Mechanisms of genome instability in Hutchinson-Gilford progeria

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder. It arises from a single point mutation in the LMNA gene. This mutation stimulates an aberrant splicing event and produces progerin, an isoform of the lamin A protein. Accumulation of progerin disrupts numerous physiological pathways and induces defects in nuclear architecture, gene expression, histone modification, cell cycle regulation, mitochondrial functionality, genome integrity and much more.

Objective

Among these phenotypes, genomic instability is tightly associated with physiological aging and considered a main contributor to the premature aging phenotypes. However, our understanding of the underlying molecular mechanisms of progerin-caused genome instability is far from clear.

Results and Conclusion

In this review, we summarize some of the recent findings and discuss potential mechanisms through which, progerin affects DNA damage repair and leads to genome integrity.

     

Defective Lamin A-Rb Signaling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disorder caused by a de novo heterozygous point mutation G608G (GGC>GGT) within exon 11 of LMNA gene encoding A-type nuclear lamins. This mutation elicits an internal deletion of 50 amino acids in the carboxyl-terminus of prelamin A. The truncated protein, progerin, retains a farnesylated cysteine at its carboxyl terminus, a modification involved in HGPS pathogenesis. Inhibition of protein farnesylation has been shown to improve abnormal nuclear morphology and phenotype in cellular and animal models of HGPS. We analyzed global gene expression changes in fibroblasts from human subjects with HGPS and found that a lamin A-Rb signaling network is a major defective regulatory axis. Treatment of fibroblasts with a protein farnesyltransferase inhibitor reversed the gene expression defects. Our study identifies Rb as a key factor in HGPS pathogenesis and suggests that its modulation could ameliorate premature aging and possibly complications of physiological aging.     

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy.     

Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin

A‐lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A‐lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease‐associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson‐Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose‐ and E3 ligase‐dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic–lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A‐lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2–lamin A axis in age‐related diseases such as cancer.     

Suppression of proliferative defects associated with processing-defective lamin A mutants by hTERT or inactivation of p53

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of aging-associated pathologies. It is linked to mutations in LMNA, which encodes A-type nuclear lamins. The most frequent HGPS-associated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and, unlike normal A-type lamins, stable farnesylation. The cellular consequences of progerin expression underlying the HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly, expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes to those of cell immortalization.     

In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria

Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.     

早老症(HGPS)的发病机制与治疗策略

早老症(Hutchinson-Gilford Progeria Syndrome,HGPS)是一种早发而严重的过早老化性疾病.它是由于编码A/C型核纤层蛋白的LMNA基因发生点突变而引起.这个突变激活了基因11号外显子上一个隐蔽的剪接位点,产生了一种被截短了50个氨基酸的A型核纤层蛋白.然而,一个广泛分布于核膜上结构蛋白的突变,如何引起HGPS患者的早老表现,目前还不太清楚.最近研究发现,HGPS患者的细胞核结构与功能发生了各种异常,主要表现在:progerin蓄积与核变形、细胞核机械性质的改变、组蛋白修饰方式与外遗传控制的改变、基因表达调控异常、p53信号传导通路激活和基因组不稳定等方面.目前存在机械应激假说和基因表达失控假说两种假说解释HGPS的发病机制.对于HGPS患者,尚无有效的临床干预措施,但有学者提出了一些治疗策略,如应用法尼基化的抑制剂、反义寡核苷酸和RNA干扰方法.HGPS被认为是研究正常衰老机制的一个模型.对HGPS深入研究将有助于阐明A型核纤层蛋白和核膜的正常生理功能,及其在生理衰老和疾病中的作用.     

Sulforaphane enhances progerin clearance in Hutchinson–Gilford progeria fibroblasts

Hutchinson–Gilford progeria syndrome (HGPS, OMIM 176670) is a rare multisystem childhood premature aging disorder linked to mutations in the LMNA gene. The most common HGPS mutation is found at position G608G within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl‐terminal tail of prelamin A, and the truncated protein is called progerin. Progerin only undergoes a subset of the normal post‐translational modifications and remains permanently farnesylated. Several attempts to rescue the normal cellular phenotype with farnesyltransferase inhibitors (FTIs) and other compounds have resulted in partial cellular recovery. Using proteomics, we report here that progerin induces changes in the composition of the HGPS nuclear proteome, including alterations to several components of the protein degradation pathways. Consequently, proteasome activity and autophagy are impaired in HGPS cells. To restore protein clearance in HGPS cells, we treated HGPS cultures with sulforaphane (SFN), an antioxidant derived from cruciferous vegetables. We determined that SFN stimulates proteasome activity and autophagy in normal and HGPS fibroblast cultures. Specifically, SFN enhances progerin clearance by autophagy and reverses the phenotypic changes that are the hallmarks of HGPS. Therefore, SFN is a promising therapeutic avenue for children with HGPS.     

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 Lmna^G609G/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.     

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

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) LMNAmutation 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.     

Calcitriol Prevents RAD51 Loss and cGAS‐STING‐IFN Response Triggered by Progerin

Hutchinson Gilford progeria syndrome (HGPS) is a devastating accelerated aging disease caused by LMNA gene mutation. The truncated lamin A protein produced “progerin” has a dominant toxic effect in cells, causing disruption of nuclear architecture and chromatin structure, genomic instability, gene expression changes, oxidative stress, and premature senescence. It was previously shown that progerin‐induced genomic instability involves replication stress (RS), characterized by replication fork stalling and nuclease‐mediated degradation of stalled forks. RS is accompanied by activation of cGAS/STING cytosolic DNA sensing pathway and STAT1‐regulated interferon (IFN)‐like response. It is also found that calcitriol, the active hormonal form of vitamin D, rescues RS and represses the cGAS/STING/IFN cascade. Here, the mechanisms underlying RS in progerin‐expressing cells and the rescue by calcitriol are explored. It is found that progerin elicits a marked downregulation of RAD51, concomitant with increased levels of phosphorylated‐RPA, a marker of RS. Interestingly, calcitriol prevents RS and activation of the cGAS/STING/IFN response in part through maintenance of RAD51 levels in progerin‐expressing cells. Thus, loss of RAD51 is one of the consequences of progerin expression that can contribute to RS and activation of the IFN response. Stabilization of RAD51 helps explain the beneficial effects of calcitriol in these processes.     

Transient introduction of human telomerase mRNA improves hallmarks of progeria cells

Hutchinson–Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra‐individual heterogeneity of expression of telomere‐associated proteins DNA PKcs/Ku70/Ku80, with low‐expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere‐associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by β‐galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.     

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.     

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 (Lmna^G609G/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.     

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.     

Unique preservation of neural cells in Hutchinson- Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA

One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage.     

Progerin-expressing endothelial cells are unable to adapt to shear stress

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. Patients with HGPS experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study, we examined how progerin-expressing endothelial cells adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control endothelial cells to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in endothelial cells, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that endothelial cells either overexpressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). Endothelial cells overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing endothelial cells had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, the prealignment of cells to shear stress before progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of endothelial cells to properly adapt to shear stress.