5
FIGURE 5
ROS analysis in the cochleae of the Hars2 CKO mice at P30. (A,B) Immunostaining of whole-mount cochleae using anti-3-NT or anti-4-HNE antibodies (green), Phalloidin (red), and DAPI (blue). Scale bars = 20 μm. (C,D) Quantification of the 3-NT or 4-HNE immunolabeling in hair cells. (E) qPCR analysis of sensory epithelia (without spiral ganglion neurons) showing significantly decreased expression of the antioxidant factors xCT, Nqo1, Sod2, and Gsr and increased expression of pro-oxidant factor Lpo (n = 6). NS represents P > 0.05, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.
FIGURE 6
FIGURE 6
Inner hair cell patch-clamp recording in the Hars2 CKO mice. (A) Representative of the Ca2+ current in IHCs of the Hars2 CKO and control mice. The current response was induced by a voltage ramp from –90 to 60 mV and then leak subtracted. (B,C) The Ca2+ current amplitude (ICa) and reversal potential (Vrev) are significantly reduced. (D) The half activation potential (Vhalf) of calcium current is less negative. (E) Slope of activation (kslope) is normal. (F) Representative traces of Ca2+ current (top) and capacitance (bottom) upon 20 ms step depolarization. (G) The membrane capacitance change (ΔCm) is comparable for stimulations of 20 ms. (H) The Ca2+ efficiency of exocytosis triggering, quantified as the ratio of ΔCm/QCa, is comparable for stimulation of 20 ms. (I) Representative traces of Ca2+ current (top) and capacitance (bottom) upon 200 ms step depolarization. (J) The ΔCm is significantly reduced for stimulations of 200 ms. (K) The ΔCm/QCa is comparable for stimulation of 200 ms (control: n = 5; Hars2 CKO: n = 5). NS represents P > 0.05, * represents P < 0.05, ** represents P < 0.01.
FIGURE 7
FIGURE 7
Inner hair cell transmission electron microscopy of the Hars2 CKO mice between P7 and P45. (A) IHCs of the control mice at different ages contains abundant mitochondria with easily-identifiable cristae, whereas various morphologically abnormal mitochondria be observed in IHCs of the Hars2 CKO mice including swollen (open arrowhead), deformed (black arrowhead) mitochondria, mitochondria with deformed cristae (black arrows), or intrinsic vacuole (black asterisk). (B) Quantified proportion of abnormal mitochondria in IHCs. (C) Quantified number of mitochondrial cristae per mitochondria in IHCs. NS represents P > 0.05, ** represents P < 0.01, *** represents P < 0.001.
FIGURE 8
FIGURE 8
Outer hair cell transmission electron microscopy of the Hars2 CKO mice between P7-P45. (A) OHCs of the control mice at different ages contains abundant mitochondria with easily-identifiable cristae, whereas various morphologically abnormal mitochondria be observed in OHCs of the Hars2 CKO mice including swollen (open arrowhead), deformed (black arrowhead) mitochondria, mitochondria with deformed cristae (black arrows), or intrinsic vacuole (black asterisk). (B) Quantified proportion of abnormal mitochondria in OHCs. (C) Quantified number of mitochondrial cristae per mitochondria in OHCs. NS represents P > 0.05, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.
FIGURE 9
FIGURE 9
Schematic diagram of the hearing phenotype and hair cell maintenance of the Hars2 CKO mice between P14 and P60 based on the current morphological and functional studies.
See this image and copyright information in PMC

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