RESUMO
Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.
Assuntos
Lesão Pulmonar Aguda/imunologia , Tetracloreto de Carbono/efeitos adversos , Neutrófilos/citologia , Espécies Reativas de Oxigênio/metabolismo , Encurtamento do Telômero , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Animais , Linhagem Celular , Senescência Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Neutrófilos/metabolismo , Estresse Oxidativo , Comunicação ParácrinaRESUMO
Telomerase, consisting of the protein subunit telomerase reverse transcriptase (TERT) and RNA component TERC, is best known for maintaining and extending human telomeres, the ends of linear chromosomes, in tissues, where it is active, such as stem cells, germline cells, lymphocytes and endothelial cells. This function is considered as canonical. However, various non-canonical functions for the protein part TERT have been discovered. There are multiple such roles which can interfere with several signaling pathways, cancer development and many other processes. One of these non-canonical functions includes shuttling of the TERT protein out of the nucleus upon increased oxidative stress into the cytoplasm and organelles such as mitochondria. Mitochondrial TERT is able to protect cells from oxidative stress, DNA damage and apoptosis although the exact mechanisms are incompletely understood. Recently, a protective role for TERT was described in brain neurons. Here TERT is able to counteract effects of toxic neurodegenerative proteins via changes in gene expression, activation of neurotrophic factors as well as activation of protein degrading pathways such as autophagy. Protein degradation processes are prominently involved in degrading toxic proteins in the brain like amyloid-ß, pathological tau and α-synuclein that are responsible for various neurodegenerative diseases. These new findings can have implications for the development of novel treatment strategies for neurodegenerative diseases. The current review summarizes our knowledge on the role of the telomerase protein TERT in brain function, in particular, under the aspect of age-related neurodegenerative diseases. It also describes various strategies to increase TERT levels in the brain.
Assuntos
Doenças Neurodegenerativas , Telomerase , Humanos , Doenças Neurodegenerativas/genética , Telomerase/metabolismo , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Estresse Oxidativo , Telômero/metabolismoRESUMO
Seventeen papers published in 2019 and early 2020 demonstrate the ongoing interest and research concerning telomeres and telomerase in aging and cancer [...].
Assuntos
Neoplasias , Telomerase , Humanos , Telomerase/genética , Telomerase/metabolismo , Envelhecimento/genética , Telômero/genética , Telômero/metabolismo , Neoplasias/genéticaRESUMO
Due to their close connection with senescence, aging, and disease, telomeres and telomerase provide a unique and vital research route for boosting longevity and health span. Despite significant advances during the last three decades, earlier studies into these two biological players were impeded by the difficulty of achieving real-time changes inside living cells. As a result of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated system's (Cas) method, targeted genetic studies are now underway to change telomerase, the genes that govern it as well as telomeres. This review will discuss studies that have utilized CRISPR-related technologies to target and modify genes relevant to telomeres and telomerase as well as to develop targeted anti-cancer therapies. These studies greatly improve our knowledge and understanding of cellular and molecular mechanisms that underlie cancer development and aging.
Assuntos
Neoplasias , Telomerase , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Neoplasias/genética , Neoplasias/terapia , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Background: Iron-overload is a well-known cause for the development of chronic liver diseases and known to induce DNA damage.Material and methods: The protective effect of argan oil (AO) from the Argania spinosa fruit and olive oil (OO) (6% AO or OO for 28 days) was evaluated on a mouse model of iron overload (3.5mg Fe2+/liter) and in human fibroblasts where DNA damage was induced via culture under hyperoxia (40% oxygen).Results: Iron treatment induced DNA damage in liver tissue while both oils were able to decrease it. We confirmed this effect in vitro in MRC-5 fibroblasts under hyperoxia. A cell-free ABTS assay suggested that improvement of liver toxicity by both oils might depend on a high content in tocopherol, phytosterol and polyphenol compounds known for their antioxidant potential. The antioxidant effect of AO was confirmed in fibroblasts by reduced intracellular peroxide levels after hyperoxia. However, we could not find a significant decrease of genes encoding pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, COX-2) or senescence markers (p16 and p21) for the oils in mouse liver.Conclusion: We found a striking effect of AO by ameliorating DNA damage after iron overload in a mouse liver model and in human fibroblasts by hyperoxia adding compelling evidence to the protective mechanisms of AO and OO.
