Aging is associated with hypermethylation of autophagy genes in macrophages

Abstract

Autophagy is a biological process characterized by self-digestion and involves induction of autophagosome formation, leading to degradation of autophagic cargo. Aging is associated with the reduction of autophagy activity leading to neurodegenerative disorders, chronic inflammation, and susceptibility to infection; however, the underlying mechanism is unclear. DNA methylation by DNA methyltransferases reduces the expression of corresponding genes. Since macrophages are major players in inflammation and defense against infection we determined the differences in methylation of autophagy genes in macrophages derived from young and aged mice. We found that promoter regions of Atg5 and LC3B are hypermethylated in macrophages from aged mice and this is accompanied by low gene expression. Treatment of aged mice and their derived macrophages with methyltransferase inhibitor (2)-epigallocatechin-3-gallate (EGCG) or specific DNA methyltransferase 2 (DNMT2) siRNA restored the expression of Atg5 and LC3 in vivo and in vitro. Our study builds a foundation for the development of novel therapeutics aimed to improve autophagy in the elderly population and suggests a role for DNMT2 in DNA methylation activities.

Keywords: Aging; DNA methylation; EGCG; autophagy; macrophages.

Figure 1.

Autophagosome formation is reduced in macrophages derived from aged mice. (A) Relative expression of autophagy genes Atg5, LC3A, and LC3B in macrophages from young and aged mice. (B) Western blot analysis of Atg5-Atg12 protein complex and LC3 in the indicated macrophages. Calreticulin served as loading control. (C) Representative confocal images depicting macrophage from young and aged mice revealing the expression of LC3 (red) and DNA (blue). Student two-tailed test was used to determine the significance of autophagy related genes expression in aged macrophages (D) Quantitative analysis of detectable amount of LC3 using ImageJ software. Data is representative of three independent experiments. Error bars indicate the standard deviation (SD).

Figure 2.

Hypermethylation of autophagy promoters is associated with elevated expression of DNMT2 in macrophages from aged mice. (A) Fold changes of methylated autophagy genes Atg5, LC3A, and LC3B in macrophages from young and aged mice quantified by qRT-PCR. (B) Relative gene expression of DNMTs in aged mice macrophages compared to macrophages from young mice. (C) Representative immunofluorescent images of macrophages from young and aged mice demonstrating the expression of DNMT2 (red) and DNA (blue). Student two-tailed test was used to determine the significance of methylated genes and DNMT expression in aged macrophages. (D) Quantitative analysis of detectable amount of DNMT2 using ImageJ software Data is representative of three independent experiments. Error bars indicate the SD. (E) Western blot analysis of DNMT2 expression level in the indicated macrophages using clareticluin level as internal loading control.

Figure 3.

DNMT2 associates with LC3, (A) Representative of confocal images depicting macrophages from young and aged mice reveal the localization of autophagosomal marker LC3 (green) and methyltransferase DNMT 2 (Red). (B) Western blot analysis of immunoprecipitation samples prepared from aged and young mice macrophages reveal the expression level and protein interaction of LC3 and DNMT2. (C) Relative gene expression of DNMT2, Atg5, LC3A, and LC3B in aged mice macrophages that were transfected with indicated siRNA against DNMT2 or Luciferase gene as a control. Student two-tailed test was used to determine the significance of autophagy related genes expression in aged macrophages Error bars indicate the SD. (D) Immunoblotting analysis of conjugated Atg5-Atg12 protein complex, DNMT2 and LC3 in transfected macrophages from aged mice. Data is representative of two independent experiments.

Figure 4.

Restoration of ATG5 and LC3 expression in macrophages from aged mice in response to EGCG. (A) Relative gene expression of DNMT2, Atg5 and LC3B in aged mice macrophages that were pretreated with the indicated methylation inhibitors compared to macrophages pretreated with DMSO. Student two-tailed test was used to determine significance. Error bars indicate the SD. (B) Immunoblotting analysis of Atg5-Atg12 protein complex in macrophages from aged mice upon treatment with the indicated methylation inhibitors. Calreticulin served as loading control.

Figure 5.

The expression of Atg5 and LC3 is restored in lungs of live aged mice in response to EGCG. (A) Relative gene expression of DNMT2, Atg5, LC3A, and LC3B in lung homogenates of aged mice treated by intratracheal installation of EGCG (300 μg/day) in comparison to young and aged mice treated with DMSO (30 μl/day). Student two-tailed test was used to determine the significance of DNMT2 and autophagy related genes expression in aged macrophages. Error bars indicate the SD. (B) Immunoblotting analysis of Atg5-Atg12 protein complex and LC3 in aged mice treated with EGCG. Calreticulin served as loading control. n = 3.