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. 2014 Feb 4;9(2):e88019.
doi: 10.1371/journal.pone.0088019. eCollection 2014.

Age-related decrease in the mitochondrial sirtuin deacetylase Sirt3 expression associated with ROS accumulation in the auditory cortex of the mimetic aging rat model

Lingling Zeng  1 , Yang Yang  1 , Yujuan Hu  1 , Yu Sun  1 , Zhengde Du  2 , Zhen Xie  1 , Tao Zhou  1 , Weijia Kong  1
Affiliations

Age-related decrease in the mitochondrial sirtuin deacetylase Sirt3 expression associated with ROS accumulation in the auditory cortex of the mimetic aging rat model

Lingling Zeng et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(5):e98726

Abstract

Age-related dysfunction of the central auditory system, also known as central presbycusis, can affect speech perception and sound localization. Understanding the pathogenesis of central presbycusis will help to develop novel approaches to prevent or treat this disease. In this study, the mechanisms of central presbycusis were investigated using a mimetic aging rat model induced by chronic injection of D-galactose (D-Gal). We showed that malondialdehyde (MDA) levels were increased and manganese superoxide dismutase (SOD2) activity was reduced in the auditory cortex in natural aging and D-Gal-induced mimetic aging rats. Furthermore, mitochondrial DNA (mtDNA) 4834 bp deletion, abnormal ultrastructure and cell apoptosis in the auditory cortex were also found in natural aging and D-Gal mimetic aging rats. Sirt3, a mitochondrial NAD+-dependent deacetylase, has been shown to play a crucial role in controlling cellular reactive oxygen species (ROS) homeostasis. However, the role of Sirt3 in the pathogenesis of age-related central auditory cortex deterioration is still unclear. Here, we showed that decreased Sirt3 expression might be associated with increased SOD2 acetylation, which negatively regulates SOD2 activity. Oxidative stress accumulation was likely the result of low SOD2 activity and a decline in ROS clearance. Our findings indicate that Sirt3 might play an essential role, via the mediation of SOD2, in central presbycusis and that manipulation of Sirt3 expression might provide a new approach to combat aging and oxidative stress-related diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MDA levels and SOD2 activity in the auditory cortex.
Effects of age and D-Gal on MDA levels and SOD2 activity in the auditory cortex. The level of MDA was increased in the D-Gal groups, while the activity of SOD2 was decreased in the D-Gal groups. *Significantly different from the NS groups (*P<0.05, **P<0.01). ∧Significantly different from the 4-month-old NS group (P<0.01). #Significantly different from the 4-month-old D-Gal group (P<0.01).
Figure 2
Figure 2. mtDNA common deletion in the auditory cortex and schematic diagram.
A. Accumulation of mtDNA common deletion was measured using quantitative PCR. The levels of the mtDNA common deletion were significantly increased in the D-Gal groups compared to the corresponding NS groups. A significant difference was also found between the 4- and 18-month-old NS or D-Gal group. **Significantly different from the NS groups (P<0.01). ∧Significantly different from the 4-month-old NS group (P<0.01). # Significantly different from the 4-month-old D-Gal group (P<0.01). Data were expressed as the mean ± SEM. (n = 6 per subgroup). B. Schematic diagram and sequences of the 4834 bp mtDNA deletion (nt 8103 – nt 12936 or nt 8119 – nt 12952). Bold black letters indicate the nucleotide sequences flanking the breakpoints of the deleted mtDNA, and the bold blue letters indicate the direct repeats. Sequences of the PCR products are shown below the in schematic diagram. The arrowheads indicate the potential breakpoints.
Figure 3
Figure 3. Age-related changes in the ultrastructural morphology in the auditory cortex.
Ultrastructural changes in the auditory cortex at different ages in the NS and D-Gal groups. Normal nucleus, uniformly chromatin, and normal mitochondria (white asterisks) are shown in the 4- and 10-month-old NS group and the 4-month-old D-Gal group, irregular nucleus, condensed chromatin, and swollen and vacuolated mitochondria (black asterisks) were found in the 18-month-old NS group, 10- and 18-month-old D-Gal group. Intact and compact myelin (white arrows) was in the 4- and 10-month-old NS group, swollen and disrupted myelin (black arrows) was in the 18-month-old NS group and different D-Gal groups. Enlarged endoplasmic reticulum was found in the 18-month-old NS group. Accumulated lipofuscin (white arrowheads) were shown in the D-Gal groups.