Assuntos
Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Sobrecarga de Ferro/tratamento farmacológico , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Óleos de Plantas/farmacologia , Animais , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Mediadores da Inflamação/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Azeite de Oliva/farmacologiaRESUMO
Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1ß-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1ß deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.
Assuntos
Envelhecimento/fisiologia , Mitocôndrias/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , FenótipoRESUMO
Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = -0.35, p = 0.03 and r = -0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.
Assuntos
Carcinogênese/metabolismo , Cromossomos Humanos/metabolismo , Neoplasias do Endométrio/metabolismo , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Telômero/metabolismo , Transcrição Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Carcinogênese/patologia , Cromossomos Humanos/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Telômero/genética , Telômero/patologia , Homeostase do TelômeroRESUMO
OBJECTIVE: Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. APPROACH AND RESULTS: In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. CONCLUSIONS: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.
Assuntos
Aterosclerose/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Proliferação de Células , Ativação Linfocitária , Linfócitos T Reguladores/enzimologia , Telomerase/metabolismo , Transferência Adotiva , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Knockout , Camundongos Knockout para ApoE , Estresse Oxidativo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Telomerase/deficiência , Telomerase/genética , Homeostase do TelômeroRESUMO
Telomeres are specialised structures at the end of linear chromosomes. They consist of tandem repeats of the hexanucleotide sequence TTAGGG, as well as a protein complex called shelterin. Together, they form a protective loop structure against chromosome fusion and degradation. Shortening or damage to telomeres and opening of the loop induce an uncapped state that triggers a DNA damage response resulting in senescence or apoptosis.Average telomere length, usually measured in human blood lymphocytes, was thought to be a biomarker for ageing, survival and mortality. However, it becomes obvious that regulation of telomere length is very complex and involves multiple processes. For example, the "end replication problem" during DNA replication as well as oxidative stress are responsible for the shortening of telomeres. In contrast, telomerase activity can potentially counteract telomere shortening when it is able to access and interact with telomeres. However, while highly active during development and in cancer cells, the enzyme is down-regulated in most human somatic cells with a few exceptions such as human lymphocytes. In addition, telomeres can be transcribed, and the transcription products called TERRA are involved in telomere length regulation.Thus, telomere length and their integrity are regulated at many different levels, and we only start to understand this process under conditions of increased oxidative stress, inflammation and during diseases as well as the ageing process.This chapter aims to describe our current state of knowledge on telomeres and telomerase and their regulation in order to better understand their role for the ageing process.
Assuntos
Envelhecimento/genética , Telomerase , Telômero , Replicação do DNA , Humanos , Estresse Oxidativo , Telomerase/metabolismoRESUMO
Telomerase is best known for its function in maintaining telomeres but has also multiple additional, non-canonical functions. One of these functions is the decrease of oxidative stress and DNA damage due to localisation of the telomerase protein TERT into mitochondria under oxidative stress. However, the exact molecular mechanisms behind these protective effects are still not well understood. We had shown previously that overexpression of human telomerase reverse transcriptase (hTERT) in human fibroblasts results in a decrease of mitochondrial DNA (mtDNA) damage after oxidative stress. MtDNA damage caused by oxidative stress is removed via the base excision repair (BER) pathway. Therefore we aimed to analyse whether telomerase is able to improve this pathway. We applied different types of DNA damaging agents such as irradiation, arsenite treatment (NaAsO2) and treatment with hydrogen peroxide (H2O2). Using a PCR-based assay to evaluate mtDNA damage, we demonstrate that overexpression of hTERT in MRC-5 fibroblasts protects mtDNA from H2O2 and NaAsO2 induced damage, compared with their isogenic telomerase-negative counterparts. However, overexpression of hTERT did not seem to increase repair of mtDNA after oxidative stress, but promoted increased levels of manganese superoxide dismutase (MnSOD) and forkhead-box-protein O3 (FoxO3a) proteins during incubation in serum free medium as well as under oxidative stress, while no differences were found in protein levels of catalase. Together, our results suggest that rather than interfering with mitochondrial DNA repair mechanisms, such as BER, telomerase seems to increase antioxidant defence mechanisms to prevent mtDNA damage and to increase cellular resistance to oxidative stress. However, the result has to be reproduced in additional cellular systems in order to generalise our findings.
