Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNAmutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.     

Accelerated methylation of ribosomal RNA genes during the cellular senescence of Werner syndrome fibroblasts.

Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.     

Asymmetry of DNA replication fork progression in Werner's syndrome

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.     

综述: 人类早衰症的发病机制及干预方法

衰老是一种在细胞和组织水平逐渐发生功能衰退的过程.早衰症是一类罕见的人类遗传性疾病,以加速衰老为特征.对早衰症的研究有助于理解人类衰老的生理过程,对衰老相关疾病的防治具有借鉴意义.成人早衰症和儿童早衰症是两种著名的人类早衰症,本文将综述这两种早衰症的发病机制及干预方法.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

ATM kinase enables the functional axis of YAP,PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.     

Hypothesis: Werner syndrome and biological ageing: A molecular genetic hypothesis

Werner syndrome (WS) is an inherited disorder that produces somatic stunting, premature ageing and early onset of degenerative and neoplastic diseases. Cultured fibroblasts derived from subjects with WS are found to undergo premature replicative senescence and thus provide a cellular model system to study the disorder. Recently, several overexpressed gene sequences isolated from a WS fibroblast cDNA library have been shown to possess the capacity to inhibit DNA synthesis and disrupt many normal biochemical processes. Because a similar constellation of genes is overexpressed in WS and senescent normal fibroblasts, these data suggest the existence of a common molecular genetic pathway for replicative senescence in both types of cell. We propose that the primary defect in WS is a mutation in a gene for a trans-acting repressor protein that reduces its binding affinity for shared regulatory regions of several genes, including those that encode inhibitors of DNA synthesis (IDS). The mutant WS repressor triggers a sequence of premature expression of IDS and other genes, with resulting inhibition of DNA synthesis and early cellular senescence, events which occur much later in normal cells.     

miR‐10a restores human mesenchymal stem cell differentiation by repressing KLF4

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging‐related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR‐10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR‐10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up‐ or downregulate miR‐10a in young and old hMSCs. Upregulation of miR‐10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR‐10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full‐length 3′‐UTR region of KLF4 harboring the seed‐matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR‐10a mimic into cells. The luciferase activity was significantly repressed by the miR‐10a mimic, proving the direct binding of miR‐10a to the 3′‐UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR‐10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging‐related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging. J. Cell. Physiol. 228: 2324–2336, 2013. © The Authors. Published by Wiley Periodicals, Inc.     

Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome

DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases.     

Vitamin C alleviates aging defects in a stem cell model for Werner syndrome

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRNdeficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.     

Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products

Although immunological methods are widely used to diagnose various infectious diseases, they have rarely been employed to detect genetic diseases. In this study, we have established an immunoblot analysis system for the diagnosis of Werner syndrome (WS), a recessive genetic disorder causing premature aging and an enhanced risk of rare cancers. The method uses an immunoblot technique with specific monoclonal antibodies to WS gene product, and B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus; these cell lines express an increased level of normal WS gene product DNA helicase. The method clearly distinguishes normal from patient LCLs containing any of the mutation types found so far in Japan, primarily because of the drastically reduced levels of mutated gene products, and secondarily because of the truncated product sizes. A comparison of this immunological diagnosis with the symptom-based clinical diagnosis has narrowed down the criteria of symptoms essential for WS diagnosis. This procedure is compatible with, and has some advantage over, the genetic method, because WS patients can be diagnosed without determining the mutated gene sequences. The method exemplified in WS may also be applied to detect some other genetic diseases.     

Calorie restriction reduces biomarkers of cellular senescence in humans

Calorie restriction (CR) with adequate nutrient intake is a potential geroprotective intervention. To advance this concept in humans, we tested the hypothesis that moderate CR in healthy young-to-middle-aged individuals would reduce circulating biomarkers of cellular senescence, a fundamental mechanism of aging and aging-related conditions. Using plasma specimens from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) phase 2 study, we found that CR significantly reduced the concentrations of several senescence biomarkers at 12 and 24 months compared to an ad libitum diet. Using machine learning, changes in biomarker concentrations emerged as important predictors of the change in HOMA-IR and insulin sensitivity index at 12 and 24 months, and the change in resting metabolic rate residual at 12 months. Finally, using adipose tissue RNA-sequencing data from a subset of participants, we observed a significant reduction in a senescence-focused gene set in response to CR at both 12 and 24 months compared to baseline. Our results advance the understanding of the effects of CR in humans and further support a link between cellular senescence and metabolic health.     

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.     

Mutations in the WRN gene in mice accelerate mortality in a p53-null background

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.     

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.     

A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.