Aprataxin localizes to mitochondria and preserves mitochondrial function Peter Sykora, Deborah L. Croteau, Vilhelm A. Bohr1, and David M. Wilson III1,2 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 Edited* by James E. Cleaver, University of California, San Francisco, CA, and approved March 23, 2011 (received for review January 4, 2011) Ataxia with oculomotor apraxia 1 is caused by mutation in the APTX and flap endonuclease 1 (FEN1), has confirmed that BER in the gene, which encodes the DNA strand-break repair protein apra- mitochondria has many, if not all, proteins and pathways active in taxin. Aprataxin exhibits homology to the histidine triad superfam- nuclear BER (26–29). These facts led us to speculate that apra- ily of nucleotide hydrolases and transferases and removes 5′- taxin might have a significant role in the maintenance of mtDNA. adenylate groups from DNA that arise from aborted ligation reac- This hypothesis is also supported by similarities between AOA1 tions. We report herein that aprataxin localizes to mitochondria and diseases associated with mitochondrial dysfunction, such as in human cells and we identify an N-terminal amino acid sequence FA (30). Like AOA1, FA patients are not susceptible to cancer that targets certain isoforms of the protein to this intracellular but frequently present with peripheral neuropathy and pro- compartment. We also show that transcripts encoding this unique gressive ataxia. FA and AOA1 patients are also reported to be fi N-terminal stretch are expressed in the human brain, with highest de cient in coenzyme Q10, an essential component of the elec- tron transport chain and potent antioxidant within the mito- production in the cerebellum. Depletion of aprataxin in human SH- – SY5Y neuroblastoma cells and primary skeletal muscle myoblasts chondria (31 33). Previous studies are equivocal regarding both the biological results in mitochondrial dysfunction, which is revealed by reduced fi citrate synthase activity and mtDNA copy number. Moreover, role and cellular localization of aprataxin. To further de ne the pathology of AOA1 and the biological role of aprataxin, we ex- mtDNA, not nuclear DNA, was found to have higher levels of back- amined the subcellular localization of aprataxin in human cell ground DNA damage on aprataxin knockdown, suggesting a direct lines and characterized mitochondrial function in aprataxin- role for the enzyme in mtDNA processing. deficient neural/muscular cells, cell types of potential clinical in- terest. Our results are consistent with the hypothesis that apra- taxia with oculomotor apraxia 1 (AOA1) is an autosomal taxin plays an important role in the repair of mtDNA and that Arecessive genetic disorder caused by mutation in the APTX defects in aprataxin expression and/or function lead to mito- gene, which encodes the single strand-break DNA repair (SSBR) chondrial dysfunction and oxidative stress. protein aprataxin (1, 2). Aprataxin is a member of the histidine triad (HIT) superfamily of nucleotide hydrolases and transferases, Results ′ and it removes 5 -AMP groups that arise from aborted DNA li- Mitochondrial Localization of Aprataxin in Human Neuroblastoma gation reactions (3–6). The clinical symptoms of AOA1 include and Skeletal Muscle Cells. The intracellular distribution of apra- CELL BIOLOGY global cerebellar atrophy characterized by loss of Purkinje cells, taxin was examined by immunofluorescence detection using ocular motor apraxia, and motor and sensory neuropathy (7–13). ’ an antibody against the N-terminal portion of the protein (see The pathophysiology of AOA1 overlaps with both Friedreich s below). Aprataxin localized predominately to the nucleus of ataxia (FA) and Ataxia-Telangiectasia (A-T), rare disorders that human SH-SY5Y neuroblastoma cells, with significant cyto- present with similar neurological symptoms but different un- plasmic staining also present (Fig. S1). We note that cytoplasmic derlying genetic mutations (14). These and other autosomal re- aprataxin has been reported previously (34), although a func- cessive cerebellar ataxias can be broadly divided into two causative tional role for aprataxin in the cytoplasm has not been described. categories based on the function of the affected protein: ataxias To determine whether cytoplasmic aprataxin localized to mi- induced by DNA repair or maintenance defects that include A-T, tochondria, aprataxin antibody staining and the mitochondrial xeroderma pigmentosum (XP), and AOA1 and degenerative or – tracking dye Mitotracker Red (Molecular Probes) were covi- metabolic ataxias that include FA (15 18). sualized by confocal laser scanning microscopy in SH-SY5Y cells Although AOA1 is associated with a DNA repair defect, and a second human neuroblastoma cell line, NT2. Aprataxin lo- AOA1 patients have fewer nonneurological symptoms than calization was also investigated in primary human skeletal muscle patients with other DNA repair defects. Most strikingly, AOA1 cells (HSMM), because patient reports suggest that muscle could patients lack the cancer susceptibility