RESUMEN
DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Daño del ADN , Factores de Transcripción Forkhead/metabolismo , Longevidad , Estrés Oxidativo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Factores de Transcripción Forkhead/genética , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Senescencia Celular , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Peroxidación de Lípido , Estrés Oxidativo , Animales , Proliferación Celular , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Envejecimiento/genética , Senescencia Celular/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Mitocondrias/genética , Animales , Antioxidantes/metabolismo , Senescencia Celular/fisiología , Óxidos N-Cíclicos/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND CONTEXT: Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE: To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN: To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS: Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured. RESULTS: Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice. CONCLUSIONS: Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.
Asunto(s)
Daño del ADN/fisiología , Degeneración del Disco Intervertebral/etiología , Degeneración del Disco Intervertebral/fisiopatología , Fumar/efectos adversos , Agrecanos/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Disco Intervertebral/diagnóstico por imagen , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Proteoglicanos/metabolismo , Factores de Riesgo , Microtomografía por Rayos XRESUMEN
Lipodystrophies represent a group of heterogeneous disorders characterized by loss of fat tissue. However, the underlying mechanisms remain poorly understood. Using mice carrying an ERCC1-XPF DNA repair defect systematically or in adipocytes, we show that DNA damage signaling triggers a chronic autoinflammatory response leading to fat depletion. Ercc1-/- and aP2-Ercc1F/- fat depots show extensive gene expression similarities to lipodystrophic Pparγ(ldi/+) animals, focal areas of ruptured basement membrane, the reappearance of primary cilia, necrosis, fibrosis, and a marked decrease in adiposity. We find that persistent DNA damage in aP2-Ercc1F/- fat depots and in adipocytes ex vivo triggers the induction of proinflammatory factors by promoting transcriptionally active histone marks and the dissociation of nuclear receptor corepressor complexes from promoters; the response is cell autonomous and requires ataxia telangiectasia mutated (ATM). Thus, persistent DNA damage-driven autoinflammation plays a causative role in adipose tissue degeneration, with important ramifications for progressive lipodystrophies and natural aging.
Asunto(s)
Tejido Adiposo/metabolismo , Daño del ADN , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Células Cultivadas , Citocinas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Endonucleasas/genética , Endonucleasas/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Histonas/metabolismo , Ratones , Ratones Noqueados , PPAR gamma/genética , PPAR gamma/metabolismo , Progeria/metabolismo , Progeria/patología , Recombinasa Rad51/metabolismo , TranscriptomaRESUMEN
The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age-related spontaneous loss of CE, we characterized CE in Ercc1(-/Δ) mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5- to 6-month-old Ercc1(-/Δ) mice, age-matched wild-type (WT) littermates, and old WT mice (24- to 34-month-old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole-mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1(-/Δ) mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair-deficient mice. CD45(+) immune cells were associated with the posterior surface of CE from Ercc1(-/Δ) mice and the tissue expressed increased IL-1α, Cxcl2, and TNFα, pro-inflammatory proteins associated with senescence-associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1(-/Δ) mice offer a rapid and accurate model to study CE pathogenesis and therapy.
Asunto(s)
Envejecimiento/patología , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Reparación del ADN , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Animales , Apoptosis , Recuento de Células , Colágeno/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Lámina Limitante Posterior/metabolismo , Lámina Limitante Posterior/patología , Lámina Limitante Posterior/ultraestructura , Endonucleasas/deficiencia , Endonucleasas/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Endotelio Corneal/crecimiento & desarrollo , Endotelio Corneal/ultraestructura , Humanos , Antígenos Comunes de Leucocito/metabolismo , Ratones , FenotipoRESUMEN
Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O(2)) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O(2)). Human disc cells grown at 20% O(2) showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O(2). Importantly, we demonstrated that treatment of accelerated aging Ercc1(-/Δ) mice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1(-/Δ) mice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.
