Enhanced glutathione levels confer resistance to apoptotic and ferroptotic programmed cell death in NEIL DNA glycosylase deficient HAP1 cells.

The NTHL1 and NEIL1-3 DNA glycosylases are major enzymes in the removal of oxidative DNA base lesions, via the base excision repair (BER) pathway. It is expected that lack of these DNA glycosylases activities would render cells vulnerable to oxidative stress, promoting cell death. Intriguingly, we found that single, double, triple, and quadruple DNA glycosylase knockout HAP1 cells are, however, more resistant to oxidative stress caused by genotoxic agents than wild type cells. Furthermore, glutathione depletion in NEIL deficient cells further enhances resistance to cell death induced via apoptosis and ferroptosis. Finally, we observed higher basal level of glutathione and differential expression of NRF2-regulated genes associated with glutathione homeostasis in the NEIL triple KO cells. We propose that lack of NEIL DNA glycosylases causes aberrant transcription and subsequent errors in protein synthesis. This leads to increased endoplasmic reticulum stress and proteotoxic stress. To counteract the elevated intracellular stress, an adaptive response mediated by increased glutathione basal levels, rises in these cells. This study reveals an unforeseen role of NEIL glycosylases in regulation of resistance to oxidative stress, suggesting that modulation of NEIL glycosylase activities is a potential approach to improve the efficacy of e.g. anti-inflammatory therapies.

Safety and efficacy of plasma transfusion from exercise-trained donors in patients with early Alzheimer's disease: protocol for the ExPlas study.

INTRODUCTION: Given that exercise training reduces the risk of developing Alzheimer's disease (AD), induces changes in the blood composition and has widespread systemic benefits, it is reasonable to hypothesise that exercised plasma (ExPlas) may have rejuvenative properties. The main objective is to test safety and tolerability of transfusing ExPlas from young, healthy, fit adults to patients with mild cognitive impairment (MCI) or early AD. The study is a pilot for a future efficacy study. The key secondary objectives are examining the effect of plasma transfusions on cognitive function, fitness level, vascular risk profile, assessment of cerebral blood flow and hippocampal volume, quality of life, functional connectivity assessed by resting state functional MRI and biomarkers in blood and cerebrospinal fluid. METHODS AND ANALYSIS: ExPlas is a double-blinded, randomised controlled clinical single-centre trial. Patients up to 75 years of age with diagnosis early symptomatic phase AD will be recruited from two Norwegian hospitals. ExPlas is plasma drawn by plasmapheresis once a month for 4 months, from a total of 30 fit male donors (aged 18-40, BMI≤27 kg/m2 and maximal oxygen uptake>55 mL/kg/min). All units will be virus inactivated by the Intercept method in accordance with procedures at St. Olavs University Hospital. Comparison with isotonic saline allows differentiation from a non-blood product. The main study consists of 6 rounds of examinations in addition to 12 plasma transfusions divided over three 4-week periods during study year-1. It is also planned to conduct follow-up examinations 2 and 5 years after baseline ETHICS AND DISSEMINATION: Written informed consent will be obtained from all participants and participation is voluntary. All participants have a next of kin who will follow them throughout the study to represent the patient's interest. The study is approved by the Regional Committee for Medical and Health Research Ethics (REK 2018/702) and the Norwegian Medicines Agency (EudraCT No. 2018-000148-24). The study will be published in an open access journal and results will be presented at numerous national and international meetings as well as on social media platforms. TRIAL REGISTRATION NUMBER: EudraCT No. 2018-000148-24. CLINICALTRIALS: gov, NCT05068830.

Association of Klotho Protein Levels and KL-VS Heterozygosity With Alzheimer Disease and Amyloid and Tau Burden.

