Assessing the Formation of Purine Lesions in Mitochondrial DNA of Cockayne Syndrome Cells.

Mitochondrial (mt) DNA and nuclear (n) DNA have known structures and roles in cells; however, they are rarely compared under specific conditions such as oxidative or degenerative environments that can create damage to the DNA base moieties. Six purine lesions were ascertained in the mtDNA of wild type (wt) CSA (CS3BE-wtCSA) and wtCSB (CS1AN-wtCSB) cells and defective counterparts CS3BE and CS1AN in comparison with the corresponding total (t) DNA (t = n + mt). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. The 8-oxo-Pu levels were found to be in the range of 25-50 lesions/107 nucleotides in both the mtDNA and tDNA. The four cPu were undetectable in the mtDNA both in defective cells and in the wt counterparts (CSA and CSB), contrary to their detection in tDNA, indicating a nonappearance of hydroxyl radical (HO•) reactivity within the mtDNA. In order to assess the HO• reactivity towards purine nucleobases in the two genetic materials, we performed γ-radiolysis experiments coupled with the 8-oxo-Pu and cPu quantifications on isolated mtDNA and tDNA from wtCSB cells. In the latter experiments, all six purine lesions were detected in both of the DNA, showing a higher resistance to HO• attack in the case of mtDNA compared with tDNA, likely due to their different DNA helical topology influencing the relative abundance of the lesions.

Effects of Oxygen Tension for Membrane Lipidome Remodeling of Cockayne Syndrome Cell Models.

Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes.

DRP1 Inhibition Rescues Mitochondrial Integrity and Excessive Apoptosis in CS-A Disease Cell Models.

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.

The interplay between mitochondrial functionality and genome integrity in the prevention of human neurologic diseases.

As mitochondria are vulnerable to oxidative damage and represent the main source of reactive oxygen species (ROS), they are considered key tuners of ROS metabolism and buffering, whose dysfunction can progressively impact neuronal networks and disease. Defects in DNA repair and DNA damage response (DDR) may also affect neuronal health and lead to neuropathology. A number of congenital DNA repair and DDR defective syndromes, indeed, show neurological phenotypes, and a growing body of evidence indicate that defects in the mechanisms that control genome stability in neurons acts as aging-related modifiers of common neurodegenerative diseases such as Alzheimer, Parkinson's, Huntington diseases and Amyotrophic Lateral Sclerosis. In this review we elaborate on the established principles and recent concepts supporting the hypothesis that deficiencies in either DNA repair or DDR might contribute to neurodegeneration via mechanisms involving mitochondrial dysfunction/deranged metabolism.

Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells.

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3-6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

Correction: Krokidis, M.G., et al. Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells. Cells 2020, 9, 1671.

The originally published article [...].

Purine DNA Lesions at Different Oxygen Concentration in DNA Repair-Impaired Human Cells (EUE-siXPA).

Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5',8-cyclopurines (cPu) lesions (i.e., 5'R-cdG, 5'S-cdG, 5'R-cdA and 5'S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82-2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10-4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5'R/5'S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.

miR-200a Modulates the Expression of the DNA Repair Protein OGG1 Playing a Role in Aging of Primary Human Keratinocytes.

Oxidative DNA damage accumulation may induce cellular senescence. Notably, senescent cells accumulate in aged tissues and are present at the sites of age-related pathologies. Although the signaling of DNA strand breaks has been extensively studied, the role of oxidative base lesions has not fully investigated in primary human keratinocyte aging. In this study, we show that primary human keratinocytes from elderly donors are characterized by a significant accumulation of the oxidative base lesion 8-OH-dG, impairment of oxidative DNA repair, and increase of miR-200a levels. Notably, OGG1-2a, a critical enzyme for 8-OH-dG repair, is a direct target of miR-200a and its expression levels significantly decrease in aged keratinocytes. The 8-OH-dG accumulation displays a significant linear relationship with the aging biomarker p16 expression during keratinocyte senescence. Interestingly, we found that miR-200a overexpression down-modulates its putative target Bmi-1, a well-known p16 repressor, and up-regulates p16 itself. miR-200a overexpression also up-regulates the NLRP3 inflammasome and IL-1ß expression. Of note, primary keratinocytes from elderly donors are characterized by NRPL3 activation and IL-1ß secretion. These findings point to miR-200a as key player in primary human keratinocyte aging since it is able to reduce oxidative DNA repair activity and may induce several senescence features through p16 and IL-1ß up-regulation.

CSA and CSB play a role in the response to DNA breaks.

CS proteins have been involved in the repair of a wide variety of DNA lesions. Here, we analyse the role of CS proteins in DNA break repair by studying histone H2AX phosphorylation in different cell cycle phases and DNA break repair by comet assay in CS-A and CS-B primary and transformed cells. Following methyl methane sulphate treatment a significant accumulation of unrepaired single strand breaks was detected in CS cells as compared to normal cells, leading to accumulation of double strand breaks in S and G2 phases. A delay in DSBs repair and accumulation in S and G2 phases were also observed following IR exposure. These data confirm the role of CSB in the suppression of NHEJ in S and G2 phase cells and extend this function to CSA. However, the repair kinetics of double strand breaks showed unique features for CS-A and CS-B cells suggesting that these proteins may act at different times along DNA break repair. The involvement of CS proteins in the repair of DNA breaks may play an important role in the clinical features of CS patients.

Overexpression of parkin rescues the defective mitochondrial phenotype and the increased apoptosis of Cockayne Syndrome A cells.

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

Crosstalk between mismatch repair and base excision repair in human gastric cancer.

DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase ß (Polß). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polß or Polß active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polß were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polß were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polß is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polß that modulates the response to alkylation damage. These studies suggest that the Polß/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.

Single nucleotide polymorphisms in DNA glycosylases: From function to disease.

Oxidative stress is associated with a growing number of diseases that span from cancer to neurodegeneration. Most oxidatively induced DNA base lesions are repaired by the base excision repair (BER) pathway which involves the action of various DNA glycosylases. There are numerous genome wide studies attempting to associate single-nucleotide polymorphisms (SNPs) with predispositions to various types of disease; often, these common variants do not have significant alterations in their biochemical function and do not exhibit a convincing phenotype. Nevertheless several lines of evidence indicate that SNPs in DNA repair genes may modulate DNA repair capacity and contribute to risk of disease. This overview provides a convincing picture that SNPs of DNA glycosylases that remove oxidatively generated DNA lesions are susceptibility factors for a wide disease spectrum that includes besides cancer (particularly lung, breast and gastrointestinal tract), cochlear/ocular disorders, myocardial infarction and neurodegenerative disorders which can be all grouped under the umbrella of oxidative stress-related pathologies.

The response to oxidative stress and metallomics analysis in a twin study: The role of the environment.

Inefficient response to oxidative stress has been associated with ageing and health risk. Metals are known to inhibit DNA repair and may modify the antioxidant response. How genetic variability and lifestyle factors modulate the response to oxidative stress is poorly explored. Our study aims to disentangle the contribution of genetics and environmental exposures to oxidative stress response using data from twin pairs. The non-enzymatic antioxidant capacity (NEAC), the repair capacity of 8-oxo-7,8-dihydroguanine (OGG activity) and the levels of 12 metals were measured in blood of 64 monozygotic and 31 dizygotic twin pairs. The contributions of genetic and environmental effects were assessed using standard univariate twin modelling. NEAC and OGG activity significantly decreased with age. Gender-, age- and body mass index-associated differences were identified for some metals. Principal Component Analysis identified two groups of metals whose levels in blood were highly correlated: As, Hg, Pb, Se, Zn and Al, Co, Cr, Mn, Ni. The environmental influence was predominant on OGG activity and NEAC variance whereas for most metals the best-fitting model incorporated additive genetic and unique environmental sources of variance. NEAC and OGG activity were both inversely correlated with blood levels of various metals. The inhibition of OGG activity by Cd was largely explained by smoking. Our data show a substantial role of environmental factors in NEAC and OGG activity variance that is not explained by twins' age. Exogenous environmental factors such as metals contribute to oxidative stress by decreasing NEAC and inhibiting repair of oxidatively-induced DNA damage.

An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients.

Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O2₋• and H2O2 being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance (¹H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer risk. The increased MN frequency was not affected by inhibition of ROS to normal levels by N-acetyl-L-cysteine.

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.

The role of CSA and CSB protein in the oxidative stress response.

Cockayne syndrome (CS) is a rare hereditary disorder in which infants suffer severe developmental and neurological alterations and early death. Two genes encoding RNA polymerase II cofactors, CSA and CSB, are mutated in this syndrome. CSA and CSB proteins are known to be involved in the transcription-coupled DNA repair pathway but the sensitivity of mutant cells to a number of physical/chemical agents besides UV radiation, such as ionizing radiation, hydrogen peroxide and bioenergetic inhibitors indicate that these proteins play a pivotal role in additional pathways. In this review we will discuss the evidence that implicate CS proteins in the control of oxidative stress response with special emphasis on recent findings that show an altered redox balance and dysfunctional mitochondria in cells derived from patients. Working models of how these new functions might be key to developmental and neurological disease in CS will be discussed.

The cross talk between pathways in the repair of 8-oxo-7,8-dihydroguanine in mouse and human cells.

Although oxidatively damaged DNA is repaired primarily via the base excision repair (BER) pathway, it is now evident that multiple subpathways are needed. Yet, their relative contributions and coordination are still unclear. Here, mouse embryo fibroblasts (MEFs) from selected nucleotide excision repair (NER) and/or BER mouse mutants with severe (Csb(m/m)/Xpa(-/-) and Csb(m/m)/Xpc(-/-)), mild (Csb(m/m)), or no progeria (Xpa(-/-), Xpc(-/-), Ogg1(-/-), Csb(m/m)/Ogg1(-/-)) or wild-type phenotype were exposed to an oxidizing agent, potassium bromate, and genomic 8-oxo-7,8-dihydroguanine (8-oxoGua) levels were measured by HPLC-ED. The same oxidized DNA base was measured in NER/BER-defective human cell lines obtained after transfection with replicative plasmids encoding siRNA targeting DNA repair genes. We show that both BER and NER factors contribute to the repair of 8-oxoGua, although to different extents, and that the repair profiles are similar in human compared to mouse cells. The BER DNA glycosylase OGG1 dominates 8-oxoGua repair, whereas NER (XPC, XPA) and transcription-coupled repair proteins (CSB and CSA) are similar, but minor contributors. The comparison of DNA oxidation levels in double versus single defective MEFs indicates increased oxidatively damaged DNA only when both CSB and XPC/XPA are defective, indicating that these proteins operate in different pathways. Moreover, we provide the first evidence of an involvement of XPA in the control of oxidatively damaged DNA in human primary cells.

Reprint of: gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function.

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.

An altered redox balance mediates the hypersensitivity of Cockayne syndrome primary fibroblasts to oxidative stress.

Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS-A and CS-B present an altered redox balance with increased steady-state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K-ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild-type genes. High-resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS-A and CS-B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function.

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.