Downregulation of REST in the cochlea contributes to age-related hearing loss via the p53 apoptosis pathway

Abstract

Age-related hearing loss (AHL) is the most common sensory disorder amongst the elderly population. Although the degeneration of spiral ganglion neurons (SGNs) and hair cells (HCs) is considered to play a critical role in AHL, the mechanism has not been fully outlined. The repressor element 1-silencing transcription factor (REST) has recently been associated with mediating cell death in neurodegenerative diseases. However, whether REST induces degeneration of cochlear HCs and SGNs to contribute to AHL remains unknown. Here, we report that REST expression was decreased in HCs and SGNs in AHL mice. Conditional deletion of Rest in HCs and SGNs of 2-month-old mice resulted in hearing loss accompanied by the upregulation of p53, TNFR1(tumor necrosis factor receptor-1), and cleaved caspase-3. The p53 inhibitor pifithrin-α significantly attenuated SGN and HC damage and rescued hearing impairment in Rest cKO mice. Furthermore, downregulation of REST by H₂O₂ treatment induced apoptosis in the House Ear Institute Organ of Corti 1 cell, through the upregulation of p53. In contrast, overexpression of REST reversed the changes in p53 expression. In addition, REST was further shown to bind directly to the p53 promoter site, thereby inhibiting the effect of p53. Finally, in aged mice, the p53 inhibitor significantly reduced loss of HCs and SGNs, and subsequently improved hearing. In summary, our findings indicate that REST has a protective role in AHL, and that its deficiency upregulates p53 and induces cochlear cell apoptosis, which that leads to deafness.

Fig. 1. Degeneration of SGNs and HCs in AHL mice.

A Representative ABR waveforms, in response to clicking sound pressure levels, in 2- and 9-month-old C57 mice. B ABR thresholds were measured from 2- and 9-month-old C57 mice at different frequencies (4–32 kHz). Arrows indicate the threshold exceeding the maximum of TDT ABR system. C Representative ABR waveforms of 2- and 9-month-old BALB/c mice. D ABR thresholds of 2- and 9-month-old BALB/c mice are indicated. E Immunofluorescence showed the morphological changes of HCs in 2- and 9-month-old C57 and BALB/c mice. Scale bar, 10 µm. F Quantification of OHCs and IHCs from 2- and 9-month-old C57 mice. G DPOAE threshold measurement of 2- and 9-month-old C57 mice. Arrows indicate the threshold exceeding the maximum of TDT system. H Quantification of OHCs and IHCs from 2- and 9-month-old BALB/c mice. I Morphological changes of SGNs were observed in the apical, middle, and basal cochlea of 2- and 9-month-old BALB/c and C57 mice. Scale bar, 20 µm. J The density of SGNs was quantified in 2- and 9-month-old C57 mice and BALB/c mice. Data are represented as means ± SEM, **P < 0.01, ***P < 0.001.

Fig. 2. Downregulation of REST in SGNs and HCs in AHL mice is associated with apoptosis.

A–C REST expression was significantly decreased in the cochleae of AHL mice at the mRNA and protein levels, as determined by real-time PCR (A) and Western blotting (B, C). D, E Immunofluorescence showed REST expression in HCs (D) and SGNs (E) from young and AHL mice. Scale bar: 10 µm in (D), 20 µm in (E). F The mRNA expression levels of BID, BAX, FADD, FAS, P53, TNFR1 were significantly increased in cochleae from AHL mice. G, H The protein levels of p53, TNFR1, caspase-3 and cleaved caspase-3 were increased in AHL mice, as determined by Western blot (n = 4). I, J Expression of cleaved caspase-3 in SGNs from young and AHL mice. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 3. Deletion of REST in cochlear results in hearing impairment in mice.

A Schematic illustration of the Rest conditional knockout (Rest cKO) generation process. Two LoxP sites are inserted on either side of the coding sequence of exon 2. Expression of Cre recombinase is activated by a specific Atoh1 promoter in the cochlea, resulting in loss of REST function in the cochlea. B Modified Rest flox allele exon 2 and Atoh1-Cre insertion was confirmed by PCR genotyping of DNA extracted from cochlea of vehicle (WT), or Rest cKO mice. C Representative ABR waveforms of WT, Rest Het (Heterozygote mice), and Rest cKO (Homozygous mice). D ABR thresholds of WT, Rest Het, and Rest cKO mice in response to pure tone stimuli (8–32 kHz). E Morphological changes of SGNs from WT and Rest cKO mice. Scale bar: 20 µm. F The density of SGNs was quantified in WT and Rest cKO mice. G Morphological changes in HCs were observed from WT and Rest cKO mice. Scale bar: 10 µm. H, I Quantification of OHCs and IHCs in WT and Rest cKO mice. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 4. REST deficiency induced apoptosis of SGNs and HCs in 3-month-old mice.