Importance: Identification of proteins and genetic factors that reduce Alzheimer disease (AD) pathology is of importance when searching for novel AD treatments. Heterozygosity of the KL-VS haplotype has been associated with reduced amyloid and tau burden. Whether this association is mediated by the Klotho protein remains unclear. Objectives: To assess concentrations of Klotho in cerebrospinal fluid (CSF) and plasma among cognitively healthy controls and patients with AD and to correlate these findings with KL-VS heterozygosity status and amyloid and tau burden. Design, Setting, and Participants: This case-control study combined 2 independent case-control AD cohorts consisting of 243 referred patients with AD and volunteer controls recruited from January 1, 2009, to December 31, 2018. Klotho levels were measured in CSF and plasma and correlated with KL-VS heterozygosity status and levels of CSF amyloid-ß 42 (Aß42), total tau, and phosphorylated tau. Statistical analysis was performed from January 1, 2021, to March 1, 2022. Main Outcomes and Measures: Associations of Klotho levels in CSF and plasma with levels of CSF biomarkers were analyzed using linear regression. Association analyses were stratified separately by clinical groups, APOE4 status, and KL-VS heterozygosity. Pearson correlation was used to assess the correlation between CSF and plasma Klotho levels. Results: A total of 243 participants were included: 117 controls (45 men [38.5%]; median age, 65 years [range, 41-84 years]), 102 patients with mild cognitive impairment due to AD (AD-MCI; 59 men [57.8%]; median age, 66 years [range, 46-80 years]), and 24 patients with dementia due to AD (AD-dementia; 12 men [50.0%]; median age, 64.5 years [range, 54-75 years]). Median CSF Klotho levels were higher in controls (1236.4 pg/mL [range, 20.4-1726.3 pg/mL]; ß = 0.103; 95% CI, 0.023-0.183; P = .01) and patients with AD-MCI (1188.1 pg/mL [range, 756.3-1810.3 pg/mL]; ß = 0.095; 95% CI, 0.018-0.172; P = .02) compared with patients with AD-dementia (1073.3 pg/mL [range, 698.2-1661.4 pg/mL]). Higher levels of CSF Klotho were associated with lower CSF Aß42 burden (ß = 0.519; 95% CI, 0.201-0.836; P < .001) and tau burden (CSF total tau levels: ß = -0.884; 95% CI, 0.223 to -0.395; P < .001; CSF phosphorylated tau levels: ß = -0.672; 95% CI, -1.022 to -0.321; P < .001) independent of clinical, KL-VS heterozygosity, or APOE4 status. There was a weak correlation between Klotho CSF and plasma levels among the entire cohort (Pearson correlation r = 0.377; P < .001). Conclusions and Relevance: The findings of this case-control study suggest that Klotho protein levels were associated with clinical stages of AD, cognitive decline, and amyloid and tau burden and that these outcomes were more clearly mediated by the protein directly rather than the KL-VS heterozygosity variant. When selecting individuals at risk for clinical trials, the Klotho protein level and not only the genetic profile should be considered.

Age- and sex-dependent effects of DNA glycosylase Neil3 on amyloid pathology, adult neurogenesis, and memory in a mouse model of Alzheimer's disease.

Oxidative stress generating DNA damage has been shown to be a key characteristic in Alzheimer's disease (AD). However, how it affects the pathogenesis of AD is not yet fully understood. Neil3 is a DNA glycosylase initiating repair of oxidative DNA base lesions and with a distinct expression pattern in proliferating cells. In brain, its function has been linked to hippocampal-dependent memory and to induction of neurogenesis after stroke and in prion disease. Here, we generated a novel AD mouse model deficient for Neil3 to study the impact of impaired oxidative base lesion repair on the pathogenesis of AD. Our results demonstrate an age-dependent decrease in amyloid-ß (Aß) plaque deposition in female Neil3-deficient AD mice, whereas no significant difference was observed in male mice. Furthermore, male but not female Neil3-deficient AD mice show reduced neural stem cell proliferation in the adult hippocampus and impaired working memory compared to controls. These effects seem to be independent of DNA repair as both sexes show increased level of oxidative base lesions in the hippocampus upon loss of Neil3. Thus, our findings suggest an age- and sex-dependent role of Neil3 in the progression of AD by altering cerebral Aß accumulation and promoting adult hippocampal neurogenesis to maintain cognitive function.

NEIL1 and NEIL2 DNA glycosylases modulate anxiety and learning in a cooperative manner in mice.

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1-/-Neil2-/- mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1-/-Neil2-/- mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.

Impact of Oxidative DNA Damage and the Role of DNA Glycosylases in Neurological Dysfunction.

The human brain requires a high rate of oxygen consumption to perform intense metabolic activities, accounting for 20% of total body oxygen consumption. This high oxygen uptake results in the generation of free radicals, including reactive oxygen species (ROS), which, at physiological levels, are beneficial to the proper functioning of fundamental cellular processes. At supraphysiological levels, however, ROS and associated lesions cause detrimental effects in brain cells, commonly observed in several neurodegenerative disorders. In this review, we focus on the impact of oxidative DNA base lesions and the role of DNA glycosylase enzymes repairing these lesions on brain function and disease. Furthermore, we discuss the role of DNA base oxidation as an epigenetic mechanism involved in brain diseases, as well as potential roles of DNA glycosylases in different epigenetic contexts. We provide a detailed overview of the impact of DNA glycosylases on brain metabolism, cognition, inflammation, tissue loss and regeneration, and age-related neurodegenerative diseases based on evidence collected from animal and human models lacking these enzymes, as well as post-mortem studies on patients with neurological disorders.

DNA glycosylase Neil3 regulates vascular smooth muscle cell biology during atherosclerosis development.

