Newly Revised Quantitative PCR‐Based Assay for Mitochondrial and Nuclear DNA Damage

Sanders, Laurie H.; Rouanet, Jeremy P.; Howlett, Evan H.; Leuthner, Tess C.; Rooney, John P.; Greenamyre, J. Timothy; Meyer, Joel N.

doi:10.1002/cptx.50

Cited by 9 publications

(15 citation statements)

References 23 publications

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“…Since PINK1 has a role in mtDNA metabolism [4, 27], we analyzed mtDNA damage in lungs of PINK1 deficient mice (Fig 4A). We quantified DNA lesions in lung mtDNA using a long extension qPCR technique for DNA-damage quantification (LORD-Q) method [21, 22] and show more lesion in the PINK1 -/- mice (Fig 4B). To determine whether oxidation of mtDNA was increased and/or accumulative, we analyzed the content of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), the most frequent oxidative DNA modification, on purified mtDNA samples from PINK1 WT and KO mice at different ages.…”

Section: Resultsmentioning

confidence: 99%

“…Mitochondrial DNA oxidation was evaluated by ELISA detection of 8-hydroxy-2’-deoxyguanosine (Cayman #589320) according to the manufacturer’s protocol in 3 μg of isolated mtDNA from freshly isolated total lung tissue mitochondria. Mitochondria DNA lesions were quantified using long-run qPCR technique for DNA-damage quantification (LORD-Q) method calculating 10kb lesions using amplification efficiency from standard curve based on Ct values [21, 22].…”

Section: Methodsmentioning

confidence: 99%

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PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

Bueno¹,

Zank²,

Buendía-Roldán³

et al. 2019

PLoS ONE

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We have previously shown that endoplasmic reticulum stress (ER stress) represses the PTEN inducible kinase 1 (PINK1) in lung type II alveolar epithelial cells (AECII) reducing mitophagy and increasing the susceptibility to lung fibrosis. Although increased circulating mitochondrial DNA (mtDNA) has been reported in chronic lung diseases, the contribution of mitophagy in the modulation of mitochondrial DAMP release and activation of profibrotic responses is unknown. In this study, we show that ER stress and PINK1 deficiency in AECII led to mitochondrial stress with significant oxidation and damage of mtDNA and subsequent extracellular release. Extracellular mtDNA was recognized by TLR9 in AECII by an endocytic-dependent pathway. PINK1 deficiency-dependent mtDNA release promoted activation of TLR9 and triggered secretion of the profibrotic factor TGF-β which was rescued by PINK1 overexpression. Enhanced mtDNA oxidation and damage were found in aging and IPF human lungs and, in concordance, levels of circulating mtDNA were significantly elevated in plasma and bronchoalveolar lavage (BAL) from patients with IPF. Free mtDNA was found elevated in other ILDs with low expression of PINK1 including hypersensitivity pneumonitis and autoimmune interstitial lung diseases. These results support a role for PINK1 mediated mitophagy in the attenuation of mitochondrial damage associated molecular patterns (DAMP) release and control of TGF-β mediated profibrotic responses.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

Bueno¹,

Zank²,

Buendía-Roldán³

et al. 2019

PLoS ONE

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“…DNA was eluted with EDTA-free buffer, and quality was assessed using a Spectradrop microvolume microplate (Molecular Devices). Double-stranded DNA was quantified using Quant-iT Picogreen dsDNA assay (Thermo Fisher) as previously described 55,56 . Due to the fact that the Autogen automated system is much gentler on the DNA during extraction, it was necessary to freeze-thaw the DNA sample 3 times before PCR analysis to reduce mtDNA supercoiling.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…DNA damage in the mitochondrial genome was measured utilizing a PCR-based assay, currently the most robust way of measuring damage in mtDNA 57 . This assay to calculate mitochondrial DNA lesion frequency was performed as previously described 34,55,56 . Briefly, 15 ng of DNA was used to amplify long or short amplicons of the mitochondrial genome (as determined by primer sets).…”

