Premature Aging Is Associated With Higher Levels Of 8-Oxoguanine And Increased DNA Damage In The Polg Mutator Mouse

Background:The world population continues to undergo sustained changes due to an increase in life expectancy.In 2020,the global population of individuals 65 years of age and older was 727 million,and that proportion of the population is expected to increase from 9.3%in 2020 to 16.0%in 2050.In such a rapidly aging world,research on the fundamental mechanisms of aging as well as potential treatments for age-related maladies is likely to become an increasingly important public health priority.Aging is a multifactorial and complex biological process that affects organisms at multiple levels of molecules,cells,tissues,and systems.Mitochondrial dysfunction plays an important role in the aging process.However,the mechanism by which this dysfunction causes aging is not fully understood.The accumulation of mutations in the mitochondrial genome（or“mtDNA”）has been proposed as a contributor.Objective:One compelling piece of evidence in support of this hypothesis comes from the Polg^D257A/D257A mutator mouse(Polg^mut/mut).These mice express an error-prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations,accelerated aging,and premature death.In this paper,we have used the Polg^mut/mut model to investigate whether the age-related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome.Method:（1）To calculate predicted DNA methylation age,blood DNA samples were obtained from 42 patients with verified pathogenic mtDNA mutations（including six patients carrying the m.3243A>G mutation,five carrying mtDNA mutations related to Leigh Syndrome,and 31 carrying the m.8344A>G mutation related to myoclonic epilepsy with ragged red fibers（MERRF））and 111“control”individuals without pathogenic mitochondrial mutations.The DNA methylation age were analyzed using the method of UCLA Neuroscience Genomics Core（UNGC）and Zymo Research’s proprietary DNAge（?）predictor for humans.Moreover,the DNA methylation age of mouse blood and heart tissues in Polg^wt/wt and Polg^mut/mut mice were analyzed using the established DNA methylation detection method in mice.And the DNA methylation age were analyzed by the UNGC and Zymo Research’s proprietary DNAge（?）predictor for mice.（2）Quantified the 8-oxo Gua in urinary samples by ultraperformance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）.It was standardized with creatinine（Cre）.A mixed standard solution of 8-oxo Gua and creatinine was prepared with an internal standard（IS）solution of 8-oxo Gua-¹³C3 and Cre-D3.Concentrations of 8-oxo Gua and creatine were calculated from the constructed linear-regression curve of each compound with internal standard calibration and using the analyte-to-IS peak ratios measured from urinary sample solutions.We have examinedγH2AX levels and DNA damage in Mouse Embryonic Fibroblasts（MEFs）derived from Polg^mut/mutby immunofluorescence.The DNA damage was further analyzed by combining comet assay with Formamidopyrimidine DNA glycosylase（Fpg）incubation in different cell types（blood,liver and brain）from Polg^wt/wtand Polg^mut/mutmice.For these assays,cells are embedded in agarose before being lysed to release their DNA.Electrophoresis is then performed under alkaline conditions to convert AP sites and alkali-labile DNA adducts into single-strand breaks（SSBs）,which are subsequently convert to DSBs under the alkaline conditions.When the agarose is subsequently stained for DNA,undamaged DNA remains intact and runs as a coherent mass,while damaged DNA strand breaks migrate further than intactDNA,forming a“comet-shaped”tail.Comet tails will tend to be observed in cells with DNA damage,with the percentage of DNA in the tails representing the degree of DNA damage.（3）Quantified the average telomere length of Polg mice by real-time PCR:The telomere primers were amplified telomere sequences,single copy reference（SCR）primers were amplified a 100 bp-long region on mouse chromosome 10 and served as reference for data normalization.The reference genomic DNA sample with known telomere length was served as a reference for calculating the telomere length of target samples.Quantified the average telomere length of Polg mice by Southern Blot.To quantify the shortened telomeric DNA,boxes were drawn in each set of lanes to define the“bulk”and“tail”fractions（representing intact versus degraded telomeric DNA,respectively）,and the signal for each fraction was quantified by Image J.We further quantified telomere length per nucleus using a more sensitive method by quantitative fluorescence in situ hybridization（Q-FISH）.Splenic lymphocytes were isolated from Polg^wt/wt and Polg^mut/mutmice for Q-FISH analysis.For 3D fixation,a hypotonic solution was overlaid with 1 ml of freshly prepared fixative（methanol/acetic acid,3:1）.To analyse the interphase nuclei of telomeres,cells were fixed in 3.7%formaldehyde/1×Phosphate-buffered saline（PBS）for 10min.Incubation with 0.5%Triton X-100 for 10 min was used for removing the cell membrane and expose the cell nuclei.Then,the slides were then equilibrated in 70%formamide/2X saline sodium citrate（SSC）p H 7.0 for 1 hour at RT before hybridization with 8μl of peptide nucleic acid-telomere probe（PNA-telomere probe）,and DAPI was used as a counterstain for the DNA.To ensure the accuracy of the results,at least 30 interphase nuclei per cell line were performed.