Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia - PubMed
Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia
Nilesh K Sharma et al. DNA Repair (Amst). 2014 Jan.
Abstract
Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here, we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria is reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3.
Keywords: ATM; Ataxia Telangiectasia; DNA ligase 3; Mitochondria; Mitochondrial DNA repair.
Published by Elsevier B.V.
Conflict of interest statement
Conflict of Interest
The authors declare no conflict of interests
Figures
![Fig. 1.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/f600f046f16a/nihms-1506301-f0001.gif)
Genomic DNA was isolated from ATM-proficient and deficient samples and mtDNA integrity was analyzed using QPCR. Data were normalized to mtDNA content using amplification of the small mtDNA fragment (Santos et al., 2006). DNA was isolated 3 independent times and QPCR was performed two independent times with each DNA isolate, error bars represent ± SEM; (A) patient-derived cells and (B) mouse tissue (C) siRNA was transiently transfected in WT cells and levels of ATM analyzed by immunoblots 4 days after transfections (left). MtDNA damage was estimated in cells transfected with scrambled-control or siRNA targeted to ATM (right). QPCR was performed two independent times for each DNA sample from 3 independent experiments, error bars represent ± SEM. WT and A-T cells were submitted to 60 min treatment with 200 μM H2O2. Immediately after treatment cells were collected and: (D) mitochondrial ROS measured using Mitosox by FACS, N=3; * indicates significance between control WT and control A-T, and between A-T control and treated samples; (E) mitochondrial membrane polarization judged using JC-1 by FACS, N=3, * indicates significance using WT control as reference or between A-T control and treated cells. (F) Steady state levels of ATP were estimated using a luciferase-based assay. Experiments were reproduced at least 3 independent times; error bars represent ± SD. = p ≤0.05. * indicates significance between WT and A-T control. (G) Doubling time was calculated by following cell growth and counting cells each time they reached confluency. Results represent the mean doubling time calculated over a period of 3 weeks for two independently grown cultures for each cell type.
![Fig. 1.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/f600f046f16a/nihms-1506301-f0001.gif)
Genomic DNA was isolated from ATM-proficient and deficient samples and mtDNA integrity was analyzed using QPCR. Data were normalized to mtDNA content using amplification of the small mtDNA fragment (Santos et al., 2006). DNA was isolated 3 independent times and QPCR was performed two independent times with each DNA isolate, error bars represent ± SEM; (A) patient-derived cells and (B) mouse tissue (C) siRNA was transiently transfected in WT cells and levels of ATM analyzed by immunoblots 4 days after transfections (left). MtDNA damage was estimated in cells transfected with scrambled-control or siRNA targeted to ATM (right). QPCR was performed two independent times for each DNA sample from 3 independent experiments, error bars represent ± SEM. WT and A-T cells were submitted to 60 min treatment with 200 μM H2O2. Immediately after treatment cells were collected and: (D) mitochondrial ROS measured using Mitosox by FACS, N=3; * indicates significance between control WT and control A-T, and between A-T control and treated samples; (E) mitochondrial membrane polarization judged using JC-1 by FACS, N=3, * indicates significance using WT control as reference or between A-T control and treated cells. (F) Steady state levels of ATP were estimated using a luciferase-based assay. Experiments were reproduced at least 3 independent times; error bars represent ± SD. = p ≤0.05. * indicates significance between WT and A-T control. (G) Doubling time was calculated by following cell growth and counting cells each time they reached confluency. Results represent the mean doubling time calculated over a period of 3 weeks for two independently grown cultures for each cell type.
