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ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1's physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging., (© 2024. The Author(s).)

Recently, mutations in the RNA polymerase III subunit A (POLR3A) have been described as the cause of the neonatal progeria or Wiedemann-Rautenstrauch syndrome (WRS). POLR3A has important roles in transcription regulation of small RNAs, including tRNA, 5S rRNA, and 7SK rRNA. We aim to describe the cellular and molecular features of WRS fibroblasts. Cultures of primary fibroblasts from one WRS patient [monoallelic POLR3A variant c.3772_3773delCT (p.Leu1258Glyfs*12)] and one control patient were cultured in vitro. The mutation caused a decrease in the expression of wildtype POLR3A mRNA and POLR3A protein and a sharp increase in mutant protein expression. In addition, there was an increase in the nuclear localization of the mutant protein. These changes were associated with an increase in the number and area of nucleoli and to a high increase in the expression of pP53 and pH2AX. All these changes were associated with premature senescence. The present observations add to our understanding of the differences between Hutchinson-Gilford progeria syndrome and WRS and opens new alternatives to study cell senesce and human aging., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For systematic identification of transcription signatures of human cell aging, we carried out Weighted Gene Co-expression Network Analysis (WGCNA) with the RNA-sequencing data generated with young to old human dermal fibroblasts. By relating the modules to the donor's traits, we uncovered the natural aging- and premature aging disease-associated modules. The STRING functional association networks built with the core module memberships provided a systematic overview of genome-wide transcriptional changes upon aging. We validated the selected candidates via quantitative reverse transcription PCR (RT-qPCR) assay with young and aged human fibroblasts, and uncovered several genes involved in ECM, cell, and nuclear mechanics as a potential aging biomarker. Collectively, our study not only provides a snapshot of functional changes during human fibroblast aging but also presents potential aging markers that are relevant to cell mechanics.

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.

Cellular senescence is a hallmark of normal aging and aging-related syndromes, including the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), a rare genetic disorder caused by a single mutation in the LMNA gene that results in the constitutive expression of a truncated splicing mutant of lamin A known as progerin. Progerin accumulation leads to increased cellular stresses including unrepaired DNA damage, activation of the p53 signaling pathway and accelerated senescence. We previously established that the p53 isoforms ∆133p53 and p53β regulate senescence in normal human cells. However, their role in premature aging is unknown. Here we report that p53 isoforms are expressed in primary fibroblasts derived from HGPS patients, are associated with their accelerated senescence and that their manipulation can restore the replication capacity of HGPS fibroblasts. We found that in near-senescent HGPS fibroblasts, which exhibit low levels of ∆133p53 and high levels of p53β, restoration of Δ133p53 expression was sufficient to extend replicative lifespan and delay senescence, despite progerin levels and abnormal nuclear morphology remaining unchanged. Conversely, Δ133p53 depletion or p53β overexpression accelerated the onset of senescence in otherwise proliferative HGPS fibroblasts. Our data indicate that Δ133p53 exerts its role by modulating full-length p53 (FLp53) signaling to extend the replicative lifespan and promotes the repair of spontaneous progerin-induced DNA double-strand breaks (DSBs). We showed that Δ133p53 dominant-negative inhibition of FLp53 occurs directly at the p21/CDKN1A and miR-34a promoters, two p53 senescence-associated genes. In addition, Δ133p53 expression increased the expression of DNA repair RAD51, likely through upregulation of E2F1, a transcription factor that activates RAD51, to promote repair of DSBs. In summary, our data indicate that Δ133p53 modulates p53 signaling to repress progerin-induced early onset of senescence in HGPS cells. Therefore, restoration of ∆133p53 expression may be a novel therapeutic strategy to treat aging-associated phenotypes of HGPS in vivo.

LMNA encodes the A-type lamins that are part of the nuclear scaffold. Mutations in LMNA can cause a variety of disorders called laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), atypical Werner syndrome, and Emery-Dreifuss muscular dystrophy. Previous work has shown that treatment of HGPS cells with the mTOR inhibitor rapamycin or with the rapamycin analog everolimus corrects several of the phenotypes seen at the cellular level-at least in part by increasing autophagy and reducing the amount of progerin, the toxic form of lamin A that is overproduced in HGPS patients. Since other laminopathies also result in production of abnormal and potentially toxic lamin proteins, we hypothesized that everolimus would also be beneficial in those disorders. To test this, we applied everolimus to fibroblast cell lines from six laminopathy patients, each with a different mutation in LMNA Everolimus treatment increased proliferative ability and delayed senescence in all cell lines. In several cell lines, we observed that with treatment, there is a significant improvement in nuclear blebbing, which is a cellular hallmark of HGPS and other lamin disorders. These preclinical results suggest that everolimus might have clinical benefit for multiple laminopathy syndromes., Competing Interests: Conflict of interest statement: F.S.C. and T.W.G. are coauthors on a 2017 research article. They did not collaborate directly on this work., (Copyright © 2018 the Author(s). Published by PNAS.)

Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes. As a test case, cultured skin-fibroblast nuclei of individuals possessing LMNA splice-site mutation (c.357-2A>G), LMNA nonsense mutation (c.736 C>T, pQ246X) in exon 4, LMNA missense mutation (c.1003C>T, pR335W) in exon 6, Hutchinson-Gilford Progeria Syndrome, and no LMNA mutations were analyzed. For each cell type, the percentage of dysmorphic nuclei, and other morphological features such as average nuclear area and average eccentricity were obtained. Compared to blind observers, our procedure implemented in Matlab codes possessed similar accuracy to manual counting of dysmorphic nuclei while being significantly more consistent. The automatic quantification of nuclear defects revealed a correlation between in vitro results and age of patients for initial symptom onset. Our results demonstrate the method's utility in experimental studies of diseases affecting nuclear shape through automated, unbiased, and accurate identification of dysmorphic nuclei.

Ideally, disease modeling using patient-derived induced pluripotent stem cells (iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome-wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient-derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke. The most common mutation in HGPS is at position G608G (GGC>GGT) within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl-terminal tail of prelamin A, producing a truncated farnesylated protein called progerin. Lamins play important roles in the organization and structure of the nucleus. The nuclear build-up of progerin causes severe morphological and functional changes in interphase HGPS cells. In this study, we investigated whether progerin elicits spatiotemporal deviations in mitotic processes in HGPS fibroblasts. We analyzed the nuclear distribution of endogenous progerin during mitosis in relation to components of the nuclear lamina, nuclear envelope (NE) and nuclear pores. We found that progerin caused defects in chromosome segregation as early as metaphase, delayed NE reformation and trapped lamina components and inner NE proteins in the endoplasmic reticulum at the end of mitosis. Progerin displaced the centromere protein F (CENP-F) from metaphase chromosome kinetochores, which caused increased chromatin lagging, binucleated cells and genomic instability. This accumulation of progerin-dependent defects with each round of mitosis predisposes cells to premature senescence., Competing Interests: The authors declare no conflicts of interest.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery., (Copyright © 2016. Published by Elsevier Inc.)

The Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5'splice site (5'SS) instead of the LMNA 5'SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5'SS (LAΔ35 5'SS), inducing the production of another truncated LMNA protein (LAΔ35). Our data show that the progerin 5'SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LAΔ35 5'SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5'SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5'SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5'SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging., (© 2015 WILEY PERIODICALS, INC.)

Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging.

ATM-mediated phosphorylation of KAP-1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24(-/-) mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM-Kap-1 signaling is compromised in Zmpste24(-/-) MEFs, leading to defective DNA damage-induced chromatin remodeling. Knocking down Kap-1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24(-/-) MEFs. Thus, ATM-Kap-1-mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy., (© 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Small increases in the expression of wild-type prelamin A are sufficient to recapitulate the reduced cell proliferation and altered nuclear membrane morphology observed in cells expressing progerin, the mutant lamin A associated with progeria. We hypothesized that the manifestation of these phenotypes in cells expressing elevated levels of wild-type prelamin A or progerin is caused by the same molecular effectors, which play a central role in the onset of the progeroid phenotype. To experimentally test this hypothesis, we compared the transcriptomes of isogenic diploid fibroblasts expressing progerin or elevated levels of wild-type prelamin A with that of wild-type fibroblasts. We subsequently used the reversion towards normal of two phenotypes, reduced cell growth and dismorphic nuclei, by treatment with farnesyltransferase inhibitor (FTI) or overexpression of ZMPSTE24, as a filtering strategy to identify genes linked to the onset of these two phenotypes. Through this analysis we identified the gene encoding for the transcription factor FOXQ1, as a gene whose expression is induced in both cells expressing progerin and elevated levels of wild-type prelamin A, and subsequently reduced in both cell types upon conditions that ameliorate the phenotypes. We overexpressed FOXQ1 in normal fibroblasts and demonstrated that increased levels of this factor lead to the development of both features that were used in the filtering strategy. These findings suggest a potential link between this transcription factor and cell dysfunction induced by altered prelamin A metabolism.

