Research ArticleMOLECULAR BIOLOGY

SCAN1-TDP1 trapping on mitochondrial DNA promotes mitochondrial dysfunction and mitophagy

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Science Advances  06 Nov 2019:
Vol. 5, no. 11, eaax9778
DOI: 10.1126/sciadv.aax9778
  • Fig. 1 Mito-SN38 containing nanoparticle poisons Top1mt (Top1mtcc).

    (A) Western blot analysis of mitochondrial lysates extracted from the indicated MEFs. TDP1−/−/WT and TDP1−/−/H493R: TDP1−/− complemented with exogenous FLAG-tagged human wild-type or SCAN1 mutant (H493R) TDP1, respectively. Blots were probed with anti-TDP1, anti-FLAG, and anti–COX IV antibodies. COX IV served as a positive mitochondrial marker. (B) Representative confocal images showing accumulation of mitochondria-targeted cationic nanoparticle containing Top1 poison irinotecan (mito-SN38; intrinsic green fluorescence) inside the mitochondria (labeled with MitoTracker red). MEFs were incubated with mito-SN38 (5 μM for 20 min), and the fluorescence patterns were recorded under live-cell microscopy. Colocalization is shown in the merged image. Quantitation of the pixel intensity of fluorescence along the indicated white line in the merged image (right). The white line was drawn arbitrarily along the region of interest (ROI). a.u., arbitrary units. (C and D) Poisoning of Top1mt (Top1mtcc) in the indicated MEFs expressing TDP1 variants; either untreated (C) or treated with mito-SN38 (5 μM for 3 hours) (D). Top1mtcc was detected by ICE (immunocomplex of enzyme) bioassay. MtDNA at increasing concentrations (0.5, 1, 2, and 4 μg) was immunoblotted with an anti-Top1mt–specific antibody. The mtDNA input was probed with anti-dsDNA (double-stranded DNA) antibody. Densitometry analysis of trapped Top1mtcc band intensity was quantified and expressed as fold increase relative to mtDNA input (error bars represent means ± SEM). Asterisks denote statistically significant differences (**P < 0.01, t test). (E and F) Cell survival curves of indicated MEF variants (E) and a SCAN1 patient–derived lymphoblastoid cell line (BAB1662) and its wild-type counterpart (BAB1668) (F). Mito-SN38–induced cytotoxicity (%) was calculated with respect to the untreated control. Each point corresponds to the mean ± SD of at least three experiments. Error bars represent SDs (n = 3).

  • Fig. 2 Induction of irreversible mtDNA damage through selective trapping of TDP1H493R.

    (A) Detection of trapped TDP1-mtDNA complexes (mtTDP1cc) by ICE bioassays in the indicated cells following no treatment or treated with mito-SN38 (5 μM for 3 hours). MtDNA at increasing concentrations (0.5, 1, 2, and 4 μg) was immunoblotted with an anti-TDP1–specific antibody. The mtDNA input was probed with anti-dsDNA antibody. Densitometry analysis of the trapped mtTDP1cc band intensity was quantified and expressed as fold increase relative to mtDNA input (error bars represent means ± SEM). Asterisks denote statistically significant difference (*P < 0.1 and ***P < 0.001, t test). (B) Catalytically defective SCAN1-TDP1 was hypothesized to be trapped at the Top1mtcc binding sites; this is shown schematically. (C) Detection of TDP1H493R trapping sites on mtDNA by chromatin immunoprecipitation (ChIP) followed by mtDNA-specific quantitative polymerase chain reaction (qPCR) analysis. FLAG-TDP1-DNA adducts were immunoprecipitated with anti-FLAG antibody in the indicated cells after treatment with mito-SN38 treatment (5 μM for 3 hours), and the putative TDP1-binding site was quantified by qPCR. The mtDNA copy numbers of each cell line were concomitantly measured using primers for the ND2 (mitochondrial) and B2M (nuclear) genes. Enrichment of TDP1-bound mtDNA is expressed as percent input, which is then normalized to the mtDNA copy number of the cell line. Data represent means ± SE of independent experiments. Asterisks denote statistically significant differences (***P < 0.001, t test). (D and E) Cells were treated with mito-SN38 for the indicated times. After mito-SN38 removal (R), cells were cultured in drug-free medium for 12 hours (top). Long-range qPCR was used to evaluate mtDNA damage. (D) Induction of mito-SN38–induced mtDNA damage in indicated cell types. Representative images are shown for mtDNA long- and short-fragment PCR after treatment (mito-SN38, 20 μM) and drug removal (R) for the indicated time. Mouse and human mtDNA-specific primers were used. (E) Quantification of mtDNA damage using the ratio of the long-fragment versus short-fragment PCR products. Data represent means ± SE of independent experiments. Asterisks denote statistically significant difference (**P < 0.01 and ***P < 0.001, t test). kbp, kilobase pair; bp, base pair.