Asunto(s)
Envejecimiento/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adulto , Animales , Células Cultivadas , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/farmacología , Glicosaminoglicanos/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Degeneración del Disco Intervertebral/patología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxígeno/farmacología , Proteoglicanos/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Advanced age is one of the most important risk factors for osteoporosis. Accumulation of oxidative DNA damage has been proposed to contribute to age-related deregulation of osteoblastic and osteoclastic cells. Excision repair cross complementary group 1-xeroderma pigmentosum group F (ERCC1-XPF) is an evolutionarily conserved structure-specific endonuclease that is required for multiple DNA repair pathways. Inherited mutations affecting expression of ERCC1-XPF cause a severe progeroid syndrome in humans, including early onset of osteopenia and osteoporosis, or anomalies in skeletal development. Herein, we used progeroid ERCC1-XPF-deficient mice, including Ercc1-null (Ercc1(-/-)) and hypomorphic (Ercc1(-/Δ)) mice, to investigate the mechanism by which DNA damage leads to accelerated bone aging. Compared to their wild-type littermates, both Ercc1(-/-) and Ercc1(-/Δ) mice display severe, progressive osteoporosis caused by reduced bone formation and enhanced osteoclastogenesis. ERCC1 deficiency leads to atrophy of osteoblastic progenitors in the bone marrow stromal cell (BMSC) population. There is increased cellular senescence of BMSCs and osteoblastic cells, as characterized by reduced proliferation, accumulation of DNA damage, and a senescence-associated secretory phenotype (SASP). This leads to enhanced secretion of inflammatory cytokines known to drive osteoclastogenesis, such as interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and receptor activator of NF-κB ligand (RANKL), and thereby induces an inflammatory bone microenvironment favoring osteoclastogenesis. Furthermore, we found that the transcription factor NF-κB is activated in osteoblastic and osteoclastic cells of the Ercc1 mutant mice. Importantly, we demonstrated that haploinsufficiency of the p65 NF-κB subunit partially rescued the osteoporosis phenotype of Ercc1(-/Δ) mice. Finally, pharmacological inhibition of the NF-κB signaling via an I-κB kinase (IKK) inhibitor reversed cellular senescence and SASP in Ercc1(-/Δ) BMSCs. These results demonstrate that DNA damage drives osteoporosis through an NF-κB-dependent mechanism. Therefore, the NF-κB pathway represents a novel therapeutic target to treat aging-related bone disease.
Asunto(s)
Huesos/fisiología , Daño del ADN , FN-kappa B/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Ratones , Ratones Noqueados , Osteoblastos/patología , Osteoporosis/genéticaRESUMEN
Daily, cells incur tens of thousands of DNA lesions caused by endogenous processes. Due to their long-lived nature, adult stem cells may be particularly susceptible to the negative impact of this constant genotoxic stress. Indeed, in murine models of DNA repair deficiencies, there is accumulation of DNA damage in hematopoietic stem cells and premature loss of function. Herein, we demonstrate that mice expressing reduced levels of ERCC1-XPF DNA repair endonuclease (Ercc1-/Δ mice) spontaneously display a progressive decline in the number and function of hematopoietic stem/progenitor cells (HSPCs). This was accompanied by increased cell death, expression of senescence markers, reactive oxygen species, and DNA damage in HSPC populations, illustrating cell autonomous mechanisms that contribute to loss of function. In addition, the bone marrow microenvironment of Ercc1-/Δ mice was not permissive for the engraftment of transplanted normal stem cells. Bones from Ercc1-/Δ mice displayed excessive osteoclastic activity, which alters the microenvironment in a way that is unfavorable to HSPC maintenance. This was accompanied by increased proinflammatory cytokines in the bone marrow of Ercc1-/Δ mice. These data provide novel evidence that spontaneous, endogenous DNA damage, if not repaired, promotes progressive attrition of adult stem cells via both cell autonomous and nonautonomous mechanisms.
Asunto(s)
Reparación del ADN , Células Madre Hematopoyéticas/fisiología , Animales , Procesos de Crecimiento Celular/fisiología , Microambiente Celular/genética , Daño del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Endonucleasas/genética , Endonucleasas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.
Asunto(s)
Envejecimiento/metabolismo , Daño del ADN , Reparación del ADN , Degeneración del Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAMTS4 , Agrecanos/genética , Agrecanos/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Alquilantes/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Senescencia Celular/efectos de la radiación , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Endonucleasas/biosíntesis , Endonucleasas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Mecloretamina/farmacología , Ratones , Ratones Noqueados , Procolágeno N-Endopeptidasa/biosíntesis , Procolágeno N-Endopeptidasa/genética , Radiación IonizanteRESUMEN
Heightened production of collagen and other matrix proteins underlies the fibrotic phenotype of systemic sclerosis (SSc). Roscovitine is an inhibitor of cyclin-dependent kinases that promote cell cycling (CDK1, 2), neuronal development (CDK5) and control transcription (CDK7,9). In an in vivo glomerulonephritis model, roscovitine treatment decreased mesangial cell proliferation and matrix proteins [1]. We investigated whether roscovitine could regulate fibrotic protein production directly rather than through cell cycling. Our investigations revealed that roscovitine coordinately inhibited the expression of collagen, fibronectin, and connective tissue growth factor (CTGF) in normal and SSc fibroblasts. This effect occurred on a transcriptional basis and did not result from roscovitine-mediated cell cycle inhibition. Roscovitine-mediated suppression of matrix proteins could not be reversed by the exogenous profibrotic cytokines TGF-ß or IL-6. To our knowledge, we are the first to report that roscovitine modulates matrix protein transcription. Roscovitine may thus be a viable treatment option for SSc and other fibrosing diseases.
