Hutchinson-Gilford Progeria Syndrome Fibroblasts Research Articles

Enhancing Cellular Homeostasis: Targeted Botanical Compounds Boost Cellular Health Functions in Normal and Premature Aging Fibroblasts.

The human skin, the body's largest organ, undergoes continuous renewal but is significantly impacted by aging, which impairs its function and leads to visible changes. This study aimed to identify botanical compounds that mimic the anti-aging effects of baricitinib, a known JAK1/2 inhibitor. Through in silico screening of a botanical compound library, 14 potential candidates were identified, and 7 were further analyzed for their effects on cellular aging. The compounds were tested on both normal aged fibroblasts and premature aging fibroblasts derived from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). Results showed that these botanical compounds effectively inhibited the JAK/STAT pathway, reduced the levels of phosphorylated STAT1 and STAT3, and ameliorated phenotypic changes associated with cellular aging. Treatments improved cell proliferation, reduced senescence markers, and enhanced autophagy without inducing cytotoxicity. Compounds, such as Resveratrol, Bisdemethoxycurcumin, Pinosylvin, Methyl P-Hydroxycinnamate, cis-Pterostilbene, and (+)-Gallocatechin, demonstrated significant improvements in both control and HGPS fibroblasts. These findings suggest that these botanical compounds have the potential to mitigate age-related cellular alterations, offering promising strategies for anti-aging therapies, particularly for skin health. Further in vivo studies are warranted to validate these results and explore their therapeutic applications.

12604 Use Of A Novel Program "WordMiner" To Identify Cellular Proliferation Related Genes Induced In FSTL3 KO Mouse Testis

Abstract Disclosure: N. Mukherjee: None. R. Ballesteros: None. A. Mukherjee: None. TGFβ family ligands play crucial roles in promoting cellular proliferation. Moreover, activin and TGFβ are linked with cancer. Deletion of FSTL3, an inhibitor of a subset of TGFβ ligands, in mice leads to increased proliferation in all tissues investigated including the testis. We also find increased SMAD2/3 activation in FSTL3 knockout (KO) compared to wildtype (WT) in these tissues. Therefore, the FSTL3 KO mouse is a model of increased activity of endogenous TGFβ ligands that are inhibited by FSTL3, such as activin. It is likely that increased signalling by these ligands leads to the observed increased cellular proliferation in FSTL3 KO mice. To discover genes that might be involved in activin-dependent mitosis, we studied gene expression datasets linked to cellular proliferation. We hypothesised that genes important in cellular proliferation will be downregulated in senescent cells and upregulated in models of increased cellular proliferation and activin action such as the FSTL3 KO mice. We queried GEO datasets to identify studies related to senescence and selected Hutchinson-Gilford progeria syndrome (HGPS): fibroblast (HG-U133A), GSE3860. We found 4048 transcripts showing significantly (p<0.05) altered expression in HGPS fibroblasts compared to WT. We developed a Python-based program, “WordMiner”, that inputs a keyword and a list of terms (e.g. genes) and then searches a specified database sequentially for publications including the keyword and each term. The output is a table of search result frequencies and hyperlinks to the search result pages, compiled in an auxiliary file, produced with greater speed and efficiency than manual search methods. The greater the search result frequency, the stronger the known link between the keyword and the term. We used WordMiner to query the association of the search term “mitosis” with the 4048 genes above using PubMed as the database and identified 1235 transcripts. We refined this list further using the keyword “activin” to identify 420 transcripts linked to activin and mitosis. Importantly, by comparing against our RNAseq data, we find 20 of these genes upregulated in the FSTL3 KO postnatal day 3 transcriptome. This finding supports our hypothesis, and also demonstrates WordMiner as an efficient and versatile cross-referencing query tool. Among the 20 genes identified are transcripts known to be important in proliferation, such as Akap9 and Wnt2b. These and the remainder in the list of genes highlighted are therefore likely to be involved in pathways induced by activin, promoting cellular proliferation in FSTL3 KO testis. Currently, siRNA transfection experiments are underway to address whether knockdown of these genes in a Sertoli cell line (TM4) limits proliferation. This will help in the elucidation of the molecular pathways involved in postnatal Sertoli cell proliferation. Presentation: 6/3/2024

Cardiac and skeletal muscle manifestations in the G608G mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder resulting from de novo mutations in the lamin A gene. Children with HGPS typically pass away in their teenage years due to cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure, and stroke. In this study, we characterized the G608G HGPS mouse model and explored cardiac and skeletal muscle function, along with senescence-associated phenotypes in fibroblasts. Homozygous G608G HGPS mice exhibited cardiac dysfunction, including decreased cardiac output and stroke volume, and impaired left ventricle relaxation. Additionally, skeletal muscle exhibited decreased isometric tetanic torque, muscle atrophy, and increased fibrosis. HGPS fibroblasts showed nuclear abnormalities, decreased proliferation, and increased expression of senescence markers. These findings provide insights into the pathophysiology of the G608G HGPS mouse model and inform potential therapeutic strategies for HGPS.

