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Hair follicles, mammalian mini-organs that grow hair, miniaturize during aging, leading to hair thinning and loss. Here we report that hair follicle stem cells (HFSCs) lose their regenerative capabilities during aging owing to the adoption of an atypical cell division program. Cell fate tracing and cell division axis analyses revealed that while HFSCs in young mice undergo typical symmetric and asymmetric cell divisions to regenerate hair follicles, upon aging or stress, they adopt an atypical ‘stress-responsive’ type of asymmetric cell division. This type of division is accompanied by the destabilization of hemidesmosomal protein COL17A1 and cell polarity protein aPKCλ and generates terminally differentiating epidermal cells instead of regenerating the hair follicle niche. With the repetition of these atypical divisions, HFSCs detach from the basal membrane causing their exhaustion, elimination and organ aging. The experimentally induced stabilization of COL17A1 rescued organ homeostasis through aPKCλ stabilization. These results demonstrate that distinct stem cell division programs may govern tissue and organ aging. The authors identify an atypical type of stem cell division that regulates hair follicle organ homeostasis and aging in mice. These ‘stress-responsive-type’ asymmetrical cell divisions cause hair follicle stem cells to detach from the basement membrane leading to their exhaustion, elimination and organ aging.

BackgroundWerner syndrome (WS) is an autosomal recessive progeroid syndrome caused by variants inWRN. The International Registry of Werner Syndrome has identified biallelic pathogenic variants in 179/188 cases of classical WS. In the remaining nine cases, only one heterozygous pathogenic variant has been identified.MethodsTargeted long-read sequencing (T-LRS) on an Oxford Nanopore platform was used to search for a second pathogenic variant inWRN. Previously, T-LRS was successfully used to identify missing variants and analyse complex rearrangements.ResultsWe identified a second pathogenic variant in eight of nine unsolved WS cases. In five cases, T-LRS identified intronic splice variants that were confirmed by either RT-PCR or exon trapping to affect splicing; in one case, T-LRS identified a 339 kbp deletion, and in two cases, pathogenic missense variants. Phasing of long reads predicted all newly identified variants were on a different haplotype than the previously known variant. Finally, in one case, RT-PCR previously identified skipping of exon 20; however, T-LRS did not detect a pathogenic DNA sequence variant.ConclusionT-LRS is an effective method for identifying missing pathogenic variants. Although limitations with computational prediction algorithms can hinder the interpretation of variants, T-LRS is particularly effective in identifying intronic variants.

Adult progeria, Werner syndrome (WS), is an autosomal recessive disorder that develops accelerated aging-associated symptoms after puberty. Refractory skin ulcer of limbs, which is one of the symptoms specific to WS, is seriously painful and sometimes results in amputation. In recent years, cell therapy using mesenchymal stem cells (MSCs) has been attracting attention; however, the effect of WS-derived MSCs on skin ulcers is still unclear. In this study, we generated iPS cells from a patient with WS and a normal subject, differentiated them into MSCs (WS- and NM-iMSC, respectively), and performed cell therapy to a refractory skin ulcer mouse model. As a result, WS-iMSC recapitulated premature senescence phenotypes in vitro. Upon subcutaneous injection around the wounds of mice, WS-iMSC was significantly inferior in wound healing effect compared to NM-iMSC. Proteome and transcriptome analysis revealed altered expression of genes related to angiogenesis, inflammation, and proliferation in WS-iMSC with remarkable downregulation of VEGF, a potent angiogenic factor. In addition, simultaneous administration of recombinant human VEGF and WS-iMSC improved the wound healing effect in vivo. These results indicate that the expression of angiogenic factors is reduced in WS-iMSC, and its supplementation restores the wound healing ability. This finding may pave the way to develop the treatment of intractable skin ulcers of WS.

Stem cells underlie tissue homeostasis, but their dynamics during ageing—and the relevance of these dynamics to organ ageing—remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention. COL17A1-driven stem cell competition and symmetric cell divisions initially govern skin homeostasis, but the same mechanisms result in skin ageing later in life.

Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.

Werner syndrome (WS), also known as adult progeria, is characterized by accelerated aging symptoms from a young age. Patients with WS experience painful intractable skin ulcers with calcifications in their extremities, subcutaneous lipoatrophy, and sarcopenia. However, there is no significant abnormality in the trunk skin, where the subcutaneous fat relatively accumulates. The cause of such differences between the limbs and trunk is unknown. To investigate the underlying mechanism behind these phenomena, we established and analyzed dermal fibroblasts from the foot and trunk of two WS patients. As a result, WS foot-derived fibroblasts showed decreased proliferative potential compared to that from the trunk, which correlated with the telomere shortening. Transcriptome analysis showed increased expression of genes involved in osteogenesis in the foot fibroblasts, while adipogenic and chondrogenic genes were downregulated in comparison with the trunk. Consistent with these findings, the adipogenic and chondrogenic differentiation capacity was significantly decreased in the foot fibroblasts in vitro. On the other hand, the osteogenic potential was mutually maintained and comparable in the foot and trunk fibroblasts. These distinct phenotypes in the foot and trunk fibroblasts are consistent with the clinical symptoms of WS and may partially explain the underlying mechanism of this disease phenotype.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-β (TGF-β) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-β, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-β-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.

