Excessive processing and acetylation of OPA1 aggravate age-related hearing loss via the dysregulation of mitochondrial dynamics

Abstract

The pathogenesis of age-related hearing loss (ARHL) remains unclear. OPA1 is the sole fusion protein currently known to be situated in the inner mitochondrial membrane, which is pivotal for maintaining normal mitochondrial function. While it has already been demonstrated that mutations in OPA1 may lead to hereditary deafness, its involvement in the occurrence and development of ARHL has not been previously explored. In our study, we constructed D-gal-induced senescent HEI-OC1 cells and the cochlea of C57BL/6J mice with a mutated SUMOylation site of SIRT3 using CRISPR/Cas9 technology. We found enhanced L-OPA1 processing mediated by activated OMA1, and increased OPA1 acetylation resulting from reductions in SIRT3 levels in senescent HEI-OC1 cells. Consequently, the fusion function of OPA1 was inhibited, leading to mitochondrial fission and pyroptosis in hair cells, ultimately exacerbating the aging process of hair cells. Our results suggest that the dysregulation of mitochondrial dynamics in cochlear hair cells in aged mice can be ameliorated by activating the SIRT3/OPA1 signaling. This has the potential to alleviate the senescence of cochlear hair cells and reduce hearing loss in mice. Our study highlights the significant roles played by the quantities of long and short chains and the acetylation activity of OPA1 in the occurrence and development of ARHL. This finding offers new perspectives and potential targets for the prevention and treatment of ARHL.

Keywords: OPA1; SIRT3; age‐related hearing loss; hair cells; mitochondrial dynamics; pyroptosis.

FIGURE 1

Senescent HEI‐OC1 cells exhibit dysregulated mitochondrial dynamics and OPA1 abnormalities. (a) Protein structures of L‐OPA1 and S‐OPA1. (b) Three‐dimensional structure of human OPA1 and its two lysine acetylation sites. (c) Mitotracker staining revealed that mitochondrial morphology exhibited a tendency toward fragmentation in senescent HEI‐OC1 cells after D‐gal (40 mg/mL) treatment for 24 h. Scale bar: 5 μm. (d, e) Alterations in the expression of mitochondrial dynamics‐related proteins by western blotting suggested the dysregulation of mitochondrial dynamics. n = 5. (f) The direct interaction between SIRT3 and OPA1 in senescent HEI‐OC1 cells was discerned through immunoprecipitation. (g, h) Statistical analyses of acetylated OPA1 levels as detected through immunoprecipitation experiments confirmed the increased acetylation of OPA1 in senescent hair cells. n = 8. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group; n.s. no statistical difference; one‐way ANOVA.

FIGURE 2

L‐OPA1 inhibition aggravates mitochondrial dysfunction and cellular senescence in HEI‐OC1 cells. (a) Analyses of ATP content revealed that the addition of MYLS22 or Tyrphostin A9 reduced ATP production in senescent cells. n = 10. (b) The detection of mitochondrial membrane potential (ΔΨm) levels indicated that the ΔΨm was reduced after L‐OPA1 inhibition. n = 5. (c, d) MtROS content and mean fluorescence intensity (MFI) levels in senescent HEI‐OC1 cells were measured by flow cytometry in the PE channel, showing that mtROS accumulation increased significantly after L‐OPA1 inhibition. n = 3. (e, f) SA‐β‐gal staining revealed that L‐OPA1 inhibition aggravated hair cell senescence and elevated the proportion of senescent cells. Scale bar: 10 μm. n = 5. (g, h) Western blotting results focusing on the expression of senescence marker proteins in senescent HEI‐OC1 cells suggested that p‐P53 and P21 levels were increased significantly after L‐OPA1 inhibition. n = 6. ^## p < 0.01 and ^### p < 0.001 versus the control group; *p < 0.05, **p < 0.01, and ***p < 0.001 versus the D‐gal group; n.s. no statistical difference; one‐way ANOVA.

FIGURE 3

Reducing OPA1 acetylation levels via the kaempferol‐mediated activation of SIRT3 ameliorates aberrant mitochondrial dynamics and the senescence of cochlear hair cells. (a) CCK‐8 assays revealed that kaempferol ameliorated the cytotoxic effects of D‐gal treatment on HEI‐OC1 cells (40 mg/mL, 24 h). (b–e) Immunoprecipitation and western blotting showed that kaempferol enhanced SIRT3 function, including elevated SIRT3 expression (n = 9) and reduced acetylated SOD₂ (n = 5)_, as well as reduced acetylated OPA1 levels (n = 6) in senescent HEI‐OC1 cells. (f, g) Immunofluorescent staining for SIRT3 and analyses of mean fluorescence intensity (MFI) similarly revealed that kaempferol enhanced SIRT3 expression and improved mitochondrial morphology. Green: SIRT3 staining, located to the mitochondria. Blue: DAPI staining, located to the nucleus. Scale bar: 10 μm. n = 5. (h, i) Statistical analyses of western blotting results pertaining to the expression of senescence marker proteins in senescent HEI‐OC1 cells suggested that p‐P53 and P21 levels significantly decreased after the activation of the SIRT3/OPA1 signaling by kaempferol. n = 6. (j, k) SA‐β‐gal staining and the proportion of senescence‐positive HEI‐OC1 cells indicated that the senescence phenotype was alleviated after kaempferol treatment. Scale bar: 10 μm. n = 5. ^## p  < 0.01 and ^### p < 0.001 versus the control group; **p  < 0.01 and ***p < 0.001 versus the D‐gal group; n.s. no statistical difference; one‐way ANOVA.

FIGURE 4

Reducing levels of OPA1 acetylation by activating SIRT3 attenuates mitochondrial structural damage in the cochlear hair cells of ARHL mice. (a–c) Immunohistochemistry (IHC) staining of acetylated SOD₂ and acetylated lysine revealed that the average optical density (AOD) value and positive staining area in the K223R group were significantly reduced relative to those in the WT group. Scale bar: 100 μm. n = 5. (d–h) OPA1 staining and AOD analyses revealed that OPA1 expression in the cochlea of 24‐month‐old mice in the K223R group exhibited a pronounced increase as compared to the WT group. Scale bar: 100 μm. n = 3 per turn and n = 8 per Corti. (i) TEM was utilized to observe mitochondrial structures in cochlear hair cells of 24‐month‐old SIRT3 deSUMOylated and WT mice. In the WT group, discernible alterations were evident in the mitochondria of cochlear hair cells, which exhibited significant swelling, disrupted or absent cristae structures, and the presence of vacuolization (indicated by blue arrows). Conversely, the mitochondria of hair cells in the SIRT3 K223R group exhibited a preserved, normal morphology. Scale bar: 1 μm. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the WT group; n.s. no statistical difference; one‐way ANOVA.

FIGURE 5

Reducing levels of OPA1 acetylation by activating SIRT3 alleviates the age‐related loss of inner and outer cochlear hair cells and the degeneration of SGNs. (a–d) Immunofluorescent staining results from inner hair cells (IHCs) and outer hair cells (OHCs) in the apical, middle, and basal turns of mouse cochlear basilar membrane. Green: Prestin‐stained OHCs; red: vGlut3‐stained IHCs; blue: Hoechst‐stained nuclei. Scale bar: 40 μm. Arrows indicate the absence of hair cells, and asterisks indicate the absence of entire rows of hair cells. Statistically, the loss of OHCs in basal turns in the 6‐month‐old K223R group as well as IHCs in basal turns and OHCs in all turns in the 24‐month‐old K223R group was significantly reduced as compared to the WT group. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the WT group; n.s. no statistical difference; one‐way ANOVA. (e–h) H&E staining results for cochlear SGNs and the organ of Corti from mice in the K223R and WT groups at 6 and 24 months of age. Scale bar: 100 μm. The number of preserved SGNs exhibited varying degrees of improvement in both the 6‐month‐old and 24‐month‐old K223R groups. n = 4. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the WT group; n.s. no statistical difference; one‐way ANOVA. (i, j) Average ABR thresholds at frequencies of 4, 5.6, 8, 11.3, 16, 22, and 32 kHz in the K223R group and WT group at 6 months and 24 months of age. n = 5. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the WT group; Two‐tailed unpaired t‐test. ^### p < 0.001 indicates a significant difference between the overall ABR thresholds of the K223R and WT groups; two‐way ANOVA. (k) Example of ABR waveforms recorded at 11.3 kHz in 24‐month‐old mice.

FIGURE 6

Dysregulated mitochondrial dynamics can activate pyroptosis through the NLRP1/3‐caspase 1 inflammatory pathway. (a) Western blotting revealed changes in the expression of inflammatory pathway molecules in senescent HEI‐OC1 cells. (b–i) Statistical analyses of western blotting results indicated that the inhibition of L‐OPA1 resulted in significant increases in the expression of inflammatory factors, including NLRP1/3, caspase 1, IL‐1β, IL‐18, and TNFα. n = 4. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 versus the control group; *p < 0.05, **p < 0.01, and ***p < 0.001 versus the D‐gal group; n.s. no statistical difference; one‐way ANOVA.