Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

Aging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Markers of Mitochondrial Metabolism in Tumor Hypoxia, Systemic Inflammation, and Adverse Outcome of Rectal Cancer.

Tumor hypoxia contributes to therapy resistance and metastatic progression of locally advanced rectal cancer (LARC). We postulated that the tumor mitochondrial metabolism, manifested by reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage, reflects how hypoxic conditions connect to cancer-induced systemic inflammation and poor outcome. Levels of ROS and mtDNA damage were analyzed in three colorectal cancer (CRC) cell lines cultured for 24 hours under normoxia (21% O2) or hypoxia (0.2% O2) and serum sampled at the time of diagnosis from 35 LARC patients participating in a prospective therapy study. Compared with normoxia, ROS were significantly repressed and mtDNA damage was significantly enhanced in the hypoxic CRC cell lines; hence, a low ratio of ROS to mtDNA damage was an indicator of hypoxic conditions. In the LARC patients, low serum ROS were associated with elevated levels of circulating carcinoembryonic antigen and tumor choline concentration, both indicative of unfavorable biology, as well as adverse progression-free and overall survival. A low ratio of ROS to mtDNA damage in serum was associated with poor local tumor response to the neoadjuvant treatment and, of note, elevated systemic inflammation factors (C-reactive protein, the interleukin-1 receptor antagonist, and factors involved in tumor necrosis factor signaling), indicating that deficient treatment response locally and detrimental inflammation systemically link to a hypoxic mitochondrial metabolism. In conclusion, serum ROS and damaged mtDNA may be markers of the mitochondrial metabolism driven by the state of oxygenation of the primary tumor and possibly implicated in systemic inflammation and adverse outcome of LARC.

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.

Altered DNA base excision repair profile in brain tissue and blood in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia globally. AD is associated with increased oxidative stress, resulting from imbalance in production and clearance of reactive oxygen species (ROS). ROS can damage DNA and other macromolecules, leading to genome instability and disrupted cellular functions. Base excision repair (BER) plays a major role in repairing oxidative DNA lesions. Here, we compared the expression of BER components APE1, OGG1, PARP1 and Polß in blood and postmortem brain tissue from patients with AD, mild cognitive impairment (MCI) and healthy controls (HC). RESULTS: BER mRNA levels were correlated to clinical signs and cerebrospinal fluid biomarkers for AD. Notably, the expression of BER genes was higher in brain tissue than in blood samples. Polß mRNA and protein levels were significantly higher in the cerebellum than in the other brain regions, more so in AD patients than in HC. Blood mRNA levels of OGG1 was low and PARP1 high in MCI and AD. CONCLUSIONS: These findings reflect the oxidative stress-generating energy-consumption in the brain and the importance of BER in repairing these damage events. The data suggest that alteration in BER gene expression is an event preceding AD. The results link DNA repair in brain and blood to the etiology of AD at the molecular level and can potentially serve in establishing novel biomarkers, particularly in the AD prodromal phase.

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.

Analysis of mitochondrial DNA and RNA integrity by a real-time qPCR-based method.

This chapter describes the use of real-time qPCR to analyze the integrity of mitochondrial nucleic acids quantitatively. The method has low material requirement, is low cost, and can detect modifications with high resolution. The method is specifically designed for mitochondrial RNA and DNA, but can be easily transferred to other high-copy number cases. This procedure describes analyses of brain nucleic acids, but other tissues or cells can be analyzed similarly.

Addressing RNA integrity to determine the impact of mitochondrial DNA mutations on brain mitochondrial function with age.

Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations.

Up-regulation of CLDN1 in gastric cancer is correlated with reduced survival.

BACKGROUND: The genetic changes in gastric adenocarcinoma are extremely complex and reliable tumor markers have not yet been identified. There are also remarkable geographical differences in the distribution of this disease. Our aim was to identify the most differentially regulated genes in 20 gastric adenocarcinomas from a Norwegian selection, compared to matched normal mucosa, and we have related our findings to prognosis, survival and chronic Helicobacter pylori infection. METHODS: Biopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 20 patients immediately following surgical resection of the tumor. Whole genome, cDNA microarray analysis was performed on the RNA isolated from the sample pairs to compare the gene expression profiles between the tumor against matched mucosa. The samples were microscopically examined to classify gastritis. The presence of H. pylori was examined using microscopy and immunohistochemistry. RESULTS: 130 genes showed differential regulation above a predefined cut-off level. Interleukin-8 (IL-8) and Claudin-1 (CLDN1) were the most consistently up-regulated genes in the tumors. Very high CLDN1 expression in the tumor was identified as an independent and significant predictor gene of reduced post-operative survival. There were distinctly different expression profiles between the tumor group and the control mucosa group, and the histological subsets of mixed type, diffuse type and intestinal type cancer demonstrated further sub-clustering. Up-regulated genes were mapped to cell-adhesion, collagen-related processes and angiogenesis, whereas normal intestinal functions such as digestion and excretion were associated with down-regulated genes. We relate the current findings to our previous study on the gene response of gastric epithelial cells to H. pylori infection. CONCLUSIONS: CLDN1 was highly up-regulated in gastric cancer, and CLDN1 expression was independently associated with a poor post-operative prognosis, and may have important prognostic value. IL-8 and CLDN1 may represent central links between the gene response seen in acute H. pylori infection of gastric epithelial cells, and ultimately gastric cancer.