Doxycycline decelerates aging in progeria mice.

Beyond the antimicrobial activity, doxycycline (DOX) exhibits longevity-promoting effect in nematodes, while its effect on mammals is unclear. Here, we applied a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), Zmpste24 knockout (KO) mice, and analyzed the antiaging effect of DOX. We found that the DOX treatment prolongs lifespan and ameliorates progeroid features of Zmpste24 KO mice, including the decline of body and tissue weight, exercise capacity and cortical bone density, and the shortened colon length. DOX treatment alleviates the abnormal nuclear envelope in multiple tissues, and attenuates cellular senescence and cell death of Zmpste24 KO and HGPS fibroblasts. DOX downregulates the level of proinflammatory IL6 in both serum and tissues. Moreover, the elevated α-tubulin (K40) acetylation mediated by NAT10 in progeria, is rescued by DOX treatment in the aorta tissues in Zmpste24 KO mice and fibroblasts. Collectively, our study uncovers that DOX can decelerate aging in progeria mice via counteracting IL6 expression and NAT10-mediated acetylation of α-tubulin.

Ghrelin delays premature aging in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition that arises from a single nucleotide alteration in the LMNA gene, leading to the production of a defective lamin A protein known as progerin. The accumulation of progerin accelerates the onset of a dramatic premature aging phenotype in children with HGPS, characterized by low body weight, lipodystrophy, metabolic dysfunction, skin, and musculoskeletal age-related dysfunctions. In most cases, these children die of age-related cardiovascular dysfunction by their early teenage years. The absence of effective treatments for HGPS underscores the critical need to explore novel safe therapeutic strategies. In this study, we show that treatment with the hormone ghrelin increases autophagy, decreases progerin levels, and alleviates other cellular hallmarks of premature aging in human HGPS fibroblasts. Additionally, using a HGPS mouse model (LmnaG609G/G609G mice), we demonstrate that ghrelin administration effectively rescues molecular and histopathological progeroid features, prevents progressive weight loss in later stages, reverses the lipodystrophic phenotype, and extends lifespan of these short-lived mice. Therefore, our findings uncover the potential of modulating ghrelin signaling offers new treatment targets and translational approaches that may improve outcomes and enhance the quality of life for patients with HGPS and other age-related pathologies.

Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification.

The expression of a mutant Lamin A, progerin, in Hutchinson-Gilford Progeria Syndrome leads to alterations in genome architecture, nuclear morphology, epigenetic states, and altered phenotypes in all cells of the mesenchymal lineage. Here, we report a comprehensive analysis of the transcriptional status of patient derived HGPS fibroblasts, including nine cell lines not previously reported, in comparison with age-matched controls, adults, and old adults. We find that Progeria fibroblasts carry abnormal transcriptional signatures, centering around several functional hubs: DNA maintenance and epigenetics, bone development and homeostasis, blood vessel maturation and development, fat deposition and lipid management, and processes related to muscle growth. Stratification of patients by age revealed misregulated expression of genes related to endochondral ossification and chondrogenic commitment in children aged 4-7 years old, where this differentiation program starts in earnest. Hi-C measurements on patient fibroblasts show weakening of genome compartmentalization strength but increases in TAD strength. While the majority of gene misregulation occurs in regions which do not change spatial chromosome organization, some expression changes in key mesenchymal lineage genes coincide with lamin associated domain misregulation and shifts in genome compartmentalization.

Lamin A/C impairments cause mitochondrial dysfunction by attenuating PGC1α and the NAMPT-NAD+ pathway.

Mutations in the lamin A/C gene (LMNA) cause laminopathies such as the premature aging Hutchinson Gilford progeria syndrome (HGPS) and altered lamin A/C levels are found in diverse malignancies. The underlying lamin-associated mechanisms remain poorly understood. Here we report that lamin A/C-null mouse embryo fibroblasts (Lmna−/− MEFs) and human progerin-expressing HGPS fibroblasts both display reduced NAD+ levels, unstable mitochondrial DNA and attenuated bioenergetics. This mitochondrial dysfunction is associated with reduced chromatin recruitment (Lmna−/− MEFs) or low levels (HGPS) of PGC1α, the key transcription factor for mitochondrial homeostasis. Lmna−/− MEFs showed reduced expression of the NAD+-biosynthesis enzyme NAMPT and attenuated activity of the NAD+-dependent deacetylase SIRT1. We find high PARylation in lamin A/C-aberrant cells, further decreasing the NAD+ pool and consistent with impaired DNA base excision repair in both cell models, a condition that fuels DNA damage-induced PARylation under oxidative stress. Further, ATAC-sequencing revealed a substantially altered chromatin landscape in Lmna−/− MEFs, including aberrantly reduced accessibility at the Nampt gene promoter. Thus, we identified a new role of lamin A/C as a key modulator of mitochondrial function through impairments of PGC1α and the NAMPT-NAD+ pathway, with broader implications for the aging process.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