![Fig. 1.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/f600f046f16a/nihms-1506301-f0001.gif)
Genomic DNA was isolated from ATM-proficient and deficient samples and mtDNA integrity was analyzed using QPCR. Data were normalized to mtDNA content using amplification of the small mtDNA fragment (Santos et al., 2006). DNA was isolated 3 independent times and QPCR was performed two independent times with each DNA isolate, error bars represent ± SEM; (A) patient-derived cells and (B) mouse tissue (C) siRNA was transiently transfected in WT cells and levels of ATM analyzed by immunoblots 4 days after transfections (left). MtDNA damage was estimated in cells transfected with scrambled-control or siRNA targeted to ATM (right). QPCR was performed two independent times for each DNA sample from 3 independent experiments, error bars represent ± SEM. WT and A-T cells were submitted to 60 min treatment with 200 μM H2O2. Immediately after treatment cells were collected and: (D) mitochondrial ROS measured using Mitosox by FACS, N=3; * indicates significance between control WT and control A-T, and between A-T control and treated samples; (E) mitochondrial membrane polarization judged using JC-1 by FACS, N=3, * indicates significance using WT control as reference or between A-T control and treated cells. (F) Steady state levels of ATP were estimated using a luciferase-based assay. Experiments were reproduced at least 3 independent times; error bars represent ± SD. = p ≤0.05. * indicates significance between WT and A-T control. (G) Doubling time was calculated by following cell growth and counting cells each time they reached confluency. Results represent the mean doubling time calculated over a period of 3 weeks for two independently grown cultures for each cell type.
![Fig. 2.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/ad80150626b6/nihms-1506301-f0004.gif)
WT controls or A-T cells were submitted to treatment with 200 μM H2O2 for 60 min and allowed to recover for up to 48h. MtDNA integrity was analyzed using QPCR; data were normalized to mtDNA copy number. Experiments were reproduced at least 3 independent time; error bars reflect ± SEM. (A) Patient- derived fibroblasts GM07532 (WT) and GM02052 (A-T), (B) patient-derived cells AG01522 (WT) and AG04405 (A-T). (C) mtDNA repair kinetics was evaluated in GM07532 (WT) upon knock down of ATM using siRNA. (D) Kinetics of mtDNA repair was followed in GM07532 (WT) up to 24h after H2O2 exposure in the presence or absence of 1 μM triapine, a RR-inhibitor. NS = not-significant.
![Fig. 2.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/ad80150626b6/nihms-1506301-f0004.gif)
WT controls or A-T cells were submitted to treatment with 200 μM H2O2 for 60 min and allowed to recover for up to 48h. MtDNA integrity was analyzed using QPCR; data were normalized to mtDNA copy number. Experiments were reproduced at least 3 independent time; error bars reflect ± SEM. (A) Patient- derived fibroblasts GM07532 (WT) and GM02052 (A-T), (B) patient-derived cells AG01522 (WT) and AG04405 (A-T). (C) mtDNA repair kinetics was evaluated in GM07532 (WT) upon knock down of ATM using siRNA. (D) Kinetics of mtDNA repair was followed in GM07532 (WT) up to 24h after H2O2 exposure in the presence or absence of 1 μM triapine, a RR-inhibitor. NS = not-significant.
![Fig. 3.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/c69c9dfff3b5/nihms-1506301-f0006.gif)
WT and A-T cells were submitted to 200 μM H2O2 for 60 min and mitochondria isolated. pUC18 was cut with PstI and incubated with 10 μg of mitochondrial extracts at 16°C overnight. The DNA was then purified and ran on agarose gels. (A) Data obtained with WT cells (left) and with the A-T counterpart (right). Arrow indicates linearized plasmid; red asterisk indicates expected ligated product. Lower left panel shows data using 10 μg of nuclear extracts; only non-H2O2 treated cells were assayed. Right lower panel depicts Ponceau staining of lysates to assure use of comparable protein concentration in the ligase assay. N: nuclear, C: cytosol and M: mitochondria.