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis.

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh⁻/⁻/ogg1⁻/⁻ double knockout mouse. In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1month) and adult (6months) wt and myh⁻/⁻/ogg1⁻/⁻ mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh⁻/⁻/ogg1⁻/⁻ mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh⁻/⁻/ogg1⁻/⁻ mouse. The reduced respiration capacity correlated with a specific reduction (-25%) in complex I biochemical activity in brain mitochondria. Our results demonstrate that mtDNA mutations are not associated with cancer development in myh⁻/⁻/ogg1⁻/⁻ mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.

Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells.

BACKGROUND: The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA⁺ and OMPLA⁻ H. pylori variants. RESULTS: Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment. CONCLUSION: In whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA⁺/⁻ did not affect the acute inflammatory response to H. pylori.

Mitochondrial DNA damage level determines neural stem cell differentiation fate.

The mitochondrial DNA (mtDNA) of neural stem cells (NSCs) is vulnerable to oxidation damage. Subtle manipulations of the cellular redox state affect mtDNA integrity in addition to regulating the NSC differentiation lineage, suggesting a molecular link between mtDNA integrity and regulation of differentiation. Here we show that 8-oxoguanine DNA glycosylase (OGG1) is essential for repair of mtDNA damage and NSC viability during mitochondrial oxidative stress. Differentiating neural cells from ogg1(-/-) knock-out mice spontaneously accumulate mtDNA damage and concomitantly shift their differentiation direction toward an astrocytic lineage, similar to wt NSCs subjected to mtDNA damaging insults. Antioxidant treatments reversed mtDNA damage accumulation and separately increased neurogenesis in ogg1(-/-) cells. NSCs from a transgenic ogg1(-/-) mouse expressing mitochondrially targeted human OGG1 were protected from mtDNA damage during differentiation, and displayed elevated neurogenesis. The underlying mechanisms for this shift in differentiation direction involve the astrogenesis promoting Sirt1 via an increased NAD/NADH ratio in ogg1(-/-) cells. Redox manipulations to alter mtDNA damage level correspondingly activated Sirt1 in both cell types. Our results demonstrate for the first time the interdependence between mtDNA integrity and NSC differentiation fate, suggesting that mtDNA damage is the primary signal for the elevated astrogliosis and lack of neurogenesis seen during repair of neuronal injury.

Mitochondrial DNA integrity is essential for mitochondrial maturation during differentiation of neural stem cells.

Differentiation of neural stem cells (NSCs) involves the activation of aerobic metabolism, which is dependent on mitochondrial function. Here, we show that the differentiation of NSCs involves robust increases in mitochondrial mass, mitochondrial DNA (mtDNA) copy number, and respiration capacity. The increased respiration activity renders mtDNA vulnerable to oxidative damage, and NSCs defective for the mitochondrial 8-oxoguanine DNA glycosylase (OGG1) function accumulate mtDNA damage during the differentiation. The accumulated mtDNA damages in ogg1(-/-) cells inhibit the normal maturation of mitochondria that is manifested by reduced cellular levels of mitochondrial encoded complex proteins (complex I [cI], cIII, and cIV) with normal levels of the nuclear encoded cII present. The specific cI activity and inner membrane organization of respiratory complexes are similar in wt and ogg1(-/-) cells, inferring that mtDNA damage manifests itself as diminished mitochondrial biogenesis rather than the generation of dysfunctional mitochondria. Aerobic metabolism increases during differentiation in wild-type cells and to a lesser extent in ogg1(-/-) cells, whereas anaerobic rates of metabolism are constant and similar in both cell types. Our results demonstrate that mtDNA integrity is essential for effective mitochondrial maturation during NSC differentiation.

Genome profiles in maternal blood during early onset preeclampsia and towards term.

AIMS: inflammatory processes are present during preeclampsia and in normal pregnancy. Maternal inflammatory reactions may change towards term. Our objective was to evaluate genome signaling in blood during preeclampsia and towards term using microarrays. METHODS: RNA microarrays (Illumina) were conducted on blood from preeclamptic pregnancies delivered preterm, normal pregnancies at term and normal pregnancies at gestational week 31. Two statistical methods (Q-value cut-off 1%) identified data structures in the three groups and retrieved activated genes along a time axis and a diseased-healthy axis. Signaling genes were localized within known pathways and gene sets, and genes associated with inflammation were identified. RESULTS: early onset preeclampsia and term pregnancies both showed distinct expression patterns when compared to normal pregnancy at gestational week 31. In preeclampsia, 19 genes were differentially expressed, including a down-regulation of CC-chemokine receptor 3 (CCR3). Among the 183 differentially expressed genes towards term, tumor necrosis factor superfamily member 15 (TNFSF15) was up-regulated and interferon-γ receptor 2 (IFNGR2) and CXC-chemokine receptor type 4 (CXCR4) were down-regulated. Seven of the genes were similarly changed during preeclampsia and towards term. CONCLUSIONS: a possible type 1 immune response was identified both during preeclampsia and towards term. In pre-eclampsia a premature activation of leucocytes might be present.