Senescence, regulators of alternative splicing and effects of trametinib treatment in progeroid syndromes.

Progeroid syndromes such as Hutchinson Gilford Progeroid syndrome (HGPS), Werner syndrome (WS) and Cockayne syndrome (CS), result in severely reduced lifespans and premature ageing. Normal senescent cells show splicing factor dysregulation, which has not yet been investigated in syndromic senescent cells. We sought to investigate the senescence characteristics and splicing factor expression profiles of progeroid dermal fibroblasts. Natural cellular senescence can be reversed by application of the senomorphic drug, trametinib, so we also investigated its ability to reverse senescence characteristics in syndromic cells. We found that progeroid cultures had a higher senescence burden, but did not always have differences in levels of proliferation, DNA damage repair and apoptosis. Splicing factor gene expression appeared dysregulated across the three syndromes. 10 µM trametinib reduced senescent cell load and affected other aspects of the senescence phenotype (including splicing factor expression) in HGPS and Cockayne syndromes. Werner syndrome cells did not demonstrate changes in in senescence following treatment. Splicing factor dysregulation in progeroid cells provides further evidence to support this mechanism as a hallmark of cellular ageing and highlights the use of progeroid syndrome cells in the research of ageing and age-related disease. This study suggests that senomorphic drugs such as trametinib could be a useful adjunct to therapy for progeroid diseases.

Chemical screen identifies a geroprotective role of quercetin in premature aging.

Aging increases the risk of various diseases. The main goal of aging research is to find therapies that attenuate aging and alleviate aging-related diseases. In this study, we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established. Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects. Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs. RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C. Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs. Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase (WRN). Mice lacking part of the helicase domain of the WRN ortholog exhibit several phenotypic features of WS. In this study, we generated a Wrn mutant line that, like humans, relies entirely on dietary sources of vitamin C (ascorbate) to survive, by crossing them to mice that lack the gulonolactone oxidase enzyme required for ascorbate synthesis. In the presence of 0.01% ascorbate (w/v) in drinking water, double-mutant mice exhibited a severe reduction in lifespan, small size, sterility, osteopenia, and metabolic profiles different from wild-type (WT) mice. Although increasing the dose of ascorbate to 0.4% improved dramatically the phenotypes of double-mutant mice, the metabolic and cytokine profiles were different from age-matched WT mice. Finally, double-mutant mice treated with 0.01% ascorbate revealed a permanent activation of all the 3 branches of the ER stress response pathways due to a severe chronic oxidative stress in the ER compartment. In addition, markers associated with the ubiquitin-proteasome-dependent ER-associated degradation pathway were increased. Augmenting the dose of ascorbate reversed the activation of this pathway to WT levels rendering this pathway a potential therapeutic target in WS.-Aumailley, L., Dubois, M. J., Brennan, T. A., Garand, C., Paquet, E. R., Pignolo, R. J., Marette, A., Lebel, M. Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

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 WRN-deficient 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.

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells.

Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.

Impact of vitamin C on the cardiometabolic and inflammatory profiles of mice lacking a functional Werner syndrome protein helicase.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a DNA helicase/exonuclease. Mice lacking the helicase domain of this protein exhibit metabolic abnormalities that are reversed by vitamin C. In this study, we used a targeted metabolomic approach to identify serum metabolites significantly altered in young mutant mice treated with or without vitamin C. We also measured several serum inflammatory and cardiometabolic factors. We show that young mutant mice exhibit an increase in serum hydroxyproline and plasminogen activator inhibitor-1 (PAI-1), markers of cardiovascular diseases and inflammation, before they exhibit morphological anomalies in different tissues. We also observed an increase in three very long chain lysophosphatidylcholines underlying peroxisome perturbation. Vitamin C reversed the concentrations of these metabolites and PAI-1 to wild type values. Transcriptomic analyses on the liver of mutant mice revealed a decrease in the expression of genes involved in fatty acid degradation compared to wild type animals. Vitamin C treatment increased the expression of genes involved in glutathione metabolism and the synthesis of unsaturated fatty acids in these mice. These results show that changes at the transcriptomic level concord with the alterations of several serum metabolites in these mice. Finally, we found that a mislocalization of the Wrn mutant protein in the liver endoplasmic reticulum fraction increased oxidative stress in that cellular compartment. Vitamin C reversed this oxidative stress. To conclude, this study provides novel potential predictive cardiometabolic biomarkers in WS that will allow the assessment of the impact of vitamin C on patients with WS.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies.

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality.

The Drosophila orthologue of progeroid human WRN exonuclease, DmWRNexo, cleaves replication substrates but is inhibited by uracil or abasic sites : analysis of DmWRNexo activity in vitro.

Werner syndrome (WS) is a rare late-onset premature ageing disease showing many of the phenotypes associated with normal ageing, and provides one of the best models for investigating cellular pathways that lead to normal ageing. WS is caused by mutation of WRN, which encodes a multifunctional DNA replication and repair helicase/exonuclease. To investigate the role of WRN protein's unique exonuclease domain, we have recently identified DmWRNexo, the fly orthologue of the exonuclease domain of human WRN. Here, we fully characterise DmWRNexo exonuclease activity in vitro, confirming 3'-5' polarity, demonstrating a requirement for Mg(2+), inhibition by ATP, and an ability to degrade both single-stranded DNA and duplex DNA substrates with 3' or 5' overhangs, or bubble structures, but with no activity on blunt ended DNA duplexes. We report a novel active site mutation that ablates enzyme activity. Lesional substrates containing uracil are partially cleaved by DmWRNexo, but the enzyme pauses on such substrates and is inhibited by abasic sites. These strong biochemical similarities to human WRN suggest that Drosophila can provide a valuable experimental system for analysing the importance of WRN exonuclease in cell and organismal ageing.

Werner's syndrome may be lost in the shadow of the scleroderma.

We describe three patients with Werner's syndrome (WS), two of whom had been mistakenly diagnosed as having scleroderma. We would like to discuss briefly the importance of differentiation of these two disorders from each other.

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Resveratrol improves insulin resistance hyperglycemia and hepatosteatosis but not hypertriglyceridemia, inflammation, and life span in a mouse model for Werner syndrome.

Werner syndrome is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many features of Werner syndrome, including a pro-oxidant status and a shorter mean life span. Here, we show that resveratrol supplementation improved the hyperglycemia and the insulin resistance phenotype in these Wrn mutant mice. In addition, resveratrol reversed liver steatosis, lipid peroxidaton, and the defenestration phenotypes observed in such mice. Resveratrol, however, did not improve the hypertriglyceridemia, inflammatory stress, nor extend the mean life span of these mutant mice. Microarray and biologic pathway enrichment analyses on liver tissues revealed that resveratrol mainly decreased lipidogenesis and increased genes involved in the insulin signaling pathway and the glutathione metabolism in Wrn mutant mice. Finally, resveratrol-treated mutant mice exhibited an increase in the frequency of lymphoma and of several solid tumors. These results indicate that resveratrol supplementation might exert at least metabolic benefits for Werner syndrome patients.

Estrogen prevents senescence through induction of WRN, Werner syndrome protein.

Werner syndrome is a well-known human progeria. It has been revealed that loss of human WRN is a causal factor of this disease. Since pathological features of Werner syndrome resemble those of menopausal women and become apparent during puberty, we examined the effect of estrogen on WRN gene expression. Here, we reveal that WRN is induced by estrogen but not testosterone. Treatment with estrogen can induce WRN expression at the transcription and translation level in a human breast cell line. Forced expression of the estrogen receptor can restore the responsiveness of WRN to estrogen in a non-responsive cell line. Treatment with estrogen can block DNA damage-induced senescence. Moreover, WRN is suppressed by ATR that is activated by DNA damage, whereas WRN can be induced by ATR elimination. Our results suggest that WRN is essential for prevention of senescence. In addition, our results imply that the reduction of WRN in menopause could be an important factor for menopausal syndrome.

Gram-scale synthesis of the p38α MAPK-inhibitor VX-745 for preclinical studies into Werner syndrome.

BACKGROUND: The ATP-competitive p38α MAPK inhibitor VX-745 exhibits an exquisite kinase selectivity profile, is effective in blocking p38 stress signaling in Werner syndrome dermal fibroblasts, has efficacy in clinical trials and may have therapeutic value against Werner syndrome. Previous synthetic routes, however, have only resulted in milligram quantities suitable for cell-based studies, whereas gram quantities would be required for in vivo use. RESULTS & DISCUSSION: Microwave irradiation using a stop-flow monomodal microwave reactor has been found to facilitate scale-up of the synthesis of VX-745. Ullmann-type C-S bond formation using thiophenol, chloropyridazine, copper(I) catalyst and diol ligand proceeds rapidly and efficiently in this apparatus for elaboration to the pyrimido[1,6-b]pyridazinone core of VX-745 on gram scale and with good overall yield. CONCLUSION: This method delivers the p38 inhibitor VX-745 in sufficient quantities for preclinical studies to rescue the aging phenotype in Werner syndrome.

Vitamin C restores healthy aging in a mouse model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.

Werner's syndrome: a quite rare disease for differential diagnosis of scleroderma.

Werner's syndrome (WS) is an autosomal recessive disorder characterized by premature aging. The main features of the disease are scleroderma-like skin appearance, premature atherosclerosis, short stature, diabetes mellitus, early osteoporosis and early aging. Herein, we describe a patient with WS, who has scleroderma-like skin changes and discuss the literature about WS as a disease in the differential diagnosis of systemic sclerosis.