Section: Western Blot Analysismentioning

confidence: 99%

Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson’s disease

Gonzalez‐Hunt

Thacker

Toste

et al. 2020

Sci Rep

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Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson’s disease (PD) and LRRK2 kinase inhibitors are currently being tested in early phase clinical trials. In order to ensure the highest chance of success, a biomarker-guided entry into clinical trials is key. LRRK2 phosphorylation, and phosphorylation of the LRRK2 substrate Rab10, have been proposed as target engagement biomarkers for LRRK2 kinase inhibition. However, a pharmacodynamic biomarker to demonstrate that a biological response has occurred is lacking. We previously discovered that the LRRK2 G2019S mutation causes mitochondrial DNA (mtDNA) damage and is LRRK2 kinase activity-dependent. Here, we have explored the possibility that measurement of mtDNA damage is a “surrogate” for LRRK2 kinase activity and consequently of kinase inhibitor activity. Mitochondrial DNA damage was robustly increased in PD patient-derived immune cells with LRRK2 G2019S mutations as compared with controls. Following treatment with multiple classes of LRRK2 kinase inhibitors, a full reversal of mtDNA damage to healthy control levels was observed and correlated with measures of LRRK2 dephosphorylation. Taken together, assessment of mtDNA damage levels may be a sensitive measure of altered kinase activity and provide an extended profile of LRRK2 kinase modulation in clinical studies.

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“…To determine damage and repair of nDNA and mtDNA, we used the long amplicon quantitative polymerase chain reaction (LA-QPCR) method [49,50]. In these analyses we used (400 U; 2 U/µL) KAPA Long Range Hot Start DNA Polymerase (KAPA Biosystems), which is optimized for LA-QPCR with rat DNA [50]. The PCR primers employed in this study are given in Table 1.…”

Section: Analysis Of Damage and Repair Of Mitochondrial Dna And Nuclementioning

confidence: 99%

Assessment of Nuclear and Mitochondrial DNA, Expression of Mitochondria-Related Genes in Different Brain Regions in Rats after Whole-Body X-ray Irradiation

Abdullaev

Gubina

Bulanova

et al. 2020

IJMS

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Studies of molecular changes occurred in various brain regions after whole-body irradiation showed a significant increase in terms of the importance in gaining insight into how to slow down or prevent the development of long-term side effects such as carcinogenesis, cognitive impairment and other pathologies. We have analyzed nDNA damage and repair, changes in mitochondrial DNA (mtDNA) copy number and in the level of mtDNA heteroplasmy, and also examined changes in the expression of genes involved in the regulation of mitochondrial biogenesis and dynamics in three areas of the rat brain (hippocampus, cortex and cerebellum) after whole-body X-ray irradiation. Long amplicon quantitative polymerase chain reaction (LA-QPCR) was used to detect nDNA and mtDNA damage. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. The mtDNA copy numbers and expression levels of a number of genes were determined by real-time PCR. The results showed that the repair of nDNA damage in the rat brain regions occurs slowly within 24 h; in the hippocampus, this process runs much slower. The number of mtDNA copies in three regions of the rat brain increases with a simultaneous increase in mtDNA heteroplasmy. However, in the hippocampus, the copy number of mutant mtDNAs increases significantly by the time point of 24 h after radiation exposure. Our analysis shows that in the brain regions of irradiated rats, there is a decrease in the expression of genes (ND2, CytB, ATP5O) involved in ATP synthesis, although by the same time point after irradiation, an increase in transcripts of genes regulating mitochondrial biogenesis is observed. On the other hand, analysis of genes that control the dynamics of mitochondria (Mfn1, Fis1) revealed that sharp decrease in gene expression level occurred, only in the hippocampus. Consequently, the structural and functional characteristics of the hippocampus of rats exposed to whole-body radiation can be different, most significantly from those of the other brain regions.

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Newly Revised Quantitative PCR‐Based Assay for Mitochondrial and Nuclear DNA Damage

Cited by 9 publications

References 23 publications

PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson’s disease

Assessment of Nuclear and Mitochondrial DNA, Expression of Mitochondria-Related Genes in Different Brain Regions in Rats after Whole-Body X-ray Irradiation

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