POLR3A mutations cause nucleolus abnormalities and aberrant telomerase RNA metabolism in induced pluripotent stem cells from Wiedemann-Rautenstrauch premature aging syndrome patient.

Identification of mitochondrial dysfunction in Hutchinson–Gilford progeria syndrome through use of stable isotope labeling with amino acids in cell culture

Background/Objectives: Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal genetic disease caused by a silent mutation in the LMNA gene, leading to the production of progerin, a defective prelamin A variant. Progerin accumulation disrupts nuclear integrity, alters chromatin organization, and drives systemic cellular dysfunction. While autophagy and inflammation are key dysregulated pathways in HGPS, the role of microRNAs (miRNAs) in these processes remains poorly understood. Methods: We performed an extensive literature review to identify miRNAs involved in autophagy and inflammation. Through stem-loop RT-qPCR in aging HGPS and control fibroblast strains, we identified significant miRNAs and focused on the most prominent one, miR-181a-5p, for in-depth analysis. We validated our in vitro findings with miRNA expression studies in skin biopsies from an HGPS mouse model and conducted functional assays in human fibroblasts, including immunofluorescence staining, β-Galactosidase assay, qPCR, and Western blot analysis. Transfection studies were performed using an miR-181a-5p mimic and its inhibitor. Results: We identified miR-181a-5p as a critical regulator of premature senescence in HGPS. miR-181a-5p was significantly upregulated in HGPS fibroblasts and an HGPS mouse model, correlating with Sirtuin 1 (SIRT1) suppression and induction of senescence. Additionally, we demonstrated that TGFβ1 induced miR-181a-5p expression, linking inflammation to miRNA-mediated senescence. Inhibiting miR-181a-5p restored SIRT1 levels, increased proliferation, and alleviated senescence in HGPS fibroblasts, supporting its functional relevance in disease progression. Conclusions: These findings highlight the important role of miR-181a-5p in premature aging and suggest its potential as a therapeutic target for modulating senescence in progeroid syndromes.

ABSTRACTThe nuclear lamina is a proteinaceous meshwork situated underneath the inner nuclear membrane and is composed of nuclear lamin proteins, which are type-V intermediate filaments. The LMNA gene gives rise to lamin A and lamin C through alternative splicing. Mutations in LMNA cause multiple diseases known as laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder caused by a point mutation that activates a cryptic 5′ splice site in exon 11, resulting in a 150 bp deletion in the LMNA mRNA and the production of the dominant lamin A isoform progerin. During RNA sequencing analysis of wild type and HGPS patient skin fibroblasts, we discovered two novel LMNA isoforms. LMNAΔ447 and LMNAΔ297 use an alternative 3′ splice acceptor site in the 3′ untranslated region, and either the HGPS cryptic 5′ splice site in exon 11 or the wild type 5′ splice site. Both isoforms are present at low levels in HGPS patient and wild type cells in multiple cell types. We validate and quantify the expression levels of these novel isoforms in HGPS and wild type fibroblasts. Overexpression of either LMNAΔ447 or LMNAΔ297 is not sufficient to induce the typical HGPS cellular disease phenotypes and no significant difference in the two isoforms were found between young and old fibroblasts. These results identify and characterize two novel RNA isoforms of LMNA produced through alternative splicing.

Aging is a universal and inevitable process that affects virtually all living organisms; in humans, aging is characterized by a gradual decline of physical and psychological functions that ultimately leads to death. Over the past decades, the study of progeroid syndromes, a group of premature aging disorders that recapitulates multiple features of physiological aging, has provided insightful information toward the identification of mechanisms underlying aging. In this chapter, we provide an updated description of the main progeroid syndromes affecting humans, including their clinical manifestations and the genetic and molecular basis underlying their pathogenesis. Most progeroid syndromes originate from defective DNA repair and nuclear structure systems, highlighting a key role of genome stability in aging. A special emphasis is given to Hutchinson Gilford Progeria Syndrome (HGPS), the most well-studied premature aging disorder, which is characterized by accelerated aging and early death due to cardiovascular complications. HGPS is typically caused by a silent mutation in the LMNA gene that provokes the expression of progerin, a dominant-negative mutant protein that anchors aberrantly to the nuclear envelope, thereby inducing cellular toxicity and organismal detriment. Thus, we provide a description of established cellular and animal models for HGPS and discuss the perspectives for therapeutic developments, including an updated presentation of treatment strategies that have been tested so far in vitro (human HGPS fibroblast cultures) and in vivo (HGPS mice models) and in clinical trials, with argumentation of their main limitations.

Metformin is a popular antidiabetic biguanide, which has been considered as a candidate drug for cancer treatment and ageing prevention. Hutchinson-Gilford progeria syndrome (HGPS) is a devastating disease characterized by premature ageing and severe age-associated complications leading to death. The effects of metformin on HGPS dermal fibroblasts remain largely undefined. In this study, we investigated whether metformin could exert a beneficial effect on nuclear abnormalities and delay senescence in fibroblasts derived from HGPS patients. Metformin treatment partially restored normal nuclear phenotypes, delayed senescence, activated the phosphorylation of AMP-activated protein kinase and decreased reactive oxygen species formation in HGPS dermal fibroblasts. Interestingly, metformin reduced the number of phosphorylated histone variant H2AX-positive DNA damage foci and suppressed progerin protein expression, compared to the control. Furthermore, metformin-supplemented aged mice showed higher splenocyte proliferation and mRNA expression of the antioxidant enzyme, superoxide dismutase 2 than the control mice. Collectively, our results show that metformin treatment alleviates the nuclear defects and premature ageing phenotypes in HGPS fibroblasts. Thus, metformin can be considered a promising therapeutic approach for life extension in HGPS.