Impact of MnTBAP and Baricitinib Treatment on Hutchinson-Gilford Progeria Fibroblasts.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature aging disease. It is caused by a mutation in the LMNA gene, which results in a 50-amino-acid truncation of prelamin A. The resultant truncated prelamin A (progerin) lacks the cleavage site for the zinc-metallopeptidase ZMPSTE24. Progerin is permanently farnesylated, carboxymethylated, and strongly anchored to the nuclear envelope. This leads to abnormalities, such as altered nuclear shape, mitochondrial dysfunction, and inflammation. HGPS patients display symptoms of physiological aging, including atherosclerosis, alopecia, lipodystrophy, and arthritis. Currently, no cure for HGPS exists. Here we focus on a drug combination consisting of the superoxide dismutase mimetic MnTBAP and JAK1/2 inhibitor baricitinib (Bar) to restore phenotypic alterations in HGPS fibroblasts. Treating HGPS fibroblasts with the MnTBAP/Bar combination improved mitochondrial functions and sustained Bar’s positive effects on reducing progerin and pro-inflammatory factor levels. Collectively, MnTBAP/Bar combination treatment ameliorates the aberrant phenotype of HGPS fibroblasts and is a potential treatment strategy for patients with HGPS.

Baricitinib, a JAK-STAT Inhibitor, Reduces the Cellular Toxicity of the Farnesyltransferase Inhibitor Lonafarnib in Progeria Cells.

Hutchinson–Gilford progeria syndrome (HGPS) is an ultra-rare multisystem premature aging disorder that leads to early death (mean age of 14.7 years) due to myocardial infarction or stroke. Most cases have a de novo point mutation at position G608G within exon 11 of the LMNA gene. This mutation leads to the production of a permanently farnesylated truncated prelamin A protein called “progerin” that is toxic to the cells. Recently, farnesyltransferase inhibitor (FTI) lonafarnib has been approved by the FDA for the treatment of patients with HGPS. While lonafarnib treatment irrefutably ameliorates HGPS disease, it is however not a cure. FTI has been shown to cause several cellular side effects, including genomic instability as well as binucleated and donut-shaped nuclei. We report that, in addition to these cellular stresses, FTI caused an increased frequency of cytosolic DNA fragment formation. These extranuclear DNA fragments colocalized with cGAs and activated the cGAS-STING-STAT1 signaling axis, upregulating the expression of proinflammatory cytokines in FTI-treated human HGPS fibroblasts. Treatment with lonafarnib and baricitinib, a JAK-STAT inhibitor, not only prevented the activation of the cGAS STING-STAT1 pathway, but also improved the overall HGPS cellular homeostasis. These ameliorations included progerin levels, nuclear shape, proteostasis, cellular ATP, proliferation, and the reduction of cellular inflammation and senescence. Thus, we suggest that combining lonafarnib with baricitinib might provide an opportunity to reduce FTI cellular toxicity and ameliorate HGPS symptoms further than lonafarnib alone.

Impact of Progerin Expression on Adipogenesis in Hutchinson-Gilford Progeria Skin-Derived Precursor Cells.

Hutchinson–Gilford progeria syndrome (HGPS) is a segmental premature aging disease caused by a mutation in LMNA. The mutation generates a truncated and farnesylated form of prelamin A, called progerin. Affected individuals develop several features of normal aging, including lipodystrophy caused by the loss of general subcutaneous fat. To determine whether premature cellular senescence is responsible for the altered adipogenesis in patients with HGPS, we evaluated the differentiation of HGPS skin-derived precursor stem cells (SKPs) into adipocytes. The SKPs were isolated from primary human HGPS and normal fibroblast cultures, with senescence of 5 and 30%. We observed that the presence of high numbers of senescent cells reduced SKPs’ adipogenic differentiation potential. Treatment with baricitinib, a JAK–STAT inhibitor, ameliorated the ability of HGPS SKPs to differentiate into adipocytes. Our findings suggest that the development of lipodystrophy in patients with HGPS may be associated with an increased rate of cellular senescence and chronic inflammation.

Interleukin-6 neutralization ameliorates symptoms in prematurely aged mice.

Hutchinson–Gilford progeria syndrome (HGPS) causes premature aging in children, with adipose tissue, skin and bone deterioration, and cardiovascular impairment. In HGPS cells and mouse models, high levels of interleukin‐6, an inflammatory cytokine linked to aging processes, have been detected. Here, we show that inhibition of interleukin‐6 activity by tocilizumab, a neutralizing antibody raised against interleukin‐6 receptors, counteracts progeroid features in both HGPS fibroblasts and LmnaG609G / G609G progeroid mice. Tocilizumab treatment limits the accumulation of progerin, the toxic protein produced in HGPS cells, rescues nuclear envelope and chromatin abnormalities, and attenuates the hyperactivated DNA damage response. In vivo administration of tocilizumab reduces aortic lesions and adipose tissue dystrophy, delays the onset of lipodystrophy and kyphosis, avoids motor impairment, and preserves a good quality of life in progeroid mice. This work identifies tocilizumab as a valuable tool in HGPS therapy and, speculatively, in the treatment of a variety of aging‐related disorders.

ESCRT-III controls nuclear envelope deformation induced by progerin

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome.

Hutchinson–Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle‐derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24 −/− (Z24 −/−) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin‐induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F‐actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei‐induced cGAS‐Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24− / − mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.

Peroxisomal abnormalities and catalase deficiency in Hutchinson-Gilford Progeria Syndrome.

Peroxisomes are small, membrane-enclosed eukaryotic organelles that house various enzymes with metabolic functions. One important feature in both Hutchinson-Gilford Progeria Syndrome (HGPS) and normal aging is the elevated levels of Reactive Oxygen Species (ROS), which are generated from metabolic pathways with the capacity to cause oxidative damage to macromolecules within the cells. Although peroxisomal bioreactions can generate free radicals as their byproducts, many metabolic enzymes within the peroxisomes play critical roles as ROS scavengers, in particular, catalase. Here, we observed impaired peroxisomes-targeting protein trafficking, which suggested that the poorly assembled peroxisomes might cause high oxidative stress, contributing to the premature senescent phenotype in HGPS. We then investigated the ROS clearance efficiency by peroxisomal enzymes and found a significantly decreased expression of catalase in HGPS. Furthermore, we evaluated the effects of two promising HGPS-treatment drugs Methylene Blue and RAD001 (Everolimus, a rapamycin analog) on catalase in HGPS fibroblasts. We found that both drugs effectively reduced cellular ROS levels. MB, as a well-known antioxidant, did not affect catalase expression or activity. Interestingly, RAD001 treatment significantly upregulated catalase activity in HGPS cells. Our study presents the first characterization of peroxisomal function in HGPS and provides new insights into the cellular aspects of HGPS and the ongoing clinical trial.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease—Hutchinson‐Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long‐term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells.Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT‐immortalized cell lines.

A nuclear lamina-chromatin-Ran GTPase axis modulates nuclear import and DNA damage signaling.

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.

P53 isoforms regulate premature aging in human cells.

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.

Identification of novel RNA isoforms of LMNA

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.

Intermittent treatment with farnesyltransferase inhibitor and sulforaphane improves cellular homeostasis in Hutchinson-Gilford progeria fibroblasts.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic condition associated with mutations in the LMNA gene. This disease recapitulates some aspects of normal aging, such as hair loss, thin skin, joint stiffness, and atherosclerosis. The latter leads to heart attack or stroke that causes death at an average age of 14.6 years in children with HGPS. The typical LMNA mutation results in the production of a truncated prelamin A protein, progerin, that remains permanently farnesylated and abnormally associated with the nuclear envelope. Farnesyltransferase inhibitors (FTIs) reverse nuclear structure abnormalities that are characteristic of HGPS cells. The first clinical trial using the FTI, Ionafarnib, demonstrated some improvements in HGPS children and, in particular, showed a decrease in arterial stiffness. Recently, we reported that sulforaphane, an antioxidant derived from cruciferous vegetables, efficiently stimulates autophagy and enhances progerin clearance in HGPS fibroblasts. In the present study, we investigated the effect of combined lonafarnib and sulforaphane treartment in HGPS fibroblast cultures. We report that co-administration of both drugs exerts a synergistic and additive positive effect on autophagy activity but was cytotoxic to HGPS cells. In contrast, intermittent treatment with lonafarnib followed by sulforaphane separately and in repeated cycles rescued the HGPS cellular phenotype. We propose that intermittent treatment with FTI and SFN separately might be a promising therapeutic avenue for children with HGPS.