Fibroblasts from patients with the severe laminopathy diseases, restrictive dermopathy (RD) and Hutchinson Gilford progeria syndrome (HGPS), are characterized by poor growth in culture, the presence of abnormally shaped nuclei and the accumulation of DNA double-strand breaks (DSB). Here we show that the accumulation of DSB and poor growth of the fibroblasts but not the presence of abnormally shaped nuclei are caused by elevated levels of reactive oxygen species (ROS) and greater sensitivity to oxidative stress. Basal levels of ROS and sensitivity to H(2)O(2) were compared in fibroblasts from normal, RD and HGPS individuals using fluorescence activated cell sorting-based assays. Basal levels of ROS and stimulated levels of ROS were both 5-fold higher in the progeria fibroblasts. Elevated levels of ROS were correlated with lower proliferation indices but not with the presence of abnormally shaped nuclei. DSB induced by etoposide were repaired efficiently in normal, RD and HGPS fibroblasts. In contrast, DSB induced by ROS were repaired efficiently in normal fibroblasts, but in RD and HGPS fibroblasts many ROS-induced DSB were un-repairable. The accumulation of ROS-induced DSB appeared to cause the poor growth of RD and HGPS fibroblasts, since culture in the presence of the ROS scavenger N-acetyl cysteine (NAC) reduced the basal levels of DSB, eliminated un-repairable ROS-induced DSB and greatly improved population-doubling times. Our findings suggest that un-repaired ROS-induced DSB contribute significantly to the RD and HGPS phenotypes and that inclusion of NAC in a combinatorial therapy might prove beneficial to HGPS patients.

Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.

Background: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression., Methods: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses., Results: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression., Conclusions: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics., General Significance: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development., (Copyright 2010 Elsevier B.V. All rights reserved.)

The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging., (Published by Elsevier Ireland Ltd.)

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disorder caused by mutations in the gene LMNA, which encodes the nuclear matrix protein lamin A. Previous research has shown that the average telomere length in fibroblasts from HGPS patients is shorter than in age-matched controls. How mutations in lamin A lead to shortened telomere lengths is not known nor is the contribution of individual chromosome ends to the low average length understood. To measure the telomere length of individual chromosomes, we used quantitative fluorescence in situ hybridization (Q-FISH). In agreement with previous studies, we found that the average telomere length in HPGS fibroblasts is greatly reduced; however, the telomere length at chromosome ends was variable. In contrast, the telomere length in hematopoietic cells which typically do not express lamin A, was within the normal range for three out of four HGPS patient samples. Our results suggest that mutant lamin A decreases telomere length via a direct effect and that expression of mutant LMNA is necessary for telomere loss in HGPS.

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.

Mutations of the nuclear lamins cause a wide range of human diseases, including Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome. Defects in A-type lamins reduce nuclear structural integrity and affect transcriptional regulation, but few data exist on the biological role of B-type lamins. To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts from Lmnb1(Delta/Delta) and wild-type littermate embryos by time-lapse videomicroscopy. Here, we report that Lmnb1(Delta/Delta) cells displayed striking nuclear rotation, with approximately 90% of Lmnb1(Delta/Delta) nuclei rotating at least 90 degrees during an 8-h period. The rotation involved the nuclear interior as well as the nuclear envelope. The rotation of nuclei required an intact cytoskeletal network and was eliminated by expressing lamin B1 in cells. Nuclear rotation could also be abolished by expressing larger nesprin isoforms with long spectrin repeats. These findings demonstrate that lamin B1 serves a fundamental role within the nuclear envelope: anchoring the nucleus to the cytoskeleton.

Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder that displays features of segmental aging. It is manifested predominantly in connective tissue, with most prominent histological changes occurring in the skin, cartilage, bone and cardiovascular tissues. Detailed quantitative real time reverse-transcription polymerase chain reaction studies confirmed the previous observation that platelet-derived growth factor A-chain transcripts are consistently elevated 11+/-2- to 13+/-2-fold in two HGPS dermal fibroblast lines compared with age-matched controls. Furthermore, we identified two additional genes with substantially altered transcript levels. Nucleotide pyrophosphatase transcription was virtually shut down with decreased expression of 13+/-3- to 59+/-3-fold in HGPS, whereas aggrecan mRNA was elevated to 24+/-5 times to 41+/-4 times that of chronologically age-matched controls. Aggrecan, normally a component of cartilage and not always detectable in normal fibroblasts cultures, was secreted by HGPS fibroblast lines and was produced as a proteoglycan. This demonstrates that elevated aggrecan expression and its secretion are aberrant features of HGPS. We conclude that HGPS cells can display massively altered transcript levels leading to the secretion of inappropriate protein species.

