Regulation of Oxidized Base Repair in Human Chromatin by Posttranslational Modification

Base excision repair (BER) is the major pathway for the repair of oxidized bases and apurinic/apyrimidinic (abasic; AP) sites produced by reaction with reactive oxygen/nitrogen species (ROS/RNS). These metabolites are generated spontaneously by endogenous cellular processes and also by environmental agents. Because most of these lesions are promutagenic, linked to diverse disease-associated somatic mutations, as well as heritable single nucleotide polymorphisms (SNPs) in the normal human population, their prompt repair is warranted. Impairment of repair leading to mutation, a hallmark of cancer, underscores the essentiality of BER for maintaining genome integrity in humans and other mammals. In mammals, repair of oxidized bases and other BER substrates is initiated by DNA glycosylases (DGs), which excise the damaged bases and cleave the DNA strands at the resulting AP sites, followed by sequential end processing, gap-filling DNA synthesis, and ligation. In vitro BER performed with naked DNA substrates has been extensively studied, which delineates its basic mechanistic steps and subpathways. However, recent interest is directed to unraveling BER in cell chromatin, including its regulation via posttranslational modifications (PTMs), which occurs possibly in concert with nucleosome remodeling. Emerging reports on various PTMs of BER enzymes indicate that the PTMs, while dispensable for the enzymatic activity, regulate overall repair by modulating interactions with other repair proteins and chromatin factors, assembly of BER complexes, as well as turnover of the proteins, and may ultimately dictate the cellular phenotype. Here, we discuss recent advances in the BER field by reviewing the PTMs and how they regulate BER in chromatin

The C-terminal Domain (CTD) of Human DNA GlycosylaseNEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ~8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells

Chromatin-bound oxidized α-synuclein causes strand breaks in neuronal genomes in in vitro models of Parkinson’s disease

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD

Chromatin-bound oxidized α-synuclein causes strand breaks in neuronal genomes in in vitro models of Parkinson's Disease

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD

Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen

Metabolic effects of early life stress and pre-pregnancy obesity are long lasting and sex specific in mice

Early life stress (ELS) is associated with metabolic, cognitive, and psychiatric diseases and has a very high prevalence, highlighting the urgent need for a better understanding of the versatile physiological changes and identification of predictive biomarkers. In addition to programming the hypothalamic-pituitary-adrenal (HPA) axis, ELS may also affect the gut microbiota and metabolome, opening up a promising research direction for identifying early biomarkers of ELS-induced (mal)adaptation. Other factors affecting these parameters include maternal metabolic status and diet, with maternal obesity shown to predispose offspring to later metabolic disease. The aim of the present study was to investigate the long-term effects of ELS and maternal obesity on the metabolic and stress phenotype of rodent offspring. To this end, offspring of both sexes were subjected to an adverse early-life experience, and their metabolic and stress phenotypes were examined. In addition, we assessed whether a prenatal maternal and an adult high-fat diet (HFD) stressor further shape observed ELS-induced phenotypes. We show that ELS has long-term effects on male body weight (BW) across the lifespan, whereas females more successfully counteract ELS-induced weight loss, possibly by adapting their microbiota, thereby stabilizing a balanced metabolome. Furthermore, the metabolic effects of a maternal HFD on BW are exclusively triggered by a dietary challenge in adult offspring and are more pronounced in males than in females. Overall, our study suggests that the female microbiota protects against an ELS challenge, rendering them more resilient to additional maternal- and adult nutritional stressors than males.This work was supported by the “GUTMOM” grant of the ERA-Net Cofund HDHL-INTIMIC (INtesTInal MIcrobiomics) under the JPI HDHL (Joint Programming Initiative – A healthy diet for a healthy life) umbrella (01EA1805; MVS), the SCHM2360-5-1 grant (MVS) from the German Research Foundation (DFG), the ZonMw grant from the Netherlands Organisation for Health Research and Development (project number 529051019), the DIM-ELI-2 grant of La Fundación La Marató-TV3 (ref. 2018-27/30-31), the PID2019-108973RB-C22 and PCIN2017-117 grants from the Ministerio de Ciencia e Innovación of Spain and the grants GV/2020/048 and IDIFEDER/2021/072 from the Generalitat Valenciana of Spain. Open Access funding enabled and organized by Projekt DEAL.Peer reviewe

