Hutchinson-Gilford Progeria Syndrome Cells Research Articles

Replication factor C1, the large subunit of replication factor C, is proteolytically truncated in Hutchinson–Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder because of a LMNA gene mutation that produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here, we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ~ 75 kDa, which appears to be defective in loading proliferating cell nuclear antigen (PCNA) and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Because of the crucial role of RFC in DNA replication, our findings provide a mechanistic interpretation for the observed early replicative arrest and premature aging phenotypes of HPGS and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS.

A proteomic study of Hutchinson–Gilford progeria syndrome: Application of 2D-chromotography in a premature aging disease

The Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease characterized by segmental premature aging. Applying a two-dimensional chromatographic proteomic approach, the 2D Protein Fractionation System (PF2D), we identified 30 differentially expressed proteins in cultured HGPS fibroblasts. We categorized them into five groups: methylation, calcium ion binding, cytoskeleton, duplication, and regulation of apoptosis. Among these 30 proteins, 23 were down-regulated, while seven were up-regulated in HGPS fibroblasts as compared to normal fibroblasts. Three differentially expressed cytoskeleton proteins, vimentin, actin, and tubulin, were validated via Western blotting and characterized by immunostaining that revealed densely thickened bundles and irregular structures. Furthermore in the HGPS cells, the cell cycle G1 phase was elongated and the concentration of free cytosolic calcium was increased, suggesting intracellular retention of calcium. The results that we obtained have implications for understanding the aging process.

DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

Rapamycin Reverses Cellular Phenotypes and Enhances Mutant Protein Clearance in Hutchinson-Gilford Progeria Syndrome Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic disorder characterized by premature aging. HGPS is most commonly caused by a de novo single-nucleotide substitution in the lamin A/C gene (LMNA) that partially activates a cryptic splice donor site in exon 11, producing an abnormal lamin A protein termed progerin. Accumulation of progerin in dividing cells adversely affects the integrity of the nuclear scaffold and leads to nuclear blebbing in cultured cells. Progerin is also produced in normal cells, increasing in abundance as senescence approaches. Here, we report the effect of rapamycin, a macrolide antibiotic that has been implicated in slowing cellular and organismal aging, on the cellular phenotypes of HGPS fibroblasts. Treatment with rapamycin abolished nuclear blebbing, delayed the onset of cellular senescence, and enhanced the degradation of progerin in HGPS cells. Rapamycin also decreased the formation of insoluble progerin aggregates and induced clearance through autophagic mechanisms in normal fibroblasts. Our findings suggest an additional mechanism for the beneficial effects of rapamycin on longevity and encourage the hypothesis that rapamycin treatment could provide clinical benefit for children with HGPS.

HGPS-Derived iPSCs For The Ages

In this issue of Cell Stem Cell, Zhang et al. (2011) generate patient-derived iPSCs for one of the major premature aging diseases, Hutchinson-Gilford Progeria Syndrome (HGPS). These cells are a much-needed new tool to study HGPS, and their use may lead to novel insights into mechanisms of aging.

Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells

BackgroundHutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein.ResultsWe have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells.ConclusionsThis study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.

Reversal of age-dependent nuclear morphology by inhibition of prenylation does not affect lifespan in Caenorhabditis elegans

Fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and dermal cells derived from healthy old humans in culture display age-dependent progressive changes in nuclear architecture due to accumulation of farnesylated lamin A. Treating human HGPS cells or mice expressing farnesylated lamin A with farnesyl transferase inhibitors (FTIs) reverses nuclear phenotypes and extends lifespan. Aging adult Caenorhabditis elegans show changes in nuclear architecture resembling those seen in HGPS fibroblasts, as well as a decline in motility, phenotypes which are also inhibited by the FTI gliotoxin. However, it was not clear whether these effects were due to loss of farnesylation or to side effects of this drug. Here, we used a different FTI, manumycin, or downregulated polyprenyl synthetase with RNAi to test the roles of farnesylation in C. elegans aging. Our results show that the age-dependent changes in nuclear morphology depend on farnesylation. We also demonstrate that inhibition of farnesylation does not affect motility or lifespan, suggesting that the effects of blocking protein prenylation on nuclear morphology could be separated from their effects on motility and lifespan. These results provide further understanding of the role of lamin and farnesylation in the normal aging process and in HGPS.

Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature-aging syndrome caused by a dominant mutation in the gene encoding lamin A, which leads to an aberrantly spliced and processed protein termed progerin. Previous studies have shown that progerin induces early senescence associated with increased DNA-damage signaling and that telomerase extends HGPS cellular lifespan. We demonstrate that telomerase extends HGPS cellular lifespan by decreasing progerin-induced DNA-damage signaling and activation of p53 and Rb pathways that otherwise mediate the onset of premature senescence. We show further that progerin-induced DNA-damage signaling is localized to telomeres and is associated with telomere aggregates and chromosomal aberrations. Telomerase amelioration of DNA-damage signaling is relatively rapid, requires both its catalytic and DNA-binding functions, and correlates in time with the acquisition by HGPS cells of the ability to proliferate. All of these findings establish that HGPS premature cellular senescence results from progerin-induced telomere dysfunction.

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation.In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.

Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients

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 ∼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 (β 5 and β 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.

Abstract A60: Role of telomere dysfunction in the premature cellular senescence of Hutchinson-Gilford progeria syndrome

Abstract Introduction: Telomere dysfunction promotes genomic instability and is often associated with loss of tumor suppressor gene function and tumorigenesis. Conversely, in aging, telomere dysfunction is hypothesized to mediate cellular senescence by activating intact tumor suppressor pathways. Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by a dominant mutation in the nuclear lamina intermediate filament protein lamin A that leads to a truncated and permanently farnesylated protein termed progerin. Progerin expression is associated with early cellular senescence involving shortened telomeres and increased DNA damage. Herein, we investigated whether telomere dysfunction contributes to the DNA damage accumulation and premature cellular senescence phenotypes observed with cells from HGPS patients. Methods: Using HGPS cell lines from patients or normal fibroblasts ectopically expressing progerin, we investigated the effects of telomerase on cell growth, senescence, p53 and Rb tumor suppressor pathway activation, and DNA damage. We also determined whether progerin-induced DNA damage localized to telomeres. Results: We demonstrate that addition of telomerase, which has been reported to immortalize HGPS fibroblasts, abrogated premature senescence, reduced p53 and Rb tumor suppressor pathway activation, and decreased the DNA damage phenotype. These effects of telomerase required both its catalytic and DNA binding activities. Furthermore, progerin-DNA damage signaling localized to telomeres indicating that progerin causes damage at telomeres. Conclusions: These results establish that progerin causes a defect in telomere maintenance linked to a chronic DNA damage response that activates tumor suppressor genes and induces the HGPS premature senescence phenotype. Citation Information: Cancer Res 2009;69(23 Suppl):A60.

Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

Hutchinson–Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5′ portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5′ to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease ‘HGPS splicing’. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells.

Increased expression of the Hutchinson–Gilford progeria syndrome truncated lamin A transcript during cell aging

Most cases of the segmental progeroid syndrome, Hutchinson-Gilford progeria syndrome (HGPS), are caused by a de novo dominant mutation within a single codon of the LMNA gene. This mutation leads to the increased usage of an internal splice site that generates an alternative lamin A transcript with an internal deletion of 150 nucleotides, called lamin A Delta 150. The LMNA gene encodes two major proteins of the inner nuclear lamina, lamins A and C, but not much is known about their expression levels. Determination of the overall expression levels of the LMNA gene transcripts is an important step to further the understanding of the HGPS. In this study, we have performed absolute quantification of the lamins A, C and A Delta 150 transcripts in primary dermal fibroblasts from HGPS patients and unaffected age-matched and parent controls. We show that the lamin A Delta 150 transcript is present in unaffected controls but its expression is >160-fold lower than that in samples from HGPS patients. Analysis of transcript expression during in vitro aging shows that although the levels of lamin A and lamin C transcripts remain unchanged, the lamin A Delta 150 transcript increases in late passage cells from HGPS patients and parental controls. This study provides a new method for LMNA transcript analysis and insights into the expression of the LMNA gene in HGPS and normal cells.

Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A

Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component.

Mesenchymal Stem Cells Ameliorate Murine Progeria Phenotype

Our goal is to test mesenchymal stem cell therapy in a mouse model of Hutchinson-Gilford Progeria Syndrome (HGPS), a lethal childhood disorder for which no definitive therapy is available. HGPS patients have failure to thrive, lipodystrophy, skeletal dysplasia, sclerotic skin, alopecia, and occlusive vascular disease that results in premature death from myocardial infarction or stroke in early teens. In HGPS, the accumulation of incompletely processed nuclear architectural protein lamin A (a farnesylated version of prelamin A, termed progerin) results in dysfunction of multiple organ systems, particularly those of mesenchymal origin. Consistent with this, progerin has been shown to interfere with the proliferation and differentiation of mesenchymal stem cells (MSCs). Furthermore, MSC depletion is thought to account for the specific segmental nature of human progeria. The identification of HGPS-specific MSC dysfunction, and emerging evidence about the plasticity and paracrine effects of MSCs have prompted us to test the hypothesis that MSCs might be coaxed into becoming cells able to correct widespread pathology in HGPS. To determine whether MSCs can be used to rescue progeria phenotype, MSCs from wild-type donors were tested. First, donor MSCs (0.5 × 106 per recipient) were stably transfected to express the red fluoroform DsRed2, which permitted donor cell tracking in tissues post mortem. Next, to achieve wide biodistribution DsRed2+ MSCs were injected intra-arterially into 2-week-old ZMPSTE24−/− mutant mice. Like HGPS cells, ZMPSTE24−/− mutant mice accumulate progerin and the mice exhibit many progeria-like signs, including alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, osteoporosis, retarded growth, muscle weakness, and shortened lifespan. Therefore, survival, growth (measured by weight gain), and muscle weakness (measured by grip strength) were analyzed. MSC-treated (n = 13) versus untreated (n = 23) ZMPSTE24−/− mice had significantly increasedsurvival (65% versus 100%),weight gain after 6 weeks of life, andgrip strength (judged by the ability to hang onto an upside–down grid).These data are consistent with the hypothesis that the transfer of wild-type MSCs reduces the severity of the lamin A defect. To investigate whether the benefits associated with MSC infusion were attributable to persistent tissue engraftment or to a transient paracrine effect of donor MSCs, all mice were electively harvested at 20 weeks of age. Because dermal lipodystrophy, cardiac arteriosclerosis, and skeletal myopathy are prominent pathological findings in both human and murine progeria, post-mortem analyses focused on donor cell engraftment and progerin expression in corresponding organs of pathology (skin, liver, and skeletal muscle). Donor cells were identified in skin, liver, and skeletal muscle specimens with a frequency of 1%–5%, indicating that donor MSCs can repopulate tissue niches presumed to be dysfunctional in progeria. Experiments are ongoing to evaluate tissue levels of prelamin A and mature lamin A, abnormalities in nuclear shape, and pertubations in intracellular signaling pathway genes known to be dysregulated in human progeria. Collectively, these data demonstrate the proof of principle of MSC transfer for phenotypic reversion of lamin A defect in murine progeria. This may offer a valuable approach for treatment of human HGPS with human MSCs, which are already in clinical use.

Tethering by lamin A stabilizes and targets the ING1 tumour suppressor

ING proteins interact with core histones through their plant homeodomains (PHDs) and with histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes to alter chromatin structure. Here we identify a lamin interaction domain (LID) found only in ING proteins, through which they bind to and colocalize with lamin A. Lamin knockout (LMNA(-/-)) cells show reduced levels of ING1 that mislocalize. Ectopic lamin A expression increases ING1 levels and re-targets it to the nucleus to act as an epigenetic regulator. ING1 lacking the LID does not interact with lamin A or affect apoptosis. In LMNA(-/-) cells, apoptosis is not affected by ING1. Mutation of lamin A results in several laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), a severe premature ageing disorder. HGPS cells have reduced ING1 levels that mislocalize. Expression of LID peptides to block lamin A-ING1 interaction induces phenotypes reminiscent of laminopathies including HGPS. These data show that targeting of ING1 to the nucleus by lamin A maintains ING1 levels and biological function. Known roles for ING proteins in regulating apoptosis and chromatin structure indicate that loss of lamin A-ING interaction may be an effector of lamin A loss, contributing to the HGPS phenotype.

Progerin elicits disease phenotypes of progeria in mice whether or not it is farnesylated

Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.

Research on Hutchinson-Gilford Progeria Syndrome

IN this issue of the Journal, I have included a summary of a workshop held in November 2007 on the topic of Hutchinson-Gilford Progeria Syndrome (HGPS) (1). This syndrome was first described over 120 years ago by Hutchinson (2), and although the phenotype does include some aging-like changes, biogerontologists have questioned whether it is a viable model for studying accelerated aging (3). The question has justifiably risen again with the recent identification of LMNA as the gene responsible for this sporadic autosomal dominant syndrome (4). It is now clear that the syndrome results from the accumulation of a metabolite formed during processing of the mutated pre-lamin A protein. This metabolite has been named progerin, and it induces nuclear blebbing in HGPS cells grown in culture (5), suggesting that serious disruption of DNA metabolism may be occurring in these cells. At least several outcomes are possible, including induction of cell senescence, induction of apoptosis, dysregulated gene expression, dysregulation of differentiation, and so forth. Whatever the critical defect may be, Kudlow and colleagues (6) have argued that progeroid syndromes, including HGPS, ‘‘might have partial mechanistic overlap with normal aging and therefore might provide uniquely informative opportunities to formulate and test hypotheses regarding the biology of aging and age-dependent disease.’’ Warner and Sierra (7) also have suggested such possible overlap. Scaffidi and Misteli (8) have recently shown that small amounts of progerin are produced in normal cells by occasional use of the aberrant splice site used in HGPS cells, but it is not known whether these small amounts actually have any impact on longevity. However, progerin does accumulate in the skin with age in a subset of dermal fibroblasts, suggesting that this could be a possible biomarker of cellular aging (1,9). Kudlow and colleagues (6) suggest three general approaches to identify such putative mechanistic links between normal aging and accelerated aging models, whether human or murine. Because many mouse mutations that induce progeroid phenotypes share disruption of DNA metabolism as a common feature, they suggest that it would be useful to know what roles lamin A plays in the various DNA transactions important to the proper function of a mammalian cell, such as replication, transcription, repair, and recombination. Progerin has in fact been reported to interfere with mitosis (10) as well as induce DNA damage responses (11). Secondly, they suggest that persistent DNA damage signaling, cell death, and cellular changes may contribute to the pathology of HGPS, and that it is important to determine whether and why these changes are selectively targeted to specific cell lineages and tissues. Finally, a third approach is to look for single nucleotide polymorphisms (SNPs) in the gene for lamin A that associate with longevity; Francis Collins reported at the workshop that four such SNPs have been found in older people (1). Besides the interesting question of whether HGPS is or is not a valid model of accelerated human aging, the most important translational question is the development of a cure—or at the very least an effective treatment. Numerous studies have reported that farnesyltransferase inhibitors reverse the morphological defects observed in HGPS fibroblasts (12). This provides the basis for the clinical trial currently underway under the direction of Mark Kieran at Children’s Hospital in Boston. The primary endpoint is weight gain because failure to grow, starting at about age 2 years, represents a consistent and predictable characteristic of the syndrome. Secondary biomarkers such as alopecia, short stature, subcutaneous fat, bone integrity, and a variety of other abnormalities will also be followed. However, the ideal solution would be a total cure rather than an ongoing treatment. In trying to understand the underlying cause of HGPS, it may be instructive to ask what causes and ‘‘times’’ the onset of growth failure and the subsequent low weight and height gain, and why do mesenchymally derived tissues seem to be most vulnerable (13). Sharpless and DePinho (14) have reviewed mounting evidence that accumulation of damage to cellular macromolecules such as DNA could be one cause of cellular attrition with aging. Continued cellular attrition by apoptosis would result in forced regeneration of tissue cells in response to homeostatic demands, possibly resulting ultimately in an inability of stem cell pools to respond to these demands (15). Such a process could explain an onset of growth failure, at about 2 years of age, such as observed in HGPS. In addition to DNA damage accumulation in HGPS cells, the aberrant morphology of HGPS nuclei might also trigger the cellular attrition reported by Bridger and Kill (16). HGPS stem cells may also be subject to apoptosis, which would compound the attrition problem. Scaffidi and Misteli (17) have recently reported that progerin ‘‘interferes with the

Perturbation of wild‐type lamin A metabolism results in a progeroid phenotype

Mutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of disease-linked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.