Asunto(s)
Fibroblastos/patología , Purinas/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/patología , Animales , Colágeno/genética , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/efectos de los fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Humanos , Interleucina-6/biosíntesis , Ratones , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Roscovitina , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.
Asunto(s)
Envejecimiento/efectos de los fármacos , Senescencia Celular , Daño del ADN , Quinasa I-kappa B/antagonistas & inhibidores , Factor de Transcripción ReIA/metabolismo , Envejecimiento/genética , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/fisiología , Quinasa I-kappa B/metabolismo , Ratones , Ratones Transgénicos , Estrés Oxidativo , Péptidos/farmacología , Fosforilación , Progeria/tratamiento farmacológico , Progeria/patología , Unión Proteica , Transducción de Señal , Factor de Transcripción ReIA/genética , Activación TranscripcionalRESUMEN
STUDY DESIGN: NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. OBJECTIVE: To study the mediatory role of NF-κB-signaling pathway in age-dependent intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA: Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1 mice) previously been reported to exhibit age-related IDD. METHODS: Systemic inhibition of NF-κB activation was achieved either genetically by deletion of 1 allele of the NF-κB subunit p65 (Ercc1p65 mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1 mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. RESULTS.: Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1 mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1 mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1p65 mice. CONCLUSION: These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.
Asunto(s)
Envejecimiento , Degeneración del Disco Intervertebral/prevención & control , FN-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Distinciones y Premios , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Endonucleasas/deficiencia , Endonucleasas/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Datos de Secuencia Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Proteoglicanos/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: The excision repair cross complementing (ERCC1) gene product plays a vital role in the nucleotide excision repair (NER) and DNA interstrand crosslink repair pathways, which protect the genome from mutations and chromosomal aberrations, respectively. Genetic deletion of Ercc1 in the mouse causes dramatically accelerated aging. We examined the effect of Ercc1 deletion in the development of prostate cancer in a prostate recapitulation model as Ercc1 deficient mice die within four weeks of birth. METHODS: Prostate tissues from Ercc1(-/-) mice or wild-type littermates were combined with embryonic rat urogenital mesenchyme and grown as renal grafts for a total of 8, 16, and 24 weeks before histological, expression and proliferative evaluation. RESULTS: Invasive adenocarcinoma was observed in Ercc1(-/-) tissue recombinants but not wild-type as early as 8 weeks post-grafting. PIN-like lesions in Ercc1(-/-) tissue recombinants had more cytologic and architectural atypia than wild-type (P = 0.02, P = 0.0065, and P = 0.0003 at the 8, 16, and 24 weeks, respectively), as well as more proliferative cells (P = 0.022 and P = 0.033 at 8 and 16 weeks, respectively). With serial grafting, Ercc1(-/-) tissue recombinants progressed to a more severe histopathological phenotype more rapidly than wild-type (P = 0.011). CONCLUSIONS: Results show that ERCC1 and by implication the NER and/or interstrand crosslink repair mechanisms protect against prostate carcinogenesis and mutations or polymorphisms affecting these DNA repair pathways may predispose prostate epithelial cells to transformation.