Rotation of neuronal masses derived from hippocampi of four day-old mouse pups

<p>Video 1: Rotation of neuronal masses in different magnifications (20X and 40X).<br>a) 0-0.22 min.: Shows one rotating neuronal mass at 20X. 0.24-0.46 focuses on the same mass at 40X.<br>b) 0.51-0.56 min: Moves magnification at 20X over the same mass before scanning the plate.<br>c) 1.16-1.19 min: Shows few neuronal masses that have attached to the plate.<br>d) 1.31-1.35 min: Focuses on a very small cluster of neuronal cells (four in a group) that are rotating (at 20X magnification).<br>e) 1.37 min-end: Focuses on the same cluster but at 40X magnification.</p> <p>Video 2: Multiple rotating neuron mass in the same culture.<br>a) 0-0.45 min: Shows a neuron mass rotating in-spite of resistance from surrounding debris.<br>b) 0.50-1.26 min: Shows two different sizes of neuronal cell clusters spinning and rotating.<br>c) 1.36 min-end: Shows one more rotating neuronal cluster.</p> <p>Video 3: One neuronal mass rotating and spinning.</p> <p> </p

Psychosocial stress induces schizophrenia-like behavior in mice with reduced MMP-9 activity

Understanding gene-environment interactions in the pathogenesis of schizophrenia remains a major research challenge. Matrix metalloproteinase-9 (MMP-9) has been previously implicated in the pathophysiology of schizophrenia. In the present study, adolescent Mmp-9 heterozygous mice, with a genetically lower level of MMP-9, were subjected to resident-intruder psychosocial stress for 3 weeks and then examined in behavioral tests that evaluated cognitive deficits and positive- and negative-like symptoms of schizophrenia. Cognitive and positive symptoms in unstressed Mmp-9 heterozygous mice were unaffected by stress exposure, whereas negative symptoms were manifested only after stress exposure. Interestingly, negative symptoms were ameliorated by treatment with the antipsychotic drug clozapine. We describe a novel gene &times; environment interaction mouse model of schizophrenia. Lower MMP-9 levels in the brain might be a risk factor for schizophrenia that, in combination with environmental factors (e.g., psychosocial stress), may evoke schizophrenia-like symptoms that are sensitive to antipsychotic treatment

Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression.

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53's DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1's stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1's negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1's another binding site in p21's proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression. These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression

Absence of Wdr13 Gene Predisposes Mice to Mild Social Isolation – Chronic Stress, Leading to Depression-Like Phenotype Associated With Differential Expression of Synaptic Proteins

We earlier reported that the male mice lacking the Wdr13 gene (Wdr13-/0) showed mild anxiety, better memory retention, and up-regulation of synaptic proteins in the hippocampus. With increasing evidences from parallel studies in our laboratory about the possible role of Wdr13 in stress response, we investigated its role in brain. We observed that Wdr13 transcript gets up-regulated in the hippocampus of the wild-type mice exposed to stress. To further dissect its function, we analyzed the behavioral and molecular phenotypes of Wdr13-/0 mice when subjected to mild chronic psychological stress, namely; mild (attenuated) social isolation. We employed iTRAQ based quantitative proteomics, real time PCR and western blotting to investigate molecular changes. Three weeks of social isolation predisposed Wdr13-/0 mice to anhedonia, heightened anxiety-measured by Open field test (OFT), increased behavior despair- measured by Forced swim test (FST) and reduced dendritic branching along with decreased spine density of hippocampal CA1 neurons as compared to wild-type counterparts. This depression-like-phenotype was however ameliorated when treated with anti-depressant imipramine. Molecular analysis revealed that out of 1002 quantified proteins [1% False discovery rate (FDR), at-least two unique peptides], strikingly, a significant proportion of synaptic proteins including, SYN1, CAMK2A, and RAB3A were down-regulated in the socially isolated Wdr13-/0 mice as compared to its wild-type counterparts. This was in contrast to the elevated levels of these proteins in non-stressed mutants as compared to the controls. We hypothesized that a de-regulated transcription factor upstream of the synaptic genes might be responsible for the observed phenotype. Indeed, in the socially isolated Wdr13-/0 mice, there was an up-regulation of GATA1 – a transcription factor that negatively regulates synaptic genes and has been associated with Major Depression (MD) in humans. The present study demonstrates significant genotype × enviornment interaction for Wdr13 gene as shown by the reversal in the expression levels of several synaptic proteins in the mutant vis-à-vis wild-type mouse when exposed to social isolation stress