Assuntos
Meios de Cultura Livres de Soro/química , Mitocôndrias/genética , Superóxido Dismutase/genética , Telomerase/genética , Arsenitos/efeitos adversos , Células Cultivadas , Reparo do DNA , DNA Mitocondrial/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Peróxido de Hidrogênio/efeitos adversos , Mitocôndrias/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Telomerase/metabolismo , Raios Ultravioleta/efeitos adversos , Regulação para CimaRESUMO
Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Macular/terapia , Raios Ultravioleta , Terapia Ultravioleta/métodos , Idoso , Animais , Fator H do Complemento/metabolismo , Humanos , Degeneração Macular/etiologia , Camundongos , Camundongos SCIDRESUMO
The telomerase reverse transcriptase protein TERT has recently been demonstrated to have a variety of functions both in vitro and in vivo, which are distinct from its canonical role in telomere extension. In different cellular systems, TERT protein has been shown to be protective through its interaction with mitochondria. TERT has previously been found in rodent neurons, and we hypothesize that it might have a protective function in adult human brain. Here, we investigated the expression of TERT at different stages of Alzheimer's disease pathology (Braak Stages I-VI) in situ and the ability of TERT to protect against oxidative damage in an in vitro model of tau pathology. Our data reveal that TERT is expressed in vitro in mouse neurons and microglia, and in vivo in the cytoplasm of mature human hippocampal neurons and activated microglia, but is absent from astrocytes. Intriguingly, hippocampal neurons expressing TERT did not contain hyperphosphorylated tau. Vice versa, neurons that expressed high levels of pathological tau did not appear to express TERT protein. TERT protein colocalized with mitochondria in the hippocampus of Alzheimer's disease brains (Braak Stage VI), as well as in cultured neurons under conditions of oxidative stress. Our in vitro data suggest that the absence of TERT increases ROS generation and oxidative damage in neurons induced by pathological tau. Together, our findings suggest that TERT protein persists in neurons of the adult human brain, where it may have a protective role against tau pathology.
Assuntos
Doença de Alzheimer/patologia , Hipocampo/metabolismo , Tauopatias/patologia , Telomerase/metabolismo , Adolescente , Adulto , Doença de Alzheimer/genética , Análise de Variância , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Embrião de Mamíferos , Feminino , Humanos , Peroxidação de Lipídeos/genética , Fígado/patologia , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mudanças Depois da Morte , Telomerase/genética , Adulto JovemRESUMO
BACKGROUND: Aging and obesity are associated with raised oxidative stress and a reduction of nitric oxide (NO) bioavailability, with subsequent decline in insulin sensitivity and endothelial function. Inorganic nitrate is converted into NO via a 2-step reduction process and may be an effective nutritional intervention to modify vascular and metabolic functions. OBJECTIVES: This study tested whether inorganic nitrate supplementation improved glucose disposal and attenuated the acute effects of hyperglycemia on oxidative stress, inflammation, and vascular function in young and old obese participants. METHODS: Ten young (aged 18-44 y) and 10 old (aged 55-70 y) obese participants consumed 75 g glucose followed by either potassium nitrate (7 mg/kg body weight) or potassium chloride (placebo) in a randomized, double-blind crossover design. Resting blood pressure (BP), endothelial function, and blood biomarkers were measured for 3 h postintervention. Biomarkers