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

Prelamin A prenylation and the treatment of progeria

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 Δ133p53 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 Δ133p53 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 expression of the 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 Δ133p53 expression may be a novel therapeutic strategy to treat aging-associated phenotypes of HGPS in vivo.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

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.

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.

The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin, an inhibitor of the mTOR pathway, reduced nuclear blebbing in HGPS fibroblasts. However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective. Here, we show a novel, automated and high throughput nuclear shape analysis that quantitatively measures curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells. We examined HGPS fibroblast cells treated with rapamycin and RAD001 (an analog to rapamycin). Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls. In addition, we find that rapamycin treatment reduces the area of the nucleus, but leaves the eccentricity unchanged. Our nuclear shape analysis provides an unbiased, multidimensional “fingerprint” for a population of cells, which can be used to quantify treatment efficacy and analyze cellular aging.

Premature aging syndromes are a group of ultra-rare heterogeneous hereditary diseases that manifest predominantly in childhood and are characterized by accelerated aging of the organism. Despite differences in pathogenesis, the diseases are characterized by multisystem changes, including lesions of the musculoskeletal system, which are represented by multiple joint contractures, deformations of the spine and limbs, and changes in bone structure. The examination data of 6 patients were analyzed: 5 children (3 boys and 2 girls) with pediatric progeria (Hutchinson-Gilford syndrome) (4 patients with classic genotype of pediatric progeria (c.1824 C>T in the LMNA gene) and 1 child with a non-classical (c.1968+1G>A in the LMNA gene)) and one girl with neonatal progeria (Wiedemann-Rautenstrauch syndrome) (c.3337- 11T>C/ c.3677T>C in the POLR3A gene). The diagnosis was made at the age of 1.9 (1.5; 4.3) years (Me (25%; 75%)). Patients were under the supervision of a pediatric endocrinologist, examined by an orthopedic traumatologist, radiologic studies, and densitometry of the lumbar spine were performed. The age at the time of the initial examination was 6.0 (3.5; 7.2) years, and at the time of the repeated examination — 7.6 (7.5; 9.3) years. Bone and joint changes in Hutchinson-Gilford syndrome were represented by contractures of interphalangeal joints of fingers and toes, wrist, elbow, hip, knee, and ankle joints, and flat-valgus feet; in a patient with a nonclassical genotype of pediatric progeria, these changes were diagnosed at the first examination at 1 year 6 months of age, which confirms the severe course of the disease in this genotype. In 2 older patients (7 years 5 months and 9 years 10 months) coxa valga on 2 sides with development of aseptic necrosis of the femoral head and closed dislocation of the left femur were also diagnosed. In neonatal progeroid syndrome, musculoskeletal lesions were manifested as multiple contractures of large and small joints and spinal deformity. Bone age either corresponded to the chronologic age or lagged behind by 18 (15; 26) months All patients were diagnosed with osteoporosis according to densitometry (Z-criterion: -3.4 (-3.0; -3.8)); no fractures were recorded. The revealed changes in bone tissue and musculoskeletal system in our patients correspond to the features described in the world literature in patients with Hutchinson-Gilford and Wiedemann-Rautenstrauch syndromes. The similarity of pathologic changes indicates the similarity of phenotypes of diseases included in the group of premature aging syndromes.

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) is a rare, premature ageing syndrome in children. HGPS is normally caused by a mutation in the LMNA gene, encoding nuclear lamin A. The classical mutation in HGPS leads to the production of a toxic truncated version of lamin A, progerin, which retains a farnesyl group. Farnesyltransferase inhibitors (FTI), pravastatin and zoledronic acid have been used in clinical trials to target the mevalonate pathway in HGPS patients to inhibit farnesylation of progerin, in order to reduce its toxicity. Some other compounds that have been suggested as treatments include rapamycin, IGF1 and N-acetyl cysteine (NAC). We have analysed the distribution of prelamin A, lamin A, lamin A/C, progerin, lamin B1 and B2 in nuclei of HGPS cells before and after treatments with these drugs, an FTI and a geranylgeranyltransferase inhibitor (GGTI) and FTI with pravastatin and zoledronic acid in combination. Confirming other studies prelamin A, lamin A, progerin and lamin B2 staining was different between control and HGPS fibroblasts. The drugs that reduced progerin staining were FTI, pravastatin, zoledronic acid and rapamycin. However, drugs affecting the mevalonate pathway increased prelamin A, with only FTI reducing internal prelamin A foci. The distribution of lamin A in HGPS cells was improved with treatments of FTI, pravastatin and FTI + GGTI. All treatments reduced the number of cells displaying internal speckles of lamin A/C and lamin B2. Drugs targeting the mevalonate pathway worked best for progerin reduction, with zoledronic acid removing internal progerin speckles. Rapamycin and NAC, which impact on the MTOR pathway, both reduced both pools of progerin without increasing prelamin A in HGPS cell nuclei.