BACKGROUND AND AIMS: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability. METHODS: Chow diet-fed atherosclerosis-prone Apoe-/- mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3. RESULTS: We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs. CONCLUSIONS: Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

HMST-Seq-Analyzer: A new python tool for differential methylation and hydroxymethylation analysis in various DNA methylation sequencing data.

DNA methylation (5mC) and hydroxymethylation (5hmC) are chemical modifications of cytosine bases which play a crucial role in epigenetic gene regulation. However, cost, data complexity and unavailability of comprehensive analytical tools is one of the major challenges in exploring these epigenetic marks. Hydroxymethylation-and Methylation-Sensitive Tag sequencing (HMST-seq) is one of the most cost-effective techniques that enables simultaneous detection of 5mC and 5hmC at single base pair resolution. We present HMST-Seq-Analyzer as a comprehensive and robust method for performing simultaneous differential methylation analysis on 5mC and 5hmC data sets. HMST-Seq-Analyzer can detect Differentially Methylated Regions (DMRs), annotate them, give a visual overview of methylation status and also perform preliminary quality check on the data. In addition to HMST-Seq, our tool can be used on whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) data sets as well. The tool is written in Python with capacity to process data in parallel and is available at (https://hmst-seq.github.io/hmst/).

DNA glycosylase Neil2 contributes to genomic responses in the spleen during clinical prion disease.

The DNA glycosylase Neil2 is a member of the base excision repair (BER) family of enzymes, which are important for repair of oxidative DNA damage. Specifically, Neil2 participates in repair of oxidized bases in single-stranded DNA of transcriptionally active genes. Mice with genetic ablation of Neil2 (Neil2-/-) display no overt phenotypes, but an age-dependent accumulation of oxidative DNA damage and increased inflammatory responsiveness. In young mice intra-cerebrally inoculated with prions, vigorous prion propagation starts rapidly in the germinal follicles of the spleen due to inoculum spillover. Here, we compare experimental prion disease in Neil2-/- mice with that in wild-type mice at disease onset and end-stage. Specifically, we investigated disease progression, accumulation of DNA damage, and mitochondrial respiratory complex activity in brain and spleen. We used genome-wide RNA sequencing of the spleen to compare the immune responses to prion propagation between the two groups of mice, at both onset and end-stage prion disease. The Neil2-/- mice deteriorated more rapidly than wild-type mice after onset of clinical signs. Levels of DNA damage in brain increased in both mouse groups, slightly more in the Neil2-/- mice. Transcriptome data from spleen at disease onset were similar between the mouse groups with moderate genomic responses. However, at end-stage a substantial response was evident in the wild-type mice but not in Neil2-/- mice. Our data show that Neil2 counteracts toxic signaling in clinical prion disease, and this is separate from gross pathological manifestations and PrPSc accumulation.

Diverse functions of DNA glycosylases processing oxidative base lesions in brain.

Endogenous and exogenous oxidative agents continuously damage genomic DNA, with the brain being particularly vulnerable. Thus, preserving genomic integrity is key for brain health and neuronal function. Accumulation of DNA damage is one of the causative factors of ageing and increases the risk of a wide range of neurological disorders. Base excision repair is the major pathway for removal of oxidized bases in the genome and initiated by DNA glycosylases. Emerging evidence suggest that DNA glycosylases have non-canonical functions important for genome regulation. Understanding canonical and non-canonical functions of DNA glycosylases processing oxidative base lesions modulating brain function will be crucial for the development of novel therapeutic strategies.

Neuromodulatory Effect of NLRP3 and ASC in Neonatal Hypoxic Ischemic Encephalopathy.

BACKGROUND: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1ß and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. OBJECTIVE: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. METHODS: C57BL/6J, NLRP3-/-, and ASC-/- mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. RESULTS: NLRP3-/- mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC-/- mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3-/- mice, while decreased in ASC-/- mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3-/- mice. Plasma levels of IL-1ß and IL-18 were not affected, but TNF was lower in NLRP3-/- and ASC-/- mice compared to Wt mice. CONCLUSION: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.

8-oxoguanine DNA glycosylase (Ogg1) controls hepatic gluconeogenesis.