included plasma nitrate/nitrite (NOx), glucose, insulin, cyclic GMP, interleukin 6, 3-nitrotyrosine, E- and P-selectins, intercellular adhesion molecule 3 (ICAM-3), and thrombomodulin, as well as superoxide in freshly isolated peripheral blood mononuclear cells (PBMCs). RESULTS: Inorganic nitrate supplementation did not affect plasma glucose (P = 0.18) or insulin (P = 0.26) responses. The increase in plasma NOx concentrations 3 h after the administration of inorganic nitrate was significantly higher in young than in old participants (234% increase compared with 149% increase, respectively, P < 0.001). Plasma 3-nitrotyrosine concentrations declined significantly after inorganic nitrate supplementation compared with placebo (3 h postdose, 46% decrease compared with 27% increase, respectively, P = 0.04), and a similar nonsignificant trend was observed for superoxide concentrations (3 h postdose, 16% decrease compared with 23% increase, respectively, P = 0.06). Plasma cyclic GMP, ICAM-3, and thrombomodulin concentrations differed between young and old participants (P < 0.01). Inorganic nitrate supplementation did not improve BP or endothelial function. CONCLUSIONS: Oral supplementation with inorganic nitrate did not improve glucose and insulin responses but reduced oxidative stress in old individuals during acute hyperglycemia. This trial was registered at www.controlled-trials.com as ISRCTN42776917.
Assuntos
Envelhecimento , Glicemia/efeitos dos fármacos , Insulina/sangue , Nitratos/farmacologia , Obesidade/metabolismo , Compostos de Potássio/farmacologia , Adolescente , Adulto , Idoso , Biomarcadores , Glicemia/metabolismo , Feminino , Humanos , Inflamação , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Nitratos/administração & dosagem , Estresse Oxidativo , Cloreto de Potássio/administração & dosagem , Cloreto de Potássio/farmacologia , Compostos de Potássio/administração & dosagem , Adulto JovemRESUMO
Fanconi anemia (FA) is a genomic instability disorder caused by mutations in genes involved in replication-dependant-repair and removal of DNA cross-links. Mouse models with targeted deletions of FA genes have been developed; however, none of these exhibit the human bone marrow aplasia. Human embryonic stem cell (hESC) differentiation recapitulates many steps of embryonic hematopoietic development and is a useful model system to investigate the early events of hematopoietic progenitor specification. It is now possible to derive patient-specific human-induced pluripotent stem cells (hiPSC); however, this approach has been rather difficult to achieve in FA cells due to a requirement for activation of FA pathway during reprogramming process which can be bypassed either by genetic complementation or reprogramming under hypoxic conditions. In this study, we report that FA-C patient-specific hiPSC lines can be derived under normoxic conditions, albeit at much reduced efficiency. These disease-specific hiPSC lines and hESC with stable knockdown of FANCC display all the in vitro hallmarks of pluripotency. Nevertheless, the disease-specific hiPSCs show a much higher frequency of chromosomal abnormalities compared to parent fibroblasts and are unable to generate teratoma composed of all three germ layers in vivo, likely due to increased genomic instability. Both FANCC-deficient hESC and hiPSC lines are capable of undergoing hematopoietic differentiation, but the hematopoietic progenitors display an increased apoptosis in culture and reduced clonogenic potential. Together these data highlight the critical requirement for FA proteins in survival of hematopoietic progenitors, cellular reprogramming, and maintenance of genomic stability.