  • Fig. 3 TDP1H493R trapping promotes mitochondrial fission.

    Mitochondrial network dynamics were analyzed in the indicated cells using live-cell microscopy and photobleaching (FRAP analysis) of cells ectopically expressing the mitochondrial targeted fluorescence protein construct (mito-YFP). Cells were either untreated (A) or pretreated with mito-SN38 (5 μM for 3 hours) (C). A submitochondrial spot indicated by a circle was bleached (BLH) with a 514-nm laser for 30 ms and imaged at regular intervals of 500 ms thereafter. Successive images taken for 60 s after bleaching illustrate the level of return of fluorescence into the bleached areas. (B and D) Quantitative mitochondrial FRAP data (n = 25). Error bars represent means ± SEM. (E and F) Mitochondrial length was measured in the indicated MEFs by staining with MitoTracker red with or without mito-SN38 (5 μM for 3 hours). Nuclei were stained with Hoechst 33342 (blue). The bar graph (n = 25) represents means ± SEM. (G and H) Western blot analysis of Mfn1 (mitofusin 1) and Drp1 (dynamin-related protein 1) in total lysates obtained from the indicated cells after treatment with mito-SN38 (5 μM for 6 hours). Actin served as the loading control. Migration of protein molecular weight markers is indicated on the right. Bar graphs represent the fold change in densitometry analysis of Mfn1 and Drp1 normalized to actin (error bars represent means ± SEM).

  • Fig. 4 TDP1H493R trapping promotes mitochondrial dysfunction in SCAN1 cells.

    (A) Fluorescence-activated cell sorting (FACS) analysis of mitochondrial membrane potential (Δψm) using TMRM before or after treatment with mito-SN38 (5 μM for 6 hours) in the indicated cells. TMRM fluorescence was plotted against cell numbers (count). Data represent means ± SE of independent experiments. (B) FACS analysis of mitochondrial volume in the indicated cells using MitoTracker green before and after treatment with mito-SN38 (5 μM for 6 hours). Data represent means ± SE of independent experiments. (C) The gene expression profile of nuclear-encoded genes [nuclear respiratory factor 1 (NRF1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), and mitochondrial transcription factor A (TFAM)] for mitochondrial biogenesis by reverse transcription PCR. Indicated cells were either not treated or treated with mito-SN38 (5 μM for 6 hours). Data represent means ± SE of three independent experiments. Asterisks denote statistically significant differences (**P < 0.01, t test). (D) Representative Western blots for nuclear-encoded NRF1, PGC1α, and TFAM in SCAN1 patient–derived lymphoblastoid cell lines (BAB1662) and their wild-type counterpart (BAB1668) before and after treatment with mito-SN38 (5 μM) for the indicated time periods. Actin is shown as the loading control. (E and F) ROS formation was measured by fluorescent dye CM-H2DCFDA in live-cell microscopy after treatment with mito-SN38 (5 μM) or with pretreatment of N-acetyl-l-cysteine (NAC) (10 mM for 2 hours) for the indicated time. The ROS intensity is shown in green, and nuclei were stained with Hoechst 33342 (blue) in the indicated cells. Plots shown on the right represent means ± SDs of at least three experiments. Asterisks denote statistically significant difference (**P < 0.01, t test). (G and H) Representative γH2AX visualization by immunofluorescence microscopy in MEFs expressing TDP1 variants after treatment with mito-SN38 (5 μM) or with pretreatment of NAC (10 mM for 2 hours) for the indicated time. The γH2AX is shown in green, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scattergrams are shown on the right for at least three experiments; means ± SDs are indicated. Asterisks denote statistically significant differences (**P < 0.01 and ***P < 0.001, t test).

  • Fig. 5 SCAN1-TDP1 in mitochondria activates autophagy.

    (A) Immunoblotting for LC3-II lipidation with an anti-LC3B–specific antibody with the indicated cell lysates before or after treatment with a sublethal dose of mito-SN38 (2.5 μM) for the indicated time periods. The positions of LC3B-I and LC3B-II are indicated. Actin is shown as the loading control. (B) Plots represent fold increase in LC3B-II band intensity in the indicated cells, normalized to actin (error bars represent means ± SEM). (C) Representative images of GFP-LC3 puncta formation using live-cell confocal microscopy in the indicated cells by ectopically expressing GFP-LC3. The cells were either untreated or treated with mito-SN38 (2.5 μM for 12 hours) or the autophagy inhibitor Baf A1 (200 nM for 4 hours) alone or in combination with Baf A1 (200 nM for 4 hours) + mito-SN38 (2.5 μM for 12 hours). The translocation of GFP-LC3 to phagosome triggers steady-state levels of autophagosomes, as indicated by GFP-LC3 puncta formation. (D) Quantification of GFP-LC3 puncta per cell after the indicated treatment obtained from immunofluorescence confocal microscopy was calculated for 20 to 25 cells (mean ± SEM) from independent experiments. Asterisks denote statistically significant differences (**P < 0.01 and ***P < 0.001, t test). (E) Representative images of differentiated neuronal cells derived from human neuroblastoma cells (SH-SY5Y) stably expressing GFP-LC3 were transfected with lentiviral constructs of TDP1WT or TDP1H493R and were analyzed for GFP-LC3 puncta formation under live-cell confocal microscopy. The cells were either kept untreated or treated with mito-SN38 (2.5 μM for 12 hours) or the autophagy inhibitor Baf A1 (200 nM for 4 hours) alone or in combination with Baf A1 (200 nM for 4 hours) + mito-SN38 (2.5 μM for 12 hours). (F) Quantification of GFP-LC3 puncta per cell after the indicated treatments was obtained from live-cell microscopy calculated for 20 to 25 cells (mean ± SEM) from independent experiments. Asterisks denote statistically significant differences (**P < 0.01 and ***P < 0.001, t test). (G and H) Cell survival of differentiated neuronal cells from SH-SY5Y cells expressing TDP1WT or TDP1H493R. Baf A1–induced cytotoxicity (%) was calculated with respect to the untreated control (G). Combination of mito-SN38 (2.5 μM for 12 hours) + Baf A1 (for the next 24 hours)–induced cytotoxicity (%) was calculated with respect to the untreated control (H). Each point corresponds to the mean ± SD of at least three experiments. Error bars represent SD (n = 3).

  • Fig. 6 TDP1H493R trapping in mitochondria activates mitophagy.

    (A) Schematic representation for mitophagy monitoring using the dual fluorescence reporter construct p–mito-mRFP-EGFP (pAT016). Lysosomal delivery of the tandem fusion protein mito-mRFP-EGFP along with entire mitochondria results in pH-dependent quenching of green fluorescence resulting in red-only fluorescence for visual analysis of mitophagic flux. (B and C) Representative confocal live images of indicated MEFs ectopically expressing mito-mRFP-EGFP targeting mitochondria. Cells were kept untreated (B) or treated with mito-SN38 (2.5 μM for 12 hours) (C) and were analyzed under live-cell microscopy. The yellow fluorescence signals denote no mitophagy (merged image); red-only fluorescence signals denote mitophagy or mitochondria inside lysosomes. The enlarged panel shows higher-magnification image. Quantification of the indicated fluorescence obtained from live-cell confocal microscopy was calculated for 20 to 25 cells (calculated value ± SEM) in at least three independent experiments. Asterisks denote statistically significant differences (**P < 0.01 and ***P < 0.001, t test). (D) Representative confocal images of live cells for the indicated MEFs showing accumulation of ectopic PTEN-induced kinase 1 (PINK1)–GFP after mito-SN38 (2.5 μM for 3 hours) treatment. Mitochondria are labeled with MitoTracker red; the colocalization of PINK1-GFP (green) in the mitochondrial network (red) is indicated in the merged image. Quantification of the indicated fluorescence obtained from live-cell confocal microscopy was calculated for 20 to 25 cells (calculated value ± SEM) obtained from independent experiments. Asterisks denote statistically significant differences (**P < 0.01, t test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax9778/DC1

    Fig. S1. Mito-SN38 does not trap nuclear-Top1cc but impairs mitochondrial metabolism through SCAN1-TDP1 trapping in the mitochondria.

    Fig. S2. Differentiation of SH-SY5Y cells showing expression of FLAG-TDP1 and lysosomal localization of SCAN1 mitochondria showing mitophagy.

    Table S1. List of primers used.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Mito-SN38 does not trap nuclear-Top1cc but impairs mitochondrial metabolism through SCAN1-TDP1 trapping in the mitochondria.
    • Fig. S2. Differentiation of SH-SY5Y cells showing expression of FLAG-TDP1 and lysosomal localization of SCAN1 mitochondria showing mitophagy.
    • Table S1. List of primers used.

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