Background: Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT), within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein., Results: We studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress., Conclusion: Dermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic abnormalities in the disease.

The effect of insulin and growth factor mediated signaling to gene regulation was investigated in cultured fibroblasts of a patient with a premature aging syndrome (metageria) and severe insulin resistance. Insulin receptor structure and function as well as major pathways activated by insulin, i.e. phosphatidyl inositol-3 kinase (PI-3 K) cascade or mitogen-activated protein kinase (MAPK) cascades, were functional. Inducibility of the proto-oncogene cfos, a representative endpoint of signaling pathways related to gene expression, by growth factors or insulin was reduced in patient cells. This reduced induction persisted in cfos promoter reporter gene studies indicating that the post receptor defect is localized proximal to the cfos promoter itself. Abundances of the transcription factors Elk-1 and SRF being major players in coupling of MAPKs to cfos promoter activation were not altered. However, basal and inducible phosphorylation of Elk-1 was impaired. In addition, basal and stimulated transcriptional activity mediated by Elk-1 was almost abolished in patient cells. Therefore these results identify a post receptor defect in cFos induction, which appears to be related to a functional alteration of Elk-1. A possible relation of this signal transduction defect to the specific premature aging syndrome remains to be elucidated.

Hutchinson-Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process.

Hutchinson-Gilford progeria (HGP) is a genetic disorder in which individuals prematurely display features of ageing. Mutations in LMNA (lamin A) have recently been shown to underlie HGP, although how such mutations lead to the complex phenotype seen in the disease remains unclear. HGP is often associated with the premature replicative senescence of dermal fibroblasts. Normally dermal fibroblast senescence is initiated by erosion of chromosomal ends (telomeres) resulting from sustained cell division. Since ectopic expression of telomerase reproducibly immortalises human dermal fibroblasts, it is of interest to determine whether HGP fibroblasts immortalise via the same route, and at the same frequency. Three strains of HGP fibroblasts (AGO6917A, AGO6297B and AGO8466) were infected with a retroviral vector expressing the catalytic subunit of telomerase (hTERT). Here we report that fibroblast clones derived from HGP donors frequently fail to immortalise with telomerase. Of the 15 independently isolated clones from the three donors, five failed to immortalise despite the restoration of telomerase activity and the stabilisation of telomere length. In contrast, out of four clones isolated from a culture of hTERT transduced control fibroblasts, no failures to immortalise were detected. This suggests a novel cellular phenotype in HGP, one whereby the HGP mutation confers resistance to 'telomerisation'.

Messenger RNA levels were measured in actively dividing fibroblasts isolated from young, middle-age, and old-age humans and humans with progeria, a rare genetic disorder characterized by accelerated aging. Genes whose expression is associated with age-related phenotypes and diseases were identified. The data also suggest that an underlying mechanism of the aging process involves increasing errors in the mitotic machinery of dividing cells in the postreproductive stage of life. We propose that this dysfunction leads to chromosomal pathologies that result in misregulation of genes involved in the aging process.

Cultured cells from human premature aging syndromes show premature replicative senescence and as such might serve as models for genetic studies of cellular replicative senescence/aging. To date, no systematic study on chromosome-specific aneuploidy in premature aging syndromes has been carried out using molecular-cytogenetic techniques. We, therefore, have performed a comparative analysis of chromosome-specific aneuploidy levels at both interphase and metaphase in earlier (younger) and later (older) passage fibroblasts from two normal male and female versus six male and female premature aging syndromes (Cockayne, Hutchinson-Gilford and Werner) by FISH. We have used seven chromosome-specific DNA probes (nos. 1, 4, 6, 8, 10, 15, X) and four DNA probes (nos. 1, 4, 6, X) for females and males, respectively. Our data on total aneuploidy of each chromosome in all cell cultures indicated that significantly higher percentages of aneuploid cells were detectable at interphase than at metaphase. Also, the interphase aneuploidy levels of all chromosomes under study were significantly higher in cells from the syndromes as compared to these of the normal controls at both earlier and later passages. In general, the interphase aneuploidy level of each of the chromosomes in both the control and experimental cell cultures increased with in vitro proliferation and aging, although to a much lesser extent in the controls. The aneuploidy levels, however, varied widely from chromosome to chromosome in each case. Since adequate numbers of mitotic cells were not always available in some later passage cells of various premature aging syndromes, metaphase chromosome analysis for all chromosomes under study was not always possible at this stage, but interphase cytogenetics was very informative in all cases. No consistent pattern of chromosome-specific aneuploidy was detected in the earlier passage cells (younger cells) of the syndromes. However, the later passage cells (older cells) from all three female syndromes consistently showed the highest aneuploidy levels for the X chromosome at interphase.

