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.

OXR1A, a Coactivator of PRMT5 Regulating Histone Arginine Methylation.

Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A-/-) develop fatty liver. RNA sequencing of male Oxr1A-/- liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A-/- pituitary gland and in rat Oxr1A-/- pituitary adenoma cell-line GH3. Oxr1A-/- male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland.

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.

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.

Neil1 is a genetic modifier of somatic and germline CAG trinucleotide repeat instability in R6/1 mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by trinucleotide repeat (TNR) expansions. We show here that somatic TNR expansions are significantly reduced in several organs of R6/1 mice lacking exon 2 of Nei-like 1 (Neil1) (R6/1/Neil1(-/-)), when compared with R6/1/Neil1(+/+) mice. Somatic TNR expansion is measured by two different methods, namely mean repeat change and instability index. Reduced somatic expansions are more pronounced in male R6/1/Neil1(-/-) mice, although expansions are also significantly reduced in brain regions of female R6/1/Neil1(-/-) mice. In addition, we show that the lack of functional Neil1 significantly reduces germline expansion in R6/1 male mice. In vitro, purified human NEIL1 protein binds and excises 5-hydroxycytosine in duplex DNA more efficiently than in hairpin substrates. NEIL1 excision of cytosine-derived oxidative lesions could therefore be involved in initiating the process of TNR expansion, although other DNA modifications might also contribute. Altogether, these results imply that Neil1 contributes to germline and somatic HD CAG repeat expansion.

Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia.

Neural stem/progenitor cell proliferation and differentiation are required to replace damaged neurons and regain brain function after hypoxic-ischemic events. DNA base lesions accumulating during hypoxic-ischemic stress are removed by DNA glycosylases in the base-excision repair pathway to prevent cytotoxicity and mutagenesis. Expression of the DNA glycosylase endonuclease VIII-like 3 (Neil3) is confined to regenerative subregions in the embryonic and perinatal brains. Here we show profound neuropathology in Neil3-knockout mice characterized by a reduced number of microglia and loss of proliferating neuronal progenitors in the striatum after hypoxia-ischemia. In vitro expansion of Neil3-deficient neural stem/progenitor cells revealed an inability to augment neurogenesis and a reduced capacity to repair for oxidative base lesions in single-stranded DNA. We propose that Neil3 exercises a highly specialized function through accurate molecular repair of DNA in rapidly proliferating cells.

Expression patterns of Neil3 during embryonic brain development and neoplasia.

BACKGROUND: The base excision repair pathway is responsible for repairing small DNA base lesions caused by endogenous and exogenous damaging agents. Repair is initiated by DNA glycosylases that recognize and remove the lesions. NEIL3 is one of 11 mammalian DNA glycosylases identified to date and it was discovered on the basis of sequence homology to the E. coli Fpg and Nei glycosylases. Difficulties in purifying the protein have limited its biochemical characterization and in contrast to the other glycosylases, its function remains unclear. RESULTS: In this study we describe the expression pattern of Neil3 during mouse embryonic development with special focus on brain development. We have also looked at the expression of NEIL3 in several normal and tumor tissues. Quantitative real-time PCR and in situ hybridization revealed that Neil3 was highly expressed at embryonic days 12-13, when neurogenesis starts. The expression decreased during development and in the adult brain,Neil3 could not be detected in any of the brain areas examined by quantitative real-time PCR. During embryogenesis and in newborn mice specific expression was observed in areas known to harbour neural stem and progenitor cells such as the subventricular zone and the dentate gyrus. Finally, NEIL3 expression was higher in tumors compared to normal tissues, except for testis and pancreas. CONCLUSION: Our findings indicate that mammalian NEIL3 is specifically expressed in brain areas where neurogenesis takes place during development and that its expression is tightly regulated both temporally and spatially. In addition, NEIL3 seems to be upregulated in tumor tissues compared to normal tissues. Altogether, mammalian NEIL3 seems to be highly expressed in cells with high proliferative potential.

Modulation of DNA glycosylase activities in mesenchymal stem cells.