（4）Linear DNA samples were digested by Exonuclease V（Exo V）,and the total mtDNA copy number（mtDNAcn）and linear mtDNAcn were measured by a quantitative PCR-based method.The mtDNAcn was quantified as the ratio of a mitochondrial gene copy number（t RNA-Val）to a single-copy nuclear gene（β2-Microglobulin,B2M）.Moreover,the t RNA-Val primers specifically amplify a sequencing that is not duplicated in the nuclear genome as nuclear mitochondrial insertion（NUMT）.The 8-oxo-2’-deoxyguanosine（8-oxod G）levels in mtDNA and telomeric DNA were measured by a quantitative PCR（qPCR）method combined with Fpg incubation for DNA samples.The 8-oxod G level in mtDNA was quantified as the ratio of a mitochondrial gene copy number（t RNA-Val）.The 8-oxod G level in telomeric DNA was quantified as the ratio of the qPCR detected telomere levels.Result:（1）The results showed thatDNA methylation age in 42 patients with pathogenic mtDNA mutations was accelerated by 2.6 to 3 years relative to their expected age,while the control samples showed essentially no DNA methylation age acceleration（on average～0.5 years less than their expected age）.The Polg^mut/mutmice also showed a similar pattern of accelerated DNA methylation age:The results show that Polg^mut/mut mice have a higher than expected DNA methylation age（2.18weeks）compared to Polg^wt/wt mice.Additional follow-up analysis using a universal epigenetic clock that can estimate age across a variety of tissues and species also confirmed that Polg^mut/mut mice shown a significant age acceleration（7.62 weeks）in heart tissue DNA relative to Polg^wt/wt control mice.（2）The UPLC-MS/MS results showed that show that the Polg^mut/mut mutation significantly increases the level of 8-oxo Gua in urinary samples.This result suggests that Polg^mut/mut likely increases the level of total oxidative stress in the cell.To determine whether the higher 8-oxod G levels observed in the DNA molecules themselves are also associated with canonical DDR linked to DNA strand breakage,we have examinedγH2AX levels and DNA damage in MEFs derived from Polg^mut/mutby immunofluorescence.The results show that Polg^mut/mut increases the number ofγH2AX-positive MEFs.Using comet assay,we show that the levels of DNA SSBs and/or DSBs are significantly higher in liver.（3）The average telomere length was measured by real-time PCR and Southern Blot.These initial results showed that there was no significant difference in telomere length.The results of our Q-FISH analysis demonstrate that Polgmut/mut mutant cells show a decrease in their average telomere intensity.This suggests that the observed increase in 8-oxod G lesions in Polg^mut/mut is associated with increased telomere instability,resulting in telomere shortening,promoting telomere damage.Compared with Polg^wt/wt,Polg^mut/mutdecreases the average intensity of telomeres signals by～8.44%and increases the percentage of telomere aggregates by～13.56%in mice 36 weeks of age or older.（4）Our results show that Polg^mut/mut increases the total mtDNAcn,the linear mtDNAcn,and increases 8-oxod G in mtDNA.Our results show that mutator mouse has significantly higher levels of 8-oxoguanine（8-oxo Gua）that are correlated with increased nuclear DNA（nuclear DNA,n DNA）strand breakage and oxidative n DNA damage,shorter average telomere length,and reduced mtDNA integrity.Based on these results,we propose a model whereby the increased level of reactive oxygen species（ROS）associated with the accumulation of mtDNA mutations in Polg^mut/mut mice results in higher levels of 8-oxo Gua,which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening.Conclusion:（1）The single nucleotide,pathogenic changes in mtDNA are sufficient to increase DNA methylation age and accelerate cellular aging in humans,and the accumulation of mtDNA mutations will also increase DNA methylation age and accelerate cellular aging in humans.（2）The increase of mtDNA mutation rate leads to the increase of total 8-oxo Gua level in Polg^mut/mutmice,which increases DNA damage and fragmentation in vivo.（3）The Polg^mut/mutmutation accelerates telomere shortening,promotes telomere damage or telomere fusion,and leads to telomere DNA instability,which accelerates telomere shortening.（4）The Polg^mut/mutmutation increases the level of 8-oxod G in mice,increases mitochondrial oxidative damage,which damages the integrity of mtDNA,increases the break of mtDNA strand,and leads to the increased compensatory ability of mtDNAcn.（5）The study in this paper provides multiple lines of evidence that mtDNA mutation associated with the accumulation of chronic oxidative stress is the center of aging by increasing n DNA and mtDNA strand breakage,and telomere shortening.These results suggest that mitochondria play a central role in aging,simultaneously influencing multiple aging hallmarks such as genomic instability,telomere attrition,epigenetic alterations,and mitochondrial dysfunction.