Objectives To determine recent trends in mutation patterns in the WRN gene, which cause Werner syndrome (WS), a rare, inheritable progeroid syndrome in Japan. Design Retrospective cohort. Setting Longitudinal survey of WS and literature search for case reports. Participants Individuals whose genetic testing their facilities had requested between 2009 and October 2016 (N = 67). Measurements A nationwide epidemiological study was conducted from 2009 to 2011 to improve understanding of the pathology of WS and develop therapeutic guidelines. Since 2009, Chiba University Hospital consecutively evaluated the WRN gene in 67 individuals throughout Japan who had requested genetic testing. A literature search was also conducted for case reports on Japanese WS reported since 1997. Results A definitive diagnosis of WS was confirmed genetically in 50 of 67 participants. Through the literature search, 16 individuals diagnosed genetically with WS were identified. Of these 66 individuals with WS, 42 were homozygous for a WRN mutation, and 21 were compound heterozygotes. One novel mutant allele was identified in an individual with the compound heterozygous genotype. The proportion of compound heterozygotes (31.8%) was significantly greater than reported previously (14.2%), indicating that the incidence of consanguineous marriage of parents has decreased. Conclusion The increased frequency of individuals with WS with the compound heterozygous genotype is a recent trend in Japan. A long-term follow-up study on WRN homozygotes and compound heterozygotes will allow the relationship between WRN genotype and clinical severity of WS to be evaluated in the future.

In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in the WRN DNA helicase. It is characterized by the graying and loss of hair, juvenile cataracts, sclerosis and ulceration of skin, insulin-resistant diabetes mellitus, dyslipidemia, abdominal adiposity, osteoporosis, atherosclerosis, and malignant neoplasm. Patients are usually diagnosed in their 30s or 40s, but the early pathophysiology of the syndrome is still not fully understood. Here we report a 29-year-old female patient who displayed cataracts, hair graying, and tendinous calcinosis. Her parents were first cousins. Interestingly, the patient lacked the metabolic signs typical for WS, including glucose intolerance, dyslipidemia, and visceral fat accumulation. A hyperinsulinemic response at 30 min was observed in an oral glucose tolerance test. Mutational analysis for the WRN gene revealed a homozygous nucleotide substitution 3190C>T in exon 24, resulting in a protein product with replacement of an arginine residue at position 573 by termination codon (Arg987Ter). The mutated WRN protein was unable to translocate into the nucleus in an in vitro cell assay. A WS patient with an Arg987Ter mutation has been previously reported in Switzerland, the present case is the first to be identified in Asia. This case demonstrates the early clinical features of WS and suggests that metabolic abnormality, including insulin resistance, is not an essential component of WS at disease onset. Moreover, a follow-up study of such case would be useful to understand how the various clinical symptoms in WS develop and progress over the years.

Accepted: 2000-06-19, 資料番号: SA1003763000

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in the WRN DNA helicase. It is characterized by the graying and loss of hair, juvenile cataracts, sclerosis and ulceration of skin, insulin-resistant diabetes mellitus, dyslipidemia, abdominal adiposity, osteoporosis, atherosclerosis, and malignant neoplasm. Patients are usually diagnosed in their 30s or 40s, but the early pathophysiology of the syndrome is still not fully understood. Here we report a 29-year-old female patient who displayed cataracts, hair graying, and tendinous calcinosis. Her parents were first cousins. Interestingly, the patient lacked the metabolic signs typical for WS, including glucose intolerance, dyslipidemia, and visceral fat accumulation. A hyperinsulinemic response at 30 min was observed in an oral glucose tolerance test. Mutational analysis for the WRN gene revealed a homozygous nucleotide substitution 3190CT in exon 24, resulting in a protein product with replacement of an arginine residue at position 573 by termination codon (Arg987Ter). The mutated WRN protein was unable to translocate into the nucleus in an in vitro cell assay. A WS patient with an Arg987Ter mutation has been previously reported in Switzerland, the present case is the first to be identified in Asia. This case demonstrates the early clinical features of WS and suggests that metabolic abnormality, including insulin resistance, is not an essential component of WS at disease onset. Moreover, a follow-up study of such case would be useful to understand how the various clinical symptoms in WS develop and progress over the years.

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in RecQ DNA helicase. Ectopic soft tissue calcification is one of the well known symptoms in WS. However, the prevalence, clinical outcome, and mechanism of such calcification remain to be elucidated. The clinical features and mechanism of ectopic calcification were examined in seven patients with WS whose diagnosis were confirmed by a genomic DNA analysis. X-ray examinations revealed subcutaneous calcification in 35 of 41 major joints (85.3%). The patients complained of dermal pain at 23 joints among 35 joints (65.7%) with calcification. Refractory skin ulcers were found at the area of the skin overlaying the calcification in 16 joints (45.7%). In contrast, no pain or ulcers were observed in the joints without calcification. The presence of ectopic calcification could not be explained by a systemic hormonal abnormality. Cultured fibroblasts from WS patients underwent spontaneous mineralization in vitro in the normal phosphate condition, and overexpressed Pit-1, a transmembrane type III Na-Pi cotransporter both at the mRNA and protein levels. Phosphonophormic acid, a specific inhibitor for Pit-1, inhibited mineralization in the WS fibroblasts. Both calcification and Pit-1 overexpression were detected in the skin of WS in situ. WS showed a high prevalence of ectopic calcification, which was associated with dermal pain and refractory skin ulcers. An overexpression of Pit-1 therefore seems to play a key role in the formation of soft tissue calcification in this syndrome.