PGC-1α regulates the interplay between oxidative stress, senescence and autophagy in the ageing retina important in age-related macular degeneration

Abstract

We previously showed that mice with knockout in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) gene encoding the PGC-1α protein, and nuclear factor erythroid 2 like 2 (NFE2L2) gene, exhibited some features of the age-related macular degeneration (AMD) phenotype. To further explore the mechanism behind the involvement of PGC-1α in AMD pathogenesis we used young (3-month) and old (12-month) mice with knockout in the PPARGC1A gene and age-matched wild-type (WT) animals. An immunohistochemical analysis showed age-dependent different expression of markers of oxidative stress defence, senescence and autophagy in the retinal pigment epithelium of KO animals as compared with their WT counterparts. Multivariate inference testing showed that senescence and autophagy proteins had the greatest impact on the discrimination between KO and WT 3-month animals, but proteins of antioxidant defence also contributed to that discrimination. A bioinformatic analysis showed that PGC-1α might coordinate the interplay between genes encoding proteins involved in antioxidant defence, senescence and autophagy in the ageing retina. These data support importance of PGC-1α in AMD pathogenesis and confirm the utility of mice with PGC-1α knockout as an animal model to study AMD pathogenesis.

Keywords: AMD; PGC‐1α; ageing retina; age‐related macular degeneration; autophagy; cellular senescence; oxidative stress; peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha.

FIGURE 1

Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) influences the expression of markers of antioxidant defence in the retinal pigment epithelium (RPE) of young and old mice. Confocal microscopy whole retina images of the immunoreactivity of the markers of antioxidant defence: APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1), OGG1 (8‐oxoguanine DNA glycosylase), PDIA2 (protein disulfide isomerase family A member 2), TDX (thioredoxin), SOD1 (superoxide dismutase 1) and H2AX (H2A.X variant histone H2AX phosphorylated at Ser139) in RPE of wild‐type (WT) and mice with the global knockout in the PPARGC1 gene encoding the PGC‐1α protein (KO) aged 3 or 12 months. Puncta specific to the label of the primary antibody to each marker have the same colour as its abbreviation displayed between image panels. DAPI was used to stain the nuclei of RPE cells (blue). ONL, outer nuclear level; INL, inner nuclear level. Scale bar: 50 μm (upper panels). Mean grey value for the markers of antioxidant defence determined in WT and KO mice. Median ± lower and upper quartile, error bars represent minimum and maximal values; the number of readings n = 76–175; *p < 0.05, **p < 0.01, ***p < 0.001 as compared with WT animals (lower panels).

FIGURE 2

Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) influences the expression of markers of senescence in the retinal pigment epithelium (RPE) of young and old mice. Confocal microscopy whole retina images of the immunoreactivity of the markers of senescence: p16 (cyclin dependent kinase inhibitor 2A), p21 (cyclin dependent kinase inhibitor 1A), p53 (tumour protein p53), DEC1 (basic helix–loop–helix family member E40) and HMGB1 (high mobility group box 1) in RPE of wild‐type (WT) and mice with the global knockout in the peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha gene (PPARGC1A) encoding the PGC‐1α protein (KO). Puncta specific to the label of the primary antibody to each marker have the same colour as their abbreviations between images. DAPI was used to stain the nuclei of RPE cells (blue). ONL, outer nuclear level; INL, inner nuclear level. Scale bar: 50 μm (upper panels). Mean grey value for the markers of antioxidant defence determined in WT and KO mice. Median ± lower and upper quartile, error bars present minimum and maximal values; the number of readings n = 51–105; **p < 0.01, ***p < 0.001 as compared with WT animals (lower panels).

FIGURE 3

Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) influences the expression of markers of autophagy in the retinal pigment epithelium (RPE) of young and old mice. Confocal microscopy whole retina images of the immunoreactivity of the markers of autophagy: UBB (ubiquitin), p62 (sequestosome 1), BECN1 (BECLIN1), LAMP2 (lysosomal associated membrane protein 2), MAP1LC3B (microtubule associated protein 1 light chain 3 beta, LC3B), p62 (SQSTM1, sequestosome 1), PINK1 (PTEN induced kinase 1) and PRKN (parkin RBR E3 ubiquitin protein ligase) in RPE of wild‐type (WT) and mice with the global knockout in the PPARGC1A gene encoding the PGC‐1α protein (KO). Puncta specific to the label of the primary antibody to each marker have the same colour as their abbreviations placed between image panels. DAPI was used to stain the nuclei of RPE cells (blue). Arrows in the PINK/PRKN image show clusters of colocalization of both markers. ONL, outer nuclear level; INL, inner nuclear level. Scale bar: 50 μm (upper panels). Mean grey value for the markers of antioxidant defence determined in WT and KO mice. Median ± lower and upper quartile, error bars present minimum and maximal values; the number of readings n = 43–81; *p < 0.05, **p < 0.01, ***p < 0.001 as compared with WT animals (lower panels).

FIGURE 4

Principal component analysis (PCA) of the expression of genes involved in antioxidant defence, senescence and autophagy in the ageing retina of mice with knockout in the peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PPARGC1A) gene encoding PGC‐1α gene and wild‐type animals. Each dot represents the expression of a single gene. PCA was performed for 3‐month (A) and 12‐month (B) mice, PGC‐1α knockout (C) and wild‐type (D) mice or data for all kinds of mice (E). PGC‐1α is abbreviated to PGC1, its expression in WT animals is designated by 1 and its lack—by 0.

FIGURE 5

The variable correlation plots of principal component analysis (PCA) on the first two component axes of the expression of genes involved in antioxidant defence, senescence and autophagy in the ageing retina of mice with knockout in the peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PPARGC1A) gene encoding PGC‐1α gene and wild‐type animals. The contribution of each gene expression level is represented by a colour of a gradient scale (contrib). PCA was performed for 3‐month (A) and 12‐month (B) mice, PGC‐1α knockout (C) and wild‐type (D) mice or data for all kinds of mice (E).

FIGURE 6

Interaction network for the peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PPARGC1A) gene encoding the PGC‐1α protein in the mouse ageing retina. The colours of the circles representing specific genes correspond to fold changes expressed by a logarithm of the ratio of the gene expression for 3‐ and 12‐month mice except PGC‐1α represented by a yellow circle and abbreviated to PGC1. The network was built based on String Cytoscape. All abbreviations are defined in the main text.