Assuntos
Reprogramação Celular/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Diferenciação Celular/fisiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Terapia Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Cernunnos (also known as XLF) deficiency syndrome is a rare recessive autosomal disorder caused by mutations in the XLF gene, a key factor involved in the end joining step of DNA during nonhomologous end joining (NHEJ) process. Human patients with XLF mutations display microcephaly, developmental and growth delays, and severe immunodeficiency. While the clinical phenotype of DNA damage disorders, including XLF Syndrome, has been described extensively, the underlying mechanisms of disease onset, are as yet, undefined. We have been able to generate an induced pluripotent stem cell (iPSC) model of XLF deficiency, which accurately replicates the double-strand break repair deficiency observed in XLF patients. XLF patient-specific iPSCs (XLF-iPSC) show typical expression of pluripotency markers, but have altered in vitro differentiation capacity and an inability to generate teratomas comprised of all three germ layers in vivo. Our results demonstrate that XLF-iPSCs possess a weak NHEJ-mediated DNA repair capacity that is incapable of coping with the DNA lesions introduced by physiological stress, normal metabolism, and ionizing radiation. XLF-iPSC lines are capable of hematopoietic differentiation; however, the more primitive subsets of hematopoietic progenitors display increased apoptosis in culture and an inability to repair DNA damage. Together, our findings highlight the importance of NHEJ-mediated-DNA repair in the maintenance of a pristine pool of hematopoietic progenitors during human embryonic development.
Assuntos
Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Dados de Sequência MolecularRESUMO
Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.
Assuntos
Telomerase , Telomerase/genética , Telomerase/análise , Telomerase/metabolismo , Telômero/química , Telômero/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox) ) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho) (x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Doença Granulomatosa Crônica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Diferenciação Celular , Linhagem Celular , Citocinas/metabolismo , Humanos , Cariotipagem , Cinética , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The DNA damage response (DDR) orchestrates the recruitment of repair proteins at sites of damage and arrests cell-cycle progression until completion of repair. Upon irreparable damage, DNA damage foci persist (long-lived foci) and this is believed to induce cellular senescence. The resolution of DNA damage foci has previously been shown to depend on proteasomal degradation and various proteasome subunits have been implicated in the DDR. In this study, we aimed to analyze the possible distinct roles of individual proteasome subunits in the DDR. We show that specific 19S subunits respond to DNA damage by increased protein levels and nuclear translocation. Importantly, two 19S subunits, Rpn7 and Rpn11, colocalize with DNA damage foci over their whole lifespan. Although silencing of Rpn11 does not affect foci stability and lifespan, silencing of Rpn7 promotes faster resolution of DNA damage foci following genotoxic insult. For the first time, we provide evidence that Rpn7 silencing specifically decreases the frequencies of long-lived DNA damage foci without, however, affecting the repair rate of short-lived foci. Therefore, we propose that interaction of Rpn7 with DDR foci in situ mediates the protection of DNA damage foci from premature resolution. We suggest that this interaction is involved in enabling cellular senescence following genotoxic insult.
Assuntos
Dano ao DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Senescência Celular , Inibidores de Cisteína Proteinase/farmacologia , Reparo do DNA , Etoposídeo , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio , Leupeptinas/farmacologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/genética , Transporte Proteico , Interferência de RNA , Regulação para CimaRESUMO
Most people associate the enzyme telomerase with its role in maintaining telomeres, which is its best-known canonical role. For this important function, two main components are required: the protein telomerase reverse transcriptase (TERT) and the telomerase RNA component. In addition, over the last decades, an ever-growing number of other, non-telomeric, non-canonical functions for the telomerase protein TERT has been established. These reach from tumor promotion to decreasing oxidative stress and apoptosis as well as activating autophagy. These functions are more and more recognized as being important in many tissues and physiological as well as pathological conditions. The role of telomerase in brain development and neuronal cells has been investigated for more than 20 years. However, the non-telomeric role in non-dividing neurons of the brain for telomerase and the TERT-protein has only recently been highlighted by extensive research. Moreover, these developments promoted the suggestion of a beneficial and protective role of TERT against neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review summarizes the most important findings in the field of telomerase in neurons and gives an outlook onto possible therapeutic applications of boosting telomerase/TERT levels with telomerase activators to prevent or ameliorate various neurodegenerative diseases.