associated with A-T, A-T– be a target in AOA1 (31, 32). In all three cell types, aprataxin like disorder, and XP, and they do not manifest immunological A fi partially colocalized with Mitotracker Red (Fig. 1 , depicted in de ciencies (or other peripheral symptoms) common in patients ’ fi r2 with A-T and A-T–like disorder. Cells from AOA1 patients are yellow). Pearson s correlation coef cient ( ) of the relative dis- not profoundly hypersensitive to the genotoxic agents methyl tribution of the two channels predicted a moderate colocalization (r2 = 0.179–0.359) when measuring the entire cell (Fig. 1B). methanesulphonate (MMS) or hydrogen peroxide (H2O2) (19, 20) or to compounds that generate DNA double strand breaks However, negating the effect of nuclear aprataxin on the corre- APTX lation coefficient by analyzing only mitochondrial regions of the (DSBs) (19, 21). Furthermore, knockout mice do not r2 display a nuclear DNA repair defect, and neural cells from these cells revealed substantially higher levels of colocalization ( = mice have normal SSBR capacity (20). Consistent with this lack of repair deficiency, aprataxin is not recruited to nuclear DNA Author contributions: P.S., D.L.C., V.A.B., and D.M.W. designed research; P.S. performed lesions in cells exposed to H2O2, ionizing radiation, mitomycin C, or MMS (22), and aprataxin-deficient human cells have no research; P.S., D.L.C., V.A.B., and D.M.W. analyzed data; and P.S., D.L.C., V.A.B., and D.M.W. wrote the paper. reported gross defect in genomic SSBR (19). The lack of any definitive DNA repair defect in aprataxin- The authors declare no conflict of interest. deficient cells is perplexing. Aprataxin has been reported to in- *This Direct Submission article had a prearranged editor. teract with and influence many proteins involved in base excision 1V.A.B. and D.M.W. contributed equally to this work. repair (BER) (19, 22–25). Of note, BER is active in both the 2To whom correspondence should be addressed. E-mail: [email protected]. nucleus and mitochondria, and recently, mitochondrial recruit- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ment of repair cofactors, such as Cockayne syndrome B (CSB) 1073/pnas.1100084108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1100084108 PNAS | May 3, 2011 | vol. 108 | no. 18 | 7437–7442 Downloaded by guest on September 26, 2021 Fig. 1. Aprataxin is detected in mitochondria. (A)Mi- tochondrial presence of aprataxin was investigated in neuroblastoma cells (SH-SY5Y and NT2) and primary human muscle myoblasts (HSMM) using antiaprataxin antibody (FITC) and Mitotracker Red counterstained with DAPI. Colocalization is a product of difference of mean (PDM) channel highlighting the positive correla- tion of voxels in the red and green channel, and merge denotes the merge of FITC, Mitotracker Red, and DAPI staining. Cells were viewed using Z-stack confocal mi- croscopy. (Scale bars: NT2, 20 μm; HSMM, 10 μm; SH- SY5Y APTX, 20 μm.) (B) Pearson correlation coefficient (r2) between green (antibody stain) and red (Mito- tracker) channels. The range of r2 values is presented for either the whole-image analysis or only a mitochondrial region of interest. Cells analyzed, antibody used, and sample size (n) are designated. For each whole-cell analysis, three mitochondrial colocalization comparisons were performed. (C) Colocalization of aprataxin with the mitochondrial transcription factor TFAM. Antibodies to aprataxin (FITC) and TFAM (CY5) were used, and vi- sualization was performed as above. (Scale bar: 5 μm.) 0.631–0.788). The colocalization observed is not an artifact of to localize to the mitochondria (Fig. 2B). Consistent with this ob- cross-channel noise or bleed from the red channel. servation, previous studies have shown that isoform a, which lacks To exclude the possibility that the aprataxin antibody inter- this N-terminal extension, is largely nuclear (19, 24, 36). acted nonspecifically with Mitotracker Red, we also investigated To determine more directly whether the N terminus of apra- whether aprataxin colocalized with transcription factor A mito- taxin is involved in targeting the protein to mitochondria, iso- chondrial (TFAM), a protein with a well-established role in form b (Fig. 2B) (one of the isoforms that has the putative mtDNA maintenance (Fig. 1C). The results determined that mitochondrial localization N-terminal signal) was transiently aprataxin also colocalizes with TFAM (r2 = 0.328–0.437 or expressed as a C-terminal GFP fusion protein in SH-SY5Y and 0.561–0.702). NT2 cells. The cells were then exposed to Mitotracker Red to visualize mitochondrial distribution (Fig. 2C). GFP immunoflu- N-Terminal Mitochondrial Targeting Sequence in Aprataxin. Mito- orescence was compared in cells with or without the aprataxin chondrial localization of aprataxin was further investigated by isoform b GFP fusion protein, allowing us to detect nonspecific performing Western blot analysis on highly purified nuclear and overlap between the red mitochondrial channel and the green mitochondrial protein extracts from SH-SY5Y cells (35).