Adipose-tissue derived mesenchymal stem cells (AT-MSCs) are a promising tool for use in cell-based therapies. However, in vitro expansion is required to obtain clinically relevant cell numbers, and this might increase the chance of genomic instability. DNA repair is crucial for maintaining DNA integrity. Here we have compared the initial step of base excision repair in uncultured and cultured AT-MSCs by analysis of base removal activities and expression levels of relevant DNA glycosylases. Uracil, 5-hydroxyuracil and ethenoadenine removal activities were upregulated in cultured cells compared to uncultured cells. In contrast, both the 8-oxo-7,8-dihydroguanine (8-oxoG) removal activity and the concentration of 8-oxoG bases in the DNA were reduced in the cultured cells. Gene expression analysis showed no substantial changes in mRNA expression. The glycosylase activities remained stable through at least 12 passages, suggesting that DNA repair is proficient through the period required for in vitro expansion of AT-MSCs to clinically relevant numbers.

Human NEIL1 localizes with the centrosomes and condensed chromosomes during mitosis.

The DNA glycosylase hNEIL1 initiates base excision repair (BER) of a number of oxidized purines and pyrimidines in cellular DNA and is one of three mammalian orthologs of the Escherichia coli Nei/Fpg enzymes. Human NEIL1 has been purified and extensively characterized biochemically, however, not much is known about its intracellular distribution. In the present work, we have studied the cellular localization of hNEIL1 using both antibodies raised against the full-length recombinant protein and a stable HeLa cell line expressing hNEIL1 fused N-terminal to EGFP. The results presented reveal an intricate mitotic distribution of hNEIL1. Centrosomal localization of hNEIL1 was observed when mitotic HeLa cells were immunostained with hNEIL1 antibodies. This localization was confirmed when Western blots of isolated centrosomes from stably expressing hNEIL1-EGFP HeLa cells were probed with GFP or hNEIL1 antibodies, even though a fluorescent signal could not be detected in the centrosomes of these cells. Human NEIL1 was also shown to be associated with mitotic condensed chromosomes. Notably, the interaction of hNEIL1 with condensed chromatin was disrupted when cells were fixed with chemical fixatives that are regularly used in immunodetection techniques.

The capacity to remove 8-oxoG is enhanced in newborn neural stem/progenitor cells and decreases in juvenile mice and upon cell differentiation.

In mammalian cells, 8-oxoguanine DNA glycosylase-1 (OGG1) is the main DNA glycosylase for the removal of 8-oxoguanine (8-oxoG). 8-oxoG, one of the most common products of the oxidative attack of DNA, is a premutagenic lesion that accumulates spontaneously at high frequencies in the genome. In this study, Ogg1 mRNA expression was detected throughout embryonic development in mice. In situ hybridization showed that in the neonatal brain, Ogg1 expression was detected in a distinct layer of cells in the medial wall of the lateral ventricle, which may correspond to ependymal cells, and in some scattered cells in the subventricular zone (SVZ), a brain region rich in neural stem/progenitor cells. Using neurospheres as a model for the study of neural stem/progenitor cells, we found that both the expression and activity of Ogg1 were high in neurospheres derived from newborn mice and decreased in adults and upon induction of cell differentiation. Furthermore, Ogg1 was shown to be the major DNA glycosylase initiating 8-oxoG repair in neurospheres. Our results strongly indicate that enhanced DNA repair capacity is an important mechanism by which neural stem/progenitor cells maintain their genome.

Dynamic relocalization of hOGG1 during the cell cycle is disrupted in cells harbouring the hOGG1-Cys326 polymorphic variant.

Numerous lines of evidence support the role of oxidative stress in different types of cancer. A major DNA lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), is formed by reactive oxygen species in the genome under physiological conditions. 8-OxoG is strongly mutagenic, generating G.C-->T.A transversions, a frequent somatic mutation in cancers. hOGG1 was cloned as a gene encoding a DNA glycosylase that specifically recognizes and removes 8-oxoG from 8-oxoG:C base pairs and suppresses G.C-->T.A transversions. In this study, we investigated the subcellular localization and expression of hOGG1 during the cell cycle. Northern blots showed cell-cycle-dependent mRNA expression of the two major hOGG1 isoforms. By using a cell line constitutively expressing hOGG1 fused to enhanced green fluorescence protein (EGFP), we observed a dynamic relocalization of EGFP-hOGG1 to the nucleoli during the S-phase of the cell cycle, and this localization was shown to be linked to transcription. A C/G change that results in an amino acid substitution from serine to cysteine in codon 326 has been reported as a genetic polymorphism and a risk allele for a variety of cancers. We investigated the cellular localization of the corresponding protein, hOGG1-Cys326, fused to EGFP and observed a dramatic effect on its localization that is explained by a change in the phosphorylation status of hOGG1.