RESUMO
Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5'-3' exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit(+) cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.
Assuntos
Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Rim/patologia , Hepatopatias/genética , Animais , Medula Óssea/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Epistasia Genética , Exodesoxirribonucleases/metabolismo , Fígado/patologia , Camundongos , Enzimas Multifuncionais , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.
Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação de Sentido Incorreto/genética , Linhagem Celular , Anemia de Fanconi/patologia , Imunofluorescência , HumanosRESUMO
UNLABELLED: The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging-related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1(-/Δ) mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1(-/Δ) mice, we systematically compared liver from 5-month-old progeroid Ercc1(-/Δ) mice to old (24-36-month-old) wild-type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1(-/Δ) mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1(-/Δ) mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1(-/Δ) liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1(-/Δ) and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome-wide transcriptional changes between old WT and 16-week-old, but not 5-week-old, Ercc1(-/Δ) mice, emphasizing that the Ercc1(-/Δ) mice acquire an aging profile in early adulthood. CONCLUSION: There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging-related hepatic changes.
Assuntos
Envelhecimento/fisiologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Endonucleases/genética , Fígado/fisiopatologia , Progéria/fisiopatologia , Envelhecimento/patologia , Animais , Senescência Celular , Reparo do DNA , Perfilação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Camundongos , Estresse Oxidativo , Progéria/genética , Progéria/metabolismo , Progéria/patologiaRESUMO
5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.
Assuntos
5-Metilcitosina/metabolismo , Envelhecimento/metabolismo , Senescência Celular , Oxirredução , Animais , Biomarcadores , Senescência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Epigênese Genética , Fibroblastos , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da PolimeraseRESUMO
Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.
Assuntos
Senescência Celular , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Peroxidação de Lipídeos , Estresse Oxidativo , Animais , Proliferação de Células , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismoRESUMO
Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mitocôndrias/genética , Animais , Antioxidantes/metabolismo , Senescência Celular/fisiologia , Óxidos N-Cíclicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated ß-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 -/- murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 -/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.The accumulation of senescent cells is thought to contribute to the age-associated decline in tissue function. Here, the authors identify HSP90 inhibitors as a new class of senolytic compounds in an in vitro screening and show that administration of a HSP90 inhibitor reduces age-related symptoms in progeroid mice.
Assuntos
Envelhecimento/fisiologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Benzoquinonas/farmacologia , Bioensaio , Biomarcadores/metabolismo , Senescência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Endonucleases/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lactamas Macrocíclicas/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.
Assuntos
Envelhecimento/efeitos dos fármacos , Senescência Celular , Dano ao DNA , Quinase I-kappa B/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Envelhecimento/genética , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Peptídeos/farmacologia , Fosforilação , Progéria/tratamento farmacológico , Progéria/patologia , Ligação Proteica , Transdução de Sinais , Fator de Transcrição RelA/genética , Ativação TranscricionalRESUMO
ERCC1-XPF is a structure-specific endonuclease required for nucleotide excision repair, interstrand crosslink repair, and the repair of some double-strand breaks. Mutations in ERCC1 or XPF cause xeroderma pigmentosum, XFE progeroid syndrome or cerebro-oculo-facio-skeletal syndrome, characterized by increased risk of cancer, accelerated aging and severe developmental abnormalities, respectively. This review provides a comprehensive overview of the health impact of ERCC1-XPF deficiency, based on these rare diseases and mouse models of them. This offers an understanding of the tremendous health impact of DNA damage derived from environmental and endogenous sources.
Assuntos
Distúrbios no Reparo do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Animais , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Dano ao DNA , Distúrbios no Reparo do DNA/metabolismo , Distúrbios no Reparo do DNA/fisiopatologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Regulação da Expressão Gênica , Genótipo , Humanos , Camundongos , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Doenças Raras/genética , Doenças Raras/metabolismo , Doenças Raras/fisiopatologia , Telômero/genética , Telômero/metabolismo , Raios UltravioletaRESUMO
Computational image analysis is used in many areas of biological and medical research, but advanced techniques including machine learning remain underutilized. Here, we used automated segmentation and shape analyses, with pre-defined features and with computer generated components, to compare nuclei from various premature aging disorders caused by alterations in nuclear proteins. We considered cells from patients with Hutchinson-Gilford progeria syndrome (HGPS) with an altered nucleoskeletal protein; a mouse model of XFE progeroid syndrome caused by a deficiency of ERCC1-XPF DNA repair nuclease; and patients with Werner syndrome (WS) lacking a functional WRN exonuclease and helicase protein. Using feature space analysis, including circularity, eccentricity, and solidity, we found that XFE nuclei were larger and significantly more elongated than control nuclei. HGPS nuclei were smaller and rounder than the control nuclei with features suggesting small bumps. WS nuclei did not show any significant shape changes from control. We also performed principle component analysis (PCA) and a geometric, contour based metric. PCA allowed direct visualization of morphological changes in diseased nuclei, whereas standard, feature-based approaches required pre-defined parameters and indirect interpretation of multiple parameters. Both methods yielded similar results, but PCA proves to be a powerful pre-analysis methodology for unknown systems.