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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.

SUBMITTER: Garcia-Diaz B

PROVIDER: S-EPMC4512623 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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

Garcia-Diaz Beatriz B Barca Emanuele E Balreira Andrea A Lopez Luis C LC Tadesse Saba S Krishna Sindhu S Naini Ali A Mariotti Caterina C Castellotti Barbara B Quinzii Catarina M CM

Human molecular genetics 20150514 16

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, i ...[more]

PMID: 25976310

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Project description:PurposeOxidative stress is a risk factor for the onset and progression of primary open-angle glaucoma (POAG), but the exact molecular basis remains unknown. Here, we investigated the mechanisms for Pro370Leu mutant myocilin to induce mitochondrial dysfunction and subsequent reactive oxygen species (ROS) generation in trabecular meshwork (TM) cells obtained from POAG individuals.MethodsPrimary non-diseased human TM cultures were transfected with pIRES-EGFP (Mock), pIRES-wild-type (WT), or pIRES-Pro370Leu mutant myocilin. Transfection efficiency and myocilin subcellular localization were determined by polymerase chain reaction (PCR), western blot analysis, and confocal microscopy. ROS levels as well as free Ca(2+) concentrations in cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)]m) were examined by 2'7'-dichlorofluorescein diacetate (H(2)-DCF-DA), Fluo-3 acetoxymethyl ester (Fluo-3/AM), and Rhod-2 acetoxymethyl ester (rhod-2/AM), respectively, using flow cytometry. Mitochondrial functions were revealed by changes in mitochondrial membrane potential (DeltaPsim) and ATP production, which were found by fluorescent probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively.ResultsBoth WT and Pro370Leu mutant myocilin are localized in the mitochondria of TM cells as indicated using confocal microscopy and western blot analysis. Overexpression of WT myocilin decreases DeltaPsim, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca(2+)](c), and [Ca(2+)](m) levels, and lower ATP production, and yet, these effects are not seen in the overexpression of WT myocilin.ConclusionsOur findings suggested that Pro370Leu mutant myocilin causes mitochondrial defects, which may lead to TM cell dysfunction and even cell death. Therefore, preventive measures targeting mitochondrial protection may delay the onset of glaucoma in individuals carrying the Pro370Leu myocilin mutation.

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

Publications

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

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