Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction

Abstract

Caloric restriction (CR) extends the life span and health span of a variety of species and slows the progression of age-related hearing loss (AHL), a common age-related disorder associated with oxidative stress. Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.

Figure 1. CR Prevents AHL and Protect Cochlear Neurons in WT Mice, But not in Sirt3^−/− Mice

(A) ABR hearing thresholds were measured at 32, 16, and 8 kHz from control diet and/or calorie restricted WT (left) and Sirt3^−/− (right) mice at 2 and 12 months of age (n = 9–12). *Significantly different from 2-month-old WT or Sirt3^−/− mice (P < 0.05), **significantly different from 12-month-old WT mice (P < 0.05). CD = control diet, CR = calorie restricted diet. (B–M) Neurons in the basal cochlear regions from WT mice in control diet at 2 (B and C) and 12 (F and G) months of age, and calorie restricted diet at 12 months of age (J and K). Neurons from control diet Sirt3^−/− mice at 2 (D and E) and 12 (H and I) months of age, and calorie restricted Sirt3^−/− mice at 12 months of age (L and M)(n = 5). Arrows in the lower magnification photos indicate neuron regions. Scale bar = 100 µm (B, F, J, D, H, and L), 20 µm (C, G, J, E, I, and M). Data are means ± SEM. See also Figure S1, Figure S2, and Figure S3.

Figure 2. CR Reduces Oxidative DNA Damage and Increases Cell Survival in the Cochleae from WT Mice, But not from Sirt3^−/− Mice

(A) Oxidative damage to DNA (apurinic/apyrimidinic sites) was measured in the cochlea and neocortex from control diet and calorie restricted WT and Sirt3^−/− mice at 12 months of age (n = 4–5). AP sites= apurinic/apyrimidinic sites. *Significantly different from 12-month-old WT mice (P < 0.05). (B) Oxidative damage to DNA (8-oxodGuo) was measured in the liver from control diet and calorie restricted WT and Sirt3^−/− mice at 12 months of age (n = 4–5). (C) Neuron survival (neuron density) of basal, middle, and apical cochlear regions was measured from control diet and calorie restricted WT and Sirt3^−/− mice at 12 months of age (n = 4–5). (D) OH (outer hair) cell survival (%) of basal, middle, and apical cochlear regions was measured from control diet and calorie restricted WT and Sirt3^−/− mice at 12 months of age (n = 4–5). (E) IH (inner hair) cell survival (%) of basal, middle, and apical cochlear regions was measured from control diet and calorie restricted WT and Sirt3^−/− mice at 12 months of age (n = 4–5). Data are means ± SEM. See also Figure 1B–M.

Figure 3. Sirt3 Increases the Ratios Of GSH:GSSG in Mitochondria During CR

(A–C) Ratios of GSH:GSSG (A), GSSG (B), and GSH (C) were measured in the inner ear, brain (neocortex), and liver from control diet and calorie restricted WT and Sirt3^−/− mice at 5 months of age (n = 4–5). *Significantly different from 12- or 5-month-old WT mice (P < 0.05). Data are means ± SEM.

Figure 4. Sirt3 Increases Idh2 Activity and NADPH Levels in Mitochondria by Decreasing the Acetylation State of Idh2 During CR

(A) Top panels: Western blot analysis of Sirt3 and Idh2 levels in the liver from 5-month-old WT or Sirt3^−/− fed either control or calorie restricted diet. Lower panels: Endogenous acetylated Idh2 was isolated by immunoprecipitation with anti-Idh2 antibody followed by western blotting with anti-acetyl-lysine antibody (n = 3). (B–C) Quantification of the amounts of total Idh2 acetylation (B) and Sirt3 protein (C) from (A). Western blot was normalized with Idh2 levels or Sirt3 levels quantified and analyzed by Image software (n = 3). (D) Idh2 activities were measured in the liver, inner ear (cochlea), and brain (neocortex) from control diet and calorie restricted WT and Sirt3^−/− mice at 5 months of age (n = 3–5). (E) Ratios of NADPH:total NADP (NADP⁺ + NADPH) were measured in the liver, inner ear, and brain (neocortex) from control diet and caloric restricted WT and Sirt3^−/− mice at 5 months of age (n = 3–5). *Significantly different from control diet fed WT mice (P < 0.05). Data are means ± SEM.

Figure 5. Sirt3 Directly Deacetylates Idh2 and Stimulates Activity

(A–B) Sirt3 interacts with Idh2. Idh2 or Sirt3 were immunoprecipitated from HEK293 cell lysates with IgG antibody or FLAG beads. Precipitated Idh2-FLAG was detected by anti-FLAG antibody and co-IP Sirt3-HA was detected by anti-HA as indicated (A). Precipitated Sirt3-FLAG was detected by anti-FLAG antibody and co-IP Idh2-MYC was detected by anti-MYC as indicated (B) (n = 3). (C) Sirt3 deacetylates Idh2 in HEK293 cells. Idh2 was cotransfected with or without Sirt3, isolated by immunoprecipitation with anti-MYC antibody followed by western blotting with anti-acetyl-lysine antibody (n = 3). (D) Sirt3, but not Sirt5, deacetylates Idh2 in vitro. Acetylated Idh2 was prepared as outlined in Experimental Procedure, and was incubated with purified recombinant Sirt3 or Sirt5 with or without NAD⁺ at 37 °C for 1 hour. Acetylation status was assessed by western blotting with anti-acetyl-lysine antibody (n = 3). An anti-FLAG western shows equivalent Idh2 protein levels were used and coomassie staining shows purified Sirt3 and Sirt5. (E) In vitro deacetylation of Idh2 by Sirt3, but not Sirt5, stimulates Idh2 activity. Acetylated Idh2 in buffer (Tris (pH 7.5), with or without 1 mM NAD, and 1 mM DTT) was incubated with purified 50 nM Sirt3 or Sirt5 (Hallows et al., 2006) at 37°C for 1 hour, followed by Idh2 activity assay (n = 3). *Significantly different from Idh2 alone (P < 0.05). Data are means ± SEM. See also Figure S4.

Figure 6. Overexpression of Sirt3 and/or Idh2 Is Sufficient to Increase NADPH Levels and Protects HEK293 Cells from Oxidative Stress

(A–B) NADPH concentrations were significantly increased when either Idh2 or Sirt3 or both were stably overexpressed in HEK293 cells. Measurements with errors are shown for the four different stable cell populations from each type of transfection (vector alone, Sirt3, Idh2 and Sirt3 with Idh2) (n = 3). *Significantly different from vector alone (P < 0.05), **Significantly different from Idh2 or Sirt3 (P < 0.05), (B) Western blotting confirms Idh2 and Sirt3 stable expression. (C–D) Sirt3 and/or Idh2 overexpression is sufficient to protect HEK293 cells from the exogenous oxidants hydrogen peroxide (H₂O₂)(C) and menadione (D). The four different stable cells were transiently exposed to either 1 mM H₂O₂ or 25 µM menadione (n = 16). Data are means ± SEM.

Figure 7. A Model for the CR-mediated Prevention of AHL in Mammals

In response to CR, SIRT3 activates Idh2, thereby increasing NADPH levels in mitochondria. This in turn leads to an increased ratio of GSH:GSSG and decreased levels of ROS, thereby resulting in protection from oxidative stress and prevention of AHL in mammals.