Figure 4
Figure 4. Cell apoptosis in the auditory cortex.
Apoptotic cells were determined using TUNEL staining. No TUNEL-positive cellswere observed in the 4-month-old NS and D-Gal groups. The number of TUNEL-positive cells in the 10- and 18-month-old D-Gal group was significantly increased compared to the age-matched NS groups. **Significantly different from the NS groups (P<0.01). ∧Significantly different from the 4-month-old NS group (P<0.01). # Significantly different from the 4-month-old D-Gal group (P<0.01). Magnification: 400×. (n = 6 per subgroup).
Figure 5
Figure 5. mRNA expression of Sirt3 and SOD2 in the auditory cortex.
Quantitative RT-PCR was used to measure the effect of age and D-Gal on Sirt3 and SOD2 mRNA expression. The levels of Sirt3 mRNA expression in the three D-Gal groups were significantly lower than the NS groups, but a significant decrease of SOD2 mRNA expression was only found between the 18-month-old NS and D-Gal groups. Both gene expressions were significantly decreased with age. *Significantly different from the NS groups (*P<0.05, **P<0.01). ∧Significantly different from the 4-month-old NS group (P<0.05). # Significantly different from the 4-month-old D-Gal group (P<0.01). Data are expressed as the mean ± SEM. (n = 6 per subgroup)
Figure 6
Figure 6. Physical interaction between SOD2 and Sirt3 in the auditory cortex.
A. Endogenous SOD2 was immunopurified from the auditory cortex with anti-SOD2 antibody, followed by western blotting with anti-SIRT3, anti-Sirt4 and anti-Sirt5 antibodies. B. Endogenous Sirt3 was immunopurified from the auditory cortex with anti-Sirt3 antibody, followed by western blotting with anti-SOD2, anti-SOD1 and anti-SOD3 antibodies.
Figure 7
Figure 7. Protein levels of Sirt3 and SOD2 and acetylation levels of SOD2 in the auditory cortex.
A. Top panels: Western blotting analysis of Sirt3 and SOD2 in the auditory cortex from the 4-, 10- and 18-month-old rats in the NS and D-Gal groups. GAPDH was used as a reference. Lower panels: Endogenous acetylated SOD2 was isolated by immunoprecipitation with anti-SOD2 antibody followed by western blotting with anti-acetyl-lysine antibody. SOD2 was used as a reference. (n = 6 per subgroup) B. Quantification of the amounts of total Sirt3 protein (Fig. 7B) from (Fig. 7A).The levels of Sirt3 protein were significantly decreased in the D-Gal groups compared to the NS groups, as well as in the 18-month-old groups compared to the 4-month-old groups. C. Quantification of the amounts of total SOD2 protein (Fig. 7C) from (Fig. 7A). The levels of SOD2 protein was significantly decreased between the 18-month-old D-Gal and NS groups. Significant differences were also found between the 4- and 18-month-old groups. D. Quantification of the amounts of SOD2 acetylation (Fig. 7D) from (Fig. 7A). The levels of SOD2 acetylation were significantly increased in the D-Gal groups compared to the NS groups, as well as in the 18-month-old groups compared to the 4-month-old groups. **Significantly different from the NS groups (P<0.01). ∧Significantly different from the 4-month-old NS group (P<0.01). #Significantly different from the 4-month-old D-Gal group (P<0.01).
Figure 8
Figure 8. Sirt3 protein expression in the auditory cortex.
An immunofluorescence assay was used to measure the effects of age and D-Gal on Sirt3 protein expression in the auditory cortex. The levels of Sirt3 protein expression in the D-Gal groups were significantly lower compared to the NS groups. The levels were also decreased in the 18-month-old groups compared to the 4-month-old groups.
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This work was supported by grants from the Major State Basic Research Development Program of China (973 Program) (No. 2011CB504504), the National twelfth-Five Year Research Program of China (No. 2012BAI12B02), the National Natural Science Foundation of China (No. 81230021, No. 30872865 and No. 30672309), the National Nature Science Foundation of China (No. 81200745), National Nature Science Foundation of China (No. 81000409). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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