Aprataxin localizes to mitochondria and preserves mitochondrial function

Abstract

Ataxia with oculomotor apraxia 1 is caused by mutation in the APTX gene, which encodes the DNA strand-break repair protein aprataxin. Aprataxin exhibits homology to the histidine triad superfamily of nucleotide hydrolases and transferases and removes 5'-adenylate groups from DNA that arise from aborted ligation reactions. We report herein that aprataxin localizes to mitochondria in human cells and we identify an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. We also show that transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, which is revealed by reduced citrate synthase activity and mtDNA copy number. Moreover, mtDNA, not nuclear DNA, was found to have higher levels of background DNA damage on aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing.

Fig. 1.

Aprataxin is detected in mitochondria. (A) Mitochondrial 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 correlation 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 microscopy. (Scale bars: NT2, 20 μm; HSMM, 10 μm; SH-SY5Y APTX, 20 μm.) (B) Pearson correlation coefficient (r²) between green (antibody stain) and red (Mitotracker) 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 visualization was performed as above. (Scale bar: 5 μm.)

Fig. 2.

Targeting of aprataxin to mitochondria. (A) Western blotting of purified extracts confirms mitochondrial localization of aprataxin. Membrane was probed with antiaprataxin antibody (ab31841), which detected an ∼40-kDa band in both nuclear and mitochondrial extracts. Lamin b and GAPDH/Tubulin antibodies were used to detect nuclear or cytoplasmic contamination of mitochondrial extracts, respectively. The mitochondrial protein VDAC was used to confirm mitochondrial enrichment of the mitochondrial fraction; 20 μg protein were loaded in each lane. Blot is representative of four separate experiments. (B) Full-length protein and two relevant isoforms of aprataxin. Isoform b has a distinct 14-aa N-terminal stretch that harbors the putative mitochondrial targeting sequence (MTS). The probability of mitochondrial localization was estimated using the mitochondrial prediction software MitoProt. FHA, fork head-associated; NLS, nuclear localization signal; HIT, histidine-triad; ZNF, zinc finger. Antigenic region for the aprataxin antibody (amino acids 1–177 of isoform a) is denoted. (C) C-terminal GFP tagged isoform b aprataxin (Aprataxin GFP) localizes to the mitochondria. The location of the fusion protein was compared with the Mitotracker Red mitochondrial marker. Additional details are in Fig. 1. The nucleus is indicated with a yellow arrow. (Scale bars: SH-SY5Y, 20 μm; NT2, 10 μm.)

Fig. 3.

Total APTX- and MTS-encoding mRNA levels in human brain regions. Total APTX- (solid bars) and MTS-encoding (unfilled bars) mRNA levels were measured in the indicated brain regions and cross-compared with all other brain regions. Graph represents relative mRNA expression. RT-PCR assay was done in triplicate. Error bars represent SEM.

Fig. 4.

Mitochondrial dysfunction in aprataxin-deficient cells. (A) Increased ROS levels were seen in SH-SY5Y cells (Upper) and HSMM (Lower). Gray lines and symbols represent aprataxin-deficient cells, and black lines and symbols represent scrambled control cells. Error bars are SEM (n = 3). (B) Reduced aprataxin causes mitochondrial dysfunction, regardless of differentiation status. Western blotting was used to determine residual aprataxin (APTX) level. Citrate synthase and mtDNA copy number were measured as described in Materials and Methods. Undiff., undifferentiated. SH-SY5Y cells were differentiated (Diff.) with retinoic acid and brain-derived growth factor. The broken line represents the value compared with the scrambled control cells (percent scrambled). All assays were conducted at least in triplicate. (C) Reduced levels of aprataxin causes loss of mitochondria and a breakdown in the organization of the mitochondrial network. The HSMM mitochondrial matrix was visualized using Mitotracker Red and counterstained using DAPI. Cells were viewed using Z-stack confocal microscopy. (D) Aprataxin-deficient cells have an increased amount of endogenous mtDNA damage. PCR amplification of a mitochondrial or nuclear DNA fragment was used to estimate the amount of endogenous DNA damage compared with scrambled cells. The long amplification products were normalized against the amount of input DNA. The assay was conducted in triplicate. (B and D) *P < 0.05; ***P < 0.01. Error bars are SEM.