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

The level of heat-labile glucose-6-phosphate dehydrogenase (G6PD) has been measured in skin fibroblast cultures from premature ageing or DNA repair deficient genetic syndromes. The short in vitro longevity of Werner's syndrome, progeria, Cockayne's syndrome, ataxia telangiectasia, Fanconi's anaemia, and Bloom's syndrome cultures was correlated with the appearance of a significant fraction of heat-labile enzyme. Long-lived control cultures contain a low level of altered enzyme until they become senescent. The evidence that heat-labile G6PD molecules are derived from errors in synthesis, or from other causes, is critically assessed. It is shown that normal cells grown in medium containing the antibiotic, paromomycin, which is known to reduce the fidelity of ribosomal translation, produce a significant fraction of altered G6PD.

Fibroblast cultures were established from skin biopsies from 4 male Werner's syndrome patients, aged 45-50. Several subcultures were obtained from each primary outgrowth and these were passaged to the end of their in vitro lifespan. Their average longevity was significantly less than control skin fibroblasts, but there was extreme variability amongst parallel cultures. In the most thoroughly studied case, the longevity of 19 subcultures derived from a single biopsy varied from 5 to 26 passages, showing that there was considerable heterogeneity in the growth potential of the cells in the primary culture. Moreover, the growth rate from any one culture was not uniform, since long periods of slow growth were sometimes succeeded by much more rapid proliferation. These features of Werner's syndrome fibroblast populations are not seen in cultures from normal individuals. The longevity of fibroblasts from one progeria patient was also shown to be much shorter than controls. Metaphases from 3 Werner's patients demonstrated a much higher frequency of chromosome abnormalities than in normal fibroblasts and also provided evidence of subclones containing a characteristic 'marker' chromosome.

The insulin receptor, though understood in fine molecular detail, remains problematic with regards to its mechanism(s) of cellular signalling. To better understand the normal mechanisms of plasma membrane signalling by insulin, we studied the family of a patient with insulin resistance, hypoglycemia and leprechaunism. Skin fibroblasts were cultured from the proband and his parents. High-affinity insulin binding to the proband's cells was absent. Both parents' fibroblasts showed high-affinity insulin binding intermediate between the controls and the proband. Thus impaired binding conformed to an autosomal recessive pattern of inheritance. The transport of neutral amino acids by the proband's cells was comparable to controls and was insulin-sensitive. By contrast, hexose transport by the proband's fibroblasts was very high and insulin-insensitive. This increased uptake was due to an increased number of glucose transporters on the plasma membrane and to a posttranslational mechanism. Similar levels of glucose transporter mRNA were observed in control and mutant cells. Thus, this inborn error of the insulin receptor offers unique insight into a regulatory signal by its alpha subunit which, when altered, results in constitutively increased glucose transport.

Progeria is an autosomal recessive disorder showing precocious senility. The cultured skin fibroblast from both the homozygous affected individual and the heterozygous parents can be distinguished from normals by decreased cell growth in culture. Mitotic activity, DNA synthesis, and cloning efficiency are markedly reduced.

HL-A phenotypes were determined by a cytotoxicity assay on circulating lymphocytes of two boys with the premature aging syndrome, progeria. The antigenic phenotypes were not unusual inasmuch as they are frequently seen in the normal population. However, none of these antigens could be detected by the same assay on cultured skin fibroblasts from either individual, even when a significant mitotic potential remained before cessation of growth. Fibroblasts from normal donors were concordant with corresponding lymphocytes for HL-A antigens and maintained these antigens until mitotic division had virtually ceased. Absorption studies on fibroblasts with two HL-A2 antisera revealed that HL-A antigens are either absent or have a drastically reduced expression on progeric fibroblasts. The data are in accord with the concept of an immunological role in the pathogenesis of progeria.