Mitochondrial DNA (mtDNA) resides in close proximity to metabolic reactions, and is maintained by the 8-oxoguanine DNA glycosylase (Ogg1) and other members of the base excision repair pathway. Here, we tested the hypothesis that changes in liver metabolism as under fasting/feeding conditions would be sensed by liver mtDNA, and that Ogg1 deficient mice might unravel a metabolic phenotype. Wild type (WT) and ogg1-/- mice were either fed ad libitum or subjected to fasting for 24h, and the corresponding effects on liver gene expression, DNA damage, as well as serum values were analyzed. Ogg1 deficient mice fed ad libitum exhibited hyperglycemia, elevated insulin levels and higher liver glycogen content as well as increased accumulation of 8oxoG in mtDNA compared to age- and gender matched WT mice. Interestingly, these phenotypes were absent in ogg1-/- mice during fasting. Gene expression and functional analyses suggest that the diabetogenic phenotype in the ogg1-/- mice is due to a failure to suppress gluconeogensis in the fed state. The ogg1-/- mice exhibited reduced mitochondrial electron transport chain (ETC) capacity and a combined low activity of the pyruvate dehydrogenase (PDH), alluding to inefficient channeling of glycolytic products into the citric acid cycle. Our data demonstrate a physiological role of base excision repair that goes beyond DNA maintenance, and implies that DNA repair is involved in regulating metabolism.

No cancer predisposition or increased spontaneous mutation frequencies in NEIL DNA glycosylases-deficient mice.

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1 -/- /Neil2 -/-) double and NEIL1, NEIL2 and NEIL3 (Neil1 -/- /Neil2 -/- /Neil3 -/-) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture.

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3-/- mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3-/- ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI.

Neil3 induced neurogenesis protects against prion disease during the clinical phase.

Base excision repair (BER) is the major pathway for repair of oxidative DNA damage. Mice with genetic knockout of the BER enzyme Neil3 display compromised neurogenesis in the sub-ventricular zone of the lateral ventricle and sub-granular layer of the dentate gyrus of the hippocampus. To elucidate the impact of oxidative DNA damage-induced neurogenesis on prion disease we applied the experimental prion disease model on Neil3-deficient mice. The incubation period for the disease was similar in both wild type and Neil3-/- mice and the overall neuropathology appeared unaffected by Neil3 function. However, disease in the Neil3-/- mice was of shorter clinical duration. We observed a mildly reduced astrogliosis in the hippocampus and striatum in the Neil3-deficient mice. Brain expression levels of neuronal progenitor markers, nestin (Nestin), sex determining region Box 2 (Sox2), Class III beta-tubulin (Tuj1) decreased towards end-stage prion disease whereas doublecortin (Dcx) levels were less affected. Neuronal nuclei (NeuN), a marker for mature neurons declined during prion disease and more pronounced in the Neil3-/- group. Microglial activation was prominent and appeared unaffected by loss of Neil3. Our data suggest that neurogenesis induced by Neil3 repair of oxidative DNA damage protects against prion disease during the clinical phase.

PML regulates neuroprotective innate immunity and neuroblast commitment in a hypoxic-ischemic encephalopathy model.

Regulation of innate immune responses and activation of tissue regenerative processes are key elements in the pathophysiology of brain injuries. The promyelocytic leukemia (PML) gene was originally identified on a breakpoint of chromosomal translocation t(15;17) associated with acute PML. We have studied the role of PML protein during acute and regenerative phases after hypoxia-ischemia (HI) in brains of neonatal mice. We found that PML prevents tissue loss and apoptotic cell death selectively in subcortical regions of the brain at early stages after damage. In accordance with this, we revealed that PML is important for microglia activation and production of key inflammatory cytokines such as IL1α, IL1ß, IL1RN, CXCL10, CCL12 and TNFα. During the regenerative phase, PML-depleted mice were found to have impaired transformation of transit-amplifying precursors into migratory progenitors. This was accompanied by increased ratios of symmetric versus asymmetric neural progenitor cell divisions during tissue repair and a specific defect in tissue restoration within the striatum 42 days after HI. The data demonstrate a dual role of PML in protection and recovery after brain injury.

Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA.

BACKGROUND: Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. METHODS: BER gene expression levels were analyzed in 162 carotid plaques, 8 disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls. RESULTS: Our major findings were: (i) The BER pathway showed a global increased transcriptional response in plaques as compared to control arteries, accompanied by increased expression of several BER proteins. (ii) Whereas nuclear DNA stability was maintained within carotid plaques, mtDNA integrity and copy number were decreased. (iii) Within carotid plaques, mRNA levels of several BER genes correlated with macrophage markers. (iv) In vitro, some of the BER genes were highly expressed in the anti-inflammatory and pro-resolving M2 macrophages, showing increased expression upon exposure to modified lipids. CONCLUSIONS: The increased transcriptional response of BER genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion.

Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice.

Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe(-/-)Neil3(-/-) mice on high-fat diet showed accelerated plaque formation as compared to Apoe(-/-) mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe(-/-)Neil3(-/-) mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage.

Quantification of DNA Damage by Real-Time qPCR.

This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions.

Synergistic Actions of Ogg1 and Mutyh DNA Glycosylases Modulate Anxiety-like Behavior in Mice.

Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1(-/-)Mutyh(-/-) mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh(-/-) mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior.