Hutchinson-Gilford Progeria Syndrome Cells Research Articles

Increased mechanosensitivity and nuclear stiffness in Hutchinson–Gilford progeria cells: effects of farnesyltransferase inhibitors

Hutchinson-Gilford progeria syndrome (HGPS), reportedly a model for normal aging, is a genetic disorder in children marked by dramatic signs suggestive for premature aging. It is usually caused by de novo mutations in the nuclear envelope protein lamin A. Lamins are essential to maintaining nuclear integrity, and loss of lamin A/C results in increased cellular sensitivity to mechanical strain and defective mechanotransduction signaling. Since increased mechanical sensitivity in vascular cells could contribute to loss of smooth muscle cells and the development of arteriosclerosis--the leading cause of death in HGPS patients--we investigated the effect of mechanical stress on cells from HGPS patients. We found that skin fibroblasts from HGPS patients developed progressively stiffer nuclei with increasing passage number. Importantly, fibroblasts from HGPS patients had decreased viability and increased apoptosis under repetitive mechanical strain, as well as attenuated wound healing, and these defects preceded changes in nuclear stiffness. Treating fibroblasts with farnesyltransferase inhibitors restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing the directional persistence of migrating cells. However, farnesyltransferase inhibitors did not improve cellular sensitivity to mechanical strain. These data suggest that increased mechanical sensitivity in HGPS cells is unrelated to changes in nuclear stiffness and that increased biomechanical sensitivity could provide a potential mechanism for the progressive loss of vascular smooth muscle cells under physiological strain in HGPS patients.

Lamin A-dependent misregulation of adult stem cells associated with accelerated ageing

The premature-ageing disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. Progerin is also expressed sporadically in wild-type cells and has been linked to physiological ageing. Cells from HGPS patients exhibit extensive nuclear defects, including abnormal chromatin structure and increased DNA damage. At the organismal level, HGPS affects several tissues, particularly those of mesenchymal origin. How the cellular defects of HGPS cells lead to the organismal defects has been unclear. Here, we provide evidence that progerin interferes with the function of human mesenchymal stem cells (hMSCs). We find that expression of progerin activates major downstream effectors of the Notch signalling pathway. Induction of progerin in hMSCs changes their molecular identity and differentiation potential. Our results support a model in which accelerated ageing in HGPS patients, and possibly also physiological ageing, is the result of adult stem cell dysfunction and progressive deterioration of tissue functions.

Involvement of xeroderma pigmentosum group A (XPA) in progeria arising from defective maturation of prelamin A.

Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs), which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by gamma-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of xeroderma pigmentosum group A (XPA), a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and coimmunoprecipitation. RNA interference (RNAi) knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.

Distinct structural and mechanical properties of the nuclear lamina in Hutchinson–Gilford progeria syndrome

The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.

Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging

The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a mutant lamin A (LADelta50). Nuclei in cells expressing LADelta50 are abnormally shaped and display a loss of heterochromatin. To determine the mechanisms responsible for the loss of heterochromatin, epigenetic marks regulating either facultative or constitutive heterochromatin were examined. In cells from a female HGPS patient, histone H3 trimethylated on lysine 27 (H3K27me3), a mark for facultative heterochromatin, is lost on the inactive X chromosome (Xi). The methyltransferase responsible for this mark, EZH2, is also down-regulated. These alterations are detectable before the changes in nuclear shape that are considered to be the pathological hallmarks of HGPS cells. The results also show a down-regulation of the pericentric constitutive heterochromatin mark, histone H3 trimethylated on lysine 9, and an altered association of this mark with heterochromatin protein 1alpha (Hp1alpha) and the CREST antigen. This loss of constitutive heterochromatin is accompanied by an up-regulation of pericentric satellite III repeat transcripts. In contrast to these decreases in histone H3 methylation states, there is an increase in the trimethylation of histone H4K20, an epigenetic mark for constitutive heterochromatin. Expression of LADelta50 in normal cells induces changes in histone methylation patterns similar to those seen in HGPS cells. The epigenetic changes described most likely represent molecular mechanisms responsible for the rapid progression of premature aging in HGPS patients.

Aggrecan expression is substantially and abnormally upregulated in Hutchinson–Gilford Progeria Syndrome dermal fibroblasts

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.

Age-related changes of nuclear architecture inCaenorhabditis elegans

Mutations in lamins cause premature aging syndromes in humans, including the Hutchinson-Gilford Progeria Syndrome (HGPS) and Atypical Werner Syndrome. It has been shown that HGPS cells in culture undergo age-dependent progressive changes in nuclear architecture. However, it is unknown whether similar changes in nuclear architecture occur during the normal aging process. We have observed that major changes of nuclear architecture accompany Caenorhabditis elegans aging. We found that the nuclear architecture in most nonneuronal cell types undergoes progressive and stochastic age-dependent alterations, such as changes of nuclear shape and loss of peripheral heterochromatin. Furthermore, we show that the rate of these alterations is influenced by the insulin/IGF-1 like signaling pathway and that reducing the level of lamin and lamin-associated LEM domain proteins leads to shortening of lifespan. Our work not only provides evidence for changes of nuclear architecture during the normal aging process of a multicellular organism, but also suggests that HGPS is likely a result of acceleration of the normal aging process. Because the nucleus is vital for many cellular functions, our studies raise the possibility that the nucleus is a prominent focal point for regulating aging.

Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is characterized by dramatic premature aging and accelerated cardiovascular disease. HGPS is almost always caused by a de novo point mutation in the lamin A gene (LMNA) that activates a cryptic splice donor site, producing a truncated mutant protein termed "progerin." WT prelamin A is anchored to the nuclear envelope by a farnesyl isoprenoid lipid. Cleavage of the terminal 15 aa and the farnesyl group releases mature lamin A from this tether. In contrast, this cleavage site is deleted in progerin. We hypothesized that retention of the farnesyl group causes progerin to become permanently anchored in the nuclear membrane, disrupting proper nuclear scaffolding and causing the characteristic nuclear blebbing seen in HGPS cells. Also, we hypothesized that blocking farnesylation would decrease progerin toxicity. To test this hypothesis, the terminal CSIM sequence in progerin was mutated to SSIM, a sequence that cannot be farnesylated. SSIM progerin relocalized from the nuclear periphery into nucleoplasmic aggregates and produced no nuclear blebbing. Also, blocking farnesylation of authentic progerin in transiently transfected HeLa, HEK 293, and NIH 3T3 cells with farnesyltransferase inhibitors (FTIs) restored normal nuclear architecture. Last, treatment of both early- and late-passage human HGPS fibroblasts with FTIs resulted in significant reductions in nuclear blebbing. Our results suggest that treatment with FTIs represents a potential therapy for patients with HGPS.

Incomplete processing of mutant lamin A in Hutchinson–Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition

Hutchinson-Gilford progeria syndrome (HGPS) is typically caused by mutations in codon 608 (G608G) of the LMNA gene, which activates a cryptic splice site resulting in the in-frame loss of 150 nucleotides from the lamin A message. The deleted region includes a protein cleavage site that normally removes 15 amino acids, including a CAAX box farnesylation site, from the lamin A protein. We investigated the processing of the C-terminus of the mutant protein, 'progerin', and found that it does not undergo cleavage and, indeed, remains farnesylated. The retention of the farnesyl group may have numerous consequences, as farnesyl groups increase lipophilicity and are involved in membrane association and in protein interactions, and is likely to be an important factor in the HGPS phenotype. To further investigate this, we studied the effects of farnesylation inhibition on nuclear phenotypes in cells expressing normal and mutant lamin A. Expression of a GFP-progerin fusion protein in normal fibroblasts caused a high incidence of nuclear abnormalities, as was also seen in HGPS fibroblasts, and resulted in abnormal nuclear localization of GFP-progerin in comparison with the localization pattern of GFP-lamin A. Expression of a GFP-lamin A fusion containing a mutation preventing the final cleavage step, causing the protein to remain farnesylated, displayed identical localization patterns and nuclear abnormalities as in HGPS cells and in cells expressing GFP-progerin. Exposure to a farnesyltransferase inhibitor (FTI), PD169541, caused a significant improvement in the nuclear morphology of cells expressing GFP-progerin and in HGPS cells. These results implicate the abnormal farnesylation of progerin in the cellular phenotype in HGPS cells and suggest that FTIs may represent a therapeutic option for patients with HGPS.

Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS)

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13→q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.

Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder that causes premature, rapid aging shortly after birth. Recently, de novo point mutations in the Lmna gene have been found in individuals with HGPS. Lmna encodes lamin A and C, the A-type lamins, which are an important structural component of the nuclear envelope. The most common HGPS mutation is located at codon 608 (G608G). This mutation creates a cryptic splice site within exon 11, which deletes a proteolytic cleavage site within the expressed mutant lamin A. Incomplete processing of prelamin A results in nuclear lamina abnormalities that can be observed in immunofluorescent studies of HGPS cells. Mouse models, such as Lmna knockout, Zmpste24 knockout, and Lmna L530P knockin will help the study of progeria. Lmna mutations have also recently been found in patients with atypical forms of progeria. The discovery of the HGPS mutations brings the total number of diseases caused by mutant Lmna to nine, underscoring the astonishing spectrum of laminopathies. Future research into HGPS could also provide important clues about the general process of aging and aging-related diseases.

Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.

Reduced DNA repair in progeria cells and effects of γ-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.