A mRNA levels of apoptosis-related genes showed an increase in the cochlea of Rest cKO mice. B, C Expression of apoptosis-related protein in cochlea from WT and Rest cKO mice was measured by Western blot. D TUNEL staining showed the apoptosis of SGNs in apex, middle, and base of the cochlea from WT and Rest cKO mice. Scale bar: 20 µm. E Expression of cleaved caspase-3 in cochlea from WT and Rest cKO mice. Scale bar: 20 µm. F, G Quantification of the number of TUNEL-positive cells and cleaved-caspase-3 cells in SGNs of WT and Rest cKO mice. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 5. p53 inhibitor pifithrin-α rescues the hearing loss in Rest cKO mice.

A Timeline for ABR testing and drug treatment. Pifithrin-α was administered to 2-month-old Rest cKO mice at 2.2 mg/kg every other day, for 30 days. B Representative ABR waveforms were shown in WT, Rest cKO, and Rest cKO+ pifithrin-α (PFT-α) mice. C ABR thresholds of WT and Rest cKO mice are treated with pifithrin-α. Arrows indicate when the threshold exceeded the upper limits of TDT ABR system. D, E Morphometry of SGNs in WT, Rest cKO, and Rest cKO+ pifithrin-α mice. Scale bar: 20 µm. F–I Expression of cleaved caspase-3 in SGNs of apex, middle and base of cochlea from WT and Rest cKO mice. Scale bar: 20 µm. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 6. Upregulation of REST attenuates apoptosis by inhibiting the P53 pathway in vitro.

A, B REST knockdown by siRNA increased p53 and cleaved caspase-3 expression in HEI-OC1 cells. C, D. The expression of REST and p53 in HEI-OC1 cells treated with H₂O₂ for 1 hour. E, F Expression of REST and p53 were measured in HEI-OC1 cells transfected with REST plasmid for for 24 h and/or treated with H₂O₂ for 1 hour. G Apoptosis of HEI-OC1 cells transfected with REST plasmid for 24 h and/or treated with H₂O₂ for 1 hour was determined by Flow cytometry. H ChIP analysis indicated the direct binding of REST to the promoter regions of p53 gene in HEI-OC1 cells. I ChIP assay detecting P53 and SNAP25 confirmed REST binding to p53 by nucleic acid electrophoresis, using isotype IgG as negative control. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 7. p53 inhibitor pifithrin-α rescues the hearing loss in AHL mice.

A Timeline for ABR testing and drug treatment. Pifithrin-α was administered to 8-month-old WT mice at 1.1 or 2.2 mg/kg every other day, for 30 days. B, C ABR thresholds in response to click (B) or pure-tone stimuli (C) were applied in Young, AHL, AHL + saline, AHL + 1.1 mg/kg pifithrin-α (PFT-α), and AHL + 2.2 mg/kg (PFT-α) mice. D, E Changes in SGN morphology and density in Young, AHL, AHL + saline, AHL + 1.1 mg/kg PFT-α, and AHL + 2.2 mg/kg PFT-α mice. F Immunofluorescence images showed alterations of HCs in mice in different treatment groups. G, H Quantification of OHCs (G) and IHCs (H) at the cochlea’s apical middle and basal regions in Young, AHL, and PFT-α treated mice. Scale bar: 20 µm in D, 10 µm in (F). Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001.

Fig. 8. Model for mechanisms of REST downregulated induced apoptosis of SGNs and HCs through p53 pathway contribute to AHL.

In WT mice, REST inhibits P53 expression, which allows HCs and SGNs exert their normal functions, and thus preserve proper hearing. In contrast, in the cochleae of Rest cKO mice or AHL mice, REST expression was decreased, resulting in reduced inhibition of P53. The high level of p53 leads to apoptosis of SGNs and HCs, and therefore hearing loss in those mice. Administration of p53 inhibitor improved hearing in Rest cKO and AHL mice.