Asunto(s)
Adenocarcinoma/genética , Trastornos por Deficiencias en la Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/deficiencia , Neoplasias de la Próstata/genética , Animales , Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endonucleasas/genética , Masculino , RatonesRESUMEN
Several genetic defects of the nucleotide excision repair (NER) pathway, including deficiency of the Excision Repair Cross-Complementing rodent repair deficiency, complementation group 1 (ERCC1), result in pre-mature aging, impaired growth, microcephaly and delayed development of the cerebellum. These phenotypes are recapitulated in Ercc1-knockout mice, which survive for up to 4 weeks after birth. Therefore, we analyzed cerebellar and hippocampal transcriptomes of these animals at 3 weeks of age to identify the candidate mechanisms underlying central nervous system abnormalities caused by inherited defects in NER. In the cerebellum, the most prominent change was the upregulation of genes associated with gliosis. Although Purkinje cell degeneration has been reported in some mouse strains with NER impairment, the transcripts whose downregulation is associated with Purkinje cell loss were mostly unaffected by the knockout of Ercc1. In the hippocampus, there was extensive downregulation of genes related to cholesterol biosynthesis. Reduced expression of these genes was also present in the neocortex of adult mice with reduced expression of ERCC1. These changes were accompanied by reduced mRNA expression of the transcription factor Sterol Regulatory Element Binding Transcription Factor-2 (SREBF2) which is a master regulator of cholesterol biosynthesis. The downregulation of forebrain cholesterol biosynthesis genes is a newly identified consequence of ERCC1 deficiency. Reduced cholesterol biosynthesis may contribute to the neurodevelopmental disruption that is associated with ERCC1 defects and several other NER deficiencies including Cockayne syndrome. In addition, this reduction may negatively affect the function of mature synapses.
Asunto(s)
Colesterol/biosíntesis , Proteínas de Unión al ADN/deficiencia , Regulación hacia Abajo/genética , Endonucleasas/deficiencia , Prosencéfalo/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Animales , Calbindinas , Cerebelo/metabolismo , Colesterol/genética , Reparación del ADN/genética , Perfilación de la Expresión Génica , Gliosis/metabolismo , Hipocampo/metabolismo , Redes y Vías Metabólicas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genéticaRESUMEN
With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.
Asunto(s)
Músculos/metabolismo , Progeria/genética , Células Madre/citología , Animales , Antígenos CD34/biosíntesis , Antígenos Ly/metabolismo , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Colágeno/metabolismo , Reparación del ADN , Modelos Animales de Enfermedad , Genotipo , Humanos , Longevidad , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación , Osteocitos/citología , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Progeria/patologíaRESUMEN
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.
Asunto(s)
Envejecimiento/fisiología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Endonucleasas/genética , Hígado/fisiopatología , Progeria/fisiopatología , Envejecimiento/patología , Animales , Senescencia Celular , Reparación del ADN , Perfilación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Ratones , Estrés Oxidativo , Progeria/genética , Progeria/metabolismo , Progeria/patologíaRESUMEN
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.
Asunto(s)
Trastornos por Deficiencias en la Reparación del ADN/genética , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Animales , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Daño del ADN , Trastornos por Deficiencias en la Reparación del ADN/metabolismo , Trastornos por Deficiencias en la Reparación del ADN/fisiopatología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Regulación de la Expresión Génica , Genotipo , Humanos , Ratones , Ratones Noqueados , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Enfermedades Raras/genética , Enfermedades Raras/metabolismo , Enfermedades Raras/fisiopatología , Telómero/genética , Telómero/metabolismo , Rayos UltravioletaRESUMEN
Peripheral neuropathy is a common aging-related degenerative disorder that interferes with daily activities and leads to increased risk of falls and injury in the elderly. The etiology of most aging-related peripheral neuropathy is unknown. Inherited defects in several genome maintenance mechanisms cause tissue-specific accelerated aging, including neurodegeneration. We tested the hypothesis that a murine model of XFE progeroid syndrome, caused by reduced expression of ERCC1-XPF DNA repair endonuclease, develops peripheral neuropathy. Nerve conduction studies revealed normal nerve function in young adult (8 week) Ercc1(-/Δ) mice, but significant abnormalities in 20 week-old animals. Morphologic and ultrastructural analysis of the sciatic nerve from mutant mice revealed significant alterations at 20 but not 8 weeks of age. We conclude that Ercc1(-/Δ) mice have accelerated spontaneous peripheral neurodegeneration that mimics aging-related disease. This provides strong evidence that DNA damage can drive peripheral neuropathy and offers a rapid and novel model to test therapies.
Asunto(s)
Envejecimiento/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Endonucleasas/metabolismo , Enfermedades del Sistema Nervioso Periférico/enzimología , Progeria/enzimología , Envejecimiento/genética , Envejecimiento/patología , Animales , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Masculino , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/patología , Progeria/genética , Progeria/patologíaRESUMEN
Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1-XPF (Ercc1(-/Δ) mice). The ERCC1-deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild-type littermates, Ercc1(-/Δ) mice also exhibit other age-related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age-associated disc pathologies was further accelerated in Ercc1(-/Δ) mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1(-/Δ) mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis.