Fig. 5

Increase of mitochondrial size and dysfunction, oxidative stress and impaired SIRT1 expression and autophagic activities. a Representative transmission electron (TEM) micrographs showing the mitochondria located in the IHCs and OHCs from WT and p43^−/− mice aged 6 months. Note the increase in the number of mitochondria with the lack of their cristae (arrowheads) and enlarged mitochondria in the hair cells of p43^−/− mice. Scale bars = 0.5 μm. b The histogram represents the mean diameter of the mitochondria in the IHCs and OHCs from p43^−/− (red bars) and WT (blue bars) mice obtained by TEM imaging measurements. Data are expressed as mean ± SD (n = ~ 45 to 50 mitochondria, taken randomly from the IHCs or OHCs, 4 cochleae per age and strain). One-way ANOVA was followed by Dunn’s test (*P = 0.014, ***P ≤ 0.001, p43^−/− vs. WT of the same age; ***P ≤ 0.001, older p43^−/− vs. 1-month-old p43^−/−; **P ≤ 0.01, ***P ≤ 0.001, older WT vs. 1-month-old WT). Inset in c Schematic representation of the mitochondrial respiratory chain. Complex I (CxI, NADH dehydrogenase) is the entry point of electron transfer in the respiratory chain. The proton (H+) gradient generated at the level of complex I, III (CxIII, cytochrome-c reductase) and IV (CxIV, cytochrome-c oxidase) is used by ATP synthase for ATP synthesis. Complex II (CxII, succinate dehydrogenase) is a central driver of the reprogramming of metabolic and respiratory adaptation in response to various stimuli and abnormalities. c CxI, CxII and CxIV activities in whole cochlear extracts from WT (blue bars) and p43^−/− (red bars) mice aged 1 and 10 months. DCIP: 2,6-dichloroindophenolate. Data are expressed as mean ± SD (n = 8 mice per age and strain). One-way ANOVA was followed by Dunn’s test (*P = 0.037, **P = 0.004, p43^−/− vs. WT of the same age; *P = 0.037, ***P ≤ 0.001, older p43^−/− vs. 1-month-old p43^−/−; *P ≤ 0.05, older WT vs. 1-month-old WT). d–f Confocal images of transverse cryostat sections of the organ of Corti (d) and SGNs (e, f) from WT and p43^−/− mice at 1 and 6 months. The sections were immunolabeled for cytochrome c oxidase (green), Myosin 7A (red), and Hoechst (blue). Reduction of cytochrome c oxidase was only observed in the SGNs, hair cells, and cochlear nerve fibers of 6-month-old p43^−/− mice. The arrowheads in d indicate the cochlear nerve fibers. DCs: Deiters cells. Scale bars: a = 0.5 μm, d–f = 20 μm. g Quantitative analysis of malondialdehyde (MDA) in whole cochlear extracts from WT (blue bars) and p43^−/− (red bars) mice aged 1 and 10 months of age. Data are expressed as mean ± SD (n = 8 mice per age and strain). One-way ANOVA test was followed by Dunn’s test (***P = 0.001, p43^−/− vs. WT of the same age; ***P ≤ 0.001, older p43^−/− vs. 1-month-old p43^−/−; *P = 0.04, older WT vs. 1-month-old WT). h Representative western blot (Inset) and histograms of SIRT1 in whole cochlear extracts from WT (blue bars) and p43^−/− (red bars) mice aged 1, 6, and 12 months. i: Representative western blot analysis using antibodies against Foxo3a, LC3B, Rab7, BNIP3, and β-actin in whole cochlear extracts from WT and p43^−/− mice aged 1, 3, 6, and 12 months. j, k: Histograms representing the levels of Foxo3a, LC3-II, Rab7, and BNIP3 in WT (blue bars) and p43^−/− (red bars). β-actin served as a loading control. Data are expressed as mean ± SD (n = 24 cochleae per age and strain. Each experiment was performed with a pool of 8 cochleae per sample, and in biological and technical triplicate). One-way ANOVA test was followed by Dunn’s test (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, p43^−/− vs. WT of the same age; * P ≤ 0.05, ***P ≤ 0.001, older p43^−/− vs. 1-month-old p43^−/−; *P ≤ 0.05, older WT vs. 1-month-old WT)