![Fig. 4.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/a3e7b0fe5202/nihms-1506301-f0007.gif)
Lig3 levels were probed by Western blotting in crude (left) and isolated mitochondria (right) from (A) patient-derived fibroblasts and (B) tissue from WT and ATM KO animals. ND-1 is encoded by the mtDNA and was used as loading control for mitochondrial lysates. Graphs depict levels of Lig3 normalized to β-actin or ND-1. *=p ≤ 0.05. (C) Total RNA was isolated from WT and A-T fibroblasts and Lig3 transcript level was gauged by RT-PCR; data were normalized to actin. N=3 from independent RNA isolations; PCR was performed in triplicate. (D) WT and A-T cells were exposed to 25 μM of the proteasome inhibitor MG132 for 8h when cells were lysed and Lig3 levels were evaluated by Western blotting. Graph depicts levels of Lig3 normalized to tubulin. *=p ≤ 0.05. (E) Protein levels of the mtBER proteins Pol γ, OGG1, FEN1, EXOG and Dna2 were evaluated in WT and A-T cells prior to and after exposure to 200 μM H2O2 for 60 min. Blots are representative of 2-3 independent experiments.
![Fig. 4.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/a3e7b0fe5202/nihms-1506301-f0007.gif)
Lig3 levels were probed by Western blotting in crude (left) and isolated mitochondria (right) from (A) patient-derived fibroblasts and (B) tissue from WT and ATM KO animals. ND-1 is encoded by the mtDNA and was used as loading control for mitochondrial lysates. Graphs depict levels of Lig3 normalized to β-actin or ND-1. *=p ≤ 0.05. (C) Total RNA was isolated from WT and A-T fibroblasts and Lig3 transcript level was gauged by RT-PCR; data were normalized to actin. N=3 from independent RNA isolations; PCR was performed in triplicate. (D) WT and A-T cells were exposed to 25 μM of the proteasome inhibitor MG132 for 8h when cells were lysed and Lig3 levels were evaluated by Western blotting. Graph depicts levels of Lig3 normalized to tubulin. *=p ≤ 0.05. (E) Protein levels of the mtBER proteins Pol γ, OGG1, FEN1, EXOG and Dna2 were evaluated in WT and A-T cells prior to and after exposure to 200 μM H2O2 for 60 min. Blots are representative of 2-3 independent experiments.
![Fig. 4.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/a3e7b0fe5202/nihms-1506301-f0007.gif)
Lig3 levels were probed by Western blotting in crude (left) and isolated mitochondria (right) from (A) patient-derived fibroblasts and (B) tissue from WT and ATM KO animals. ND-1 is encoded by the mtDNA and was used as loading control for mitochondrial lysates. Graphs depict levels of Lig3 normalized to β-actin or ND-1. *=p ≤ 0.05. (C) Total RNA was isolated from WT and A-T fibroblasts and Lig3 transcript level was gauged by RT-PCR; data were normalized to actin. N=3 from independent RNA isolations; PCR was performed in triplicate. (D) WT and A-T cells were exposed to 25 μM of the proteasome inhibitor MG132 for 8h when cells were lysed and Lig3 levels were evaluated by Western blotting. Graph depicts levels of Lig3 normalized to tubulin. *=p ≤ 0.05. (E) Protein levels of the mtBER proteins Pol γ, OGG1, FEN1, EXOG and Dna2 were evaluated in WT and A-T cells prior to and after exposure to 200 μM H2O2 for 60 min. Blots are representative of 2-3 independent experiments.
![Fig. 5.](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2027/6211587/003de892342f/nihms-1506301-f0010.gif)
(A) AG04405 (A-T) cells were treated with 25 μM MG132 for 8h and the degree of proteasome inhibition was gauged up to 24h following MG132 exposure using a fluorescence assay. Results represent mean of 3 biological replicates, each of which was measured in duplicate. (B) AG01522 (WT for ATM) cells were exposed to 200 μM H2O2 for 60 min and mtDNA repair kinetics was followed using QPCR over 24h in the presence or absence of vehicle or 10 μM L189. Data were normalized to mtDNA copy number. Experiments were reproduced at least 3 independent time; error bars reflect ± SEM.
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References
-
- Shiloh Y, and Ziv Y, The ATM protein kinase: regulating the cellular response to genotoxic stress, and more, Nat. Rev. Mol. Cell. Biol. 4, (2013) 197–210. - PubMed
-
- Ambrose M, Goldstine JV, Gatti RA, Intrinsic mitochondrial dysfunction in ATM- deficient lymphoblastoid cells, Human. Mol. Gen. 16, (2007) 2154–2164. - PubMed
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