Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway

Abstract

Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases.

Figure 1.

Mitochondrial respiratory function in APTX-mutant and APTX-depleted cells. Respiratory chain enzyme activities normalized to mg protein levels in AOA1 fibroblasts with CoQ₁₀ deficiency (A–C) and in APTX-depleted Hela cells (F–H). Cell respiratory control ratio (D) of control and AOA1 fibroblasts. (E) Representative OCR of two control and two AOA1 cell lines. O, oligomycin; F, carbonyl cyanide-FCCP; RA, rotenone and antimycin. Data are expressed as mean ± SEM in relative percentage of controls (**P < 0.01).

Figure 2.

SDHA levels in APTX-mutant and APTX-depleted cells. Protein levels of SDHA are partially decreased in AOA1 fibroblasts with CoQ₁₀ deficiency (A) and are significantly reduced in APTX-depleted cells (B). Representative western blot. Values are expressed as mean ± SEM in relative percentage of control (*P < 0.05).

Figure 3.

CoQ₁₀ biosynthetic enzymes genes in APTX-mutant and APTX-depleted cells. mRNA expression level of CoQ₁₀ biosynthetic enzymes genes in AOA1 fibroblasts with CoQ₁₀ deficiency and APTX-depleted Hela cells. PDSS1 mRNA levels are decreased in AOA1 cells (A) and shAPTX (C), consistently with the reduction in PDSS1 protein levels (B and D). Representative PDSS1 western blot. Data are expressed as mean ± SEM in relative percentage of controls (*P < 0.05, **P < 0.01).

Figure 4.

APE1, NRF1 and NRF2 in AOA1 fibroblasts and APTX-depleted Hela cells. APE1 (A and D), NRF1 (B and E) and NRF2 (C and F) protein levels are reduced in APTX-mutant (A–C) and APTX-depleted cells (D–F). Representative western blot. Values are expressed as mean ± SEM in relative percentage of controls (*P < 0.05, **P < 0.01).

Figure 5.

CoQ₁₀ gene biosynthetic enzyme expression in APTX-depleted Hela cells. (A) PDSS1, PDSS2, COQ2, COQ5 and COQ9 are reduced in APTX-depleted cells. (B) COQ5 reduced expression was corroborated by decreased protein levels. (C) Representative western blot of COQ5. Values are expressed as mean ± SEM in relative percentage of control (*P < 0.05, **P < 0.01).

Figure 6.

NRF1, NRF2, PDSS1 and SDHA levels in APE1-depleted cells. NRF1, NRF2, PDSS1 and SDHA mRNA (A) and protein levels (B) are decreased in APE1-interfered clones. Values are expressed as mean ± SEM in relative percentage of controls (*P < 0.05, **P < 0.01).

Figure 7.

NRF1-expressing APTX-depleted Hela cells. Efficiency of NRF1 overexpression in APTX-depleted Hela cells assessed by the expression level of NRF1 mRNA (A) and western blot to measure the level of NRF1 protein (B). Expression level of PDSS1 and SDHA mRNA (C), and protein level of NRF1, NRF2, PDSS1, SDHA, COQ5 and TOM20 (D). Values are expressed as mean ± SEM in relative percentage of controls (*P < 0.05, **P < 0.01).

Figure 8.

Effects of AICAR (A) and rosiglitazone (B) supplementation on NRF1, NRF2, PDSS1, SDHA and TOM20 levels (B), in AOA1 fibroblasts. Values are expressed as mean ± SEM in relative percentage of controls (*P < 0.05).