Hutchinson-Gilford Progeria Syndrome: Genetic Insights, Clinical Challenges, and Innovative Therapeutic Approaches.

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24-deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24-deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24-deficient mouse cells lacking Icmt, indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24-deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24-deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24-deficient mouse cells lacking Icmt, indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.

Prelamin A prenylation and the treatment of progeria

Objective To assess clinicopathological features of and genetic factors in Hutchinson-Gilford progeria syndrome（HGPS）in a family. Methods General information was collected from 3 patients with Hutchinson-Gilford progeria syndrome in a family, which included 5 members over 2 generations with all the 3 children affected by HGPS. All the 3 patients underwent clinical investigation, image analysis of hands, lungs and mandibles, as well as karyotype analysis of chromosomes. LMNA gene mutations were analyzed in these family members. Results All the 3 patients developed skin sclerosis with severe growth retardation and appearance of extreme aging at about 6 months of age. Image analysis showed osteoporosis and mandibular hypoplasia in the elder patient. Karyotype analysis showed no abnormality in the patients or their parents. Mutation analysis revealed a homozygous mutation 1579 C > T（R527C）in exon 9 of the LMNA gene in all the patients, but a heterozygous mutation R527C in the LMNA gene in their parents. Conclusions The patients in this family present characteristic manifestations of HGPS, which may be caused by the homozygous LMNA mutation R527C. Key words: Progeria; LMNA gene; DNA mutational analysis; Hutchinson-Gilford syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal childhood premature aging disorder caused by a pre-messenger RNA (mRNA) splicing defect in the LMNA gene. We used combined in vitro screening and in vivo validation to systematically explore the effects of target sequence, backbone chemistry and mechanism of action to identify optimized antisense oligonucleotides (ASOs) for therapeutic use in HGPS. In a library of 198 ASOs, the most potent ASOs targeted the LMNA exon 12 junction and acted via non-RNase H-mediated mechanisms. Treatment with an optimized lead candidate resulted in extension of lifespan in a mouse model of HGPS. Progerin mRNA levels were robustly reduced in vivo, but the extent of progerin protein reduction differed between tissues, suggesting a long half-life and tissue-specific turnover of progerin in vivo. These results identify a novel therapeutic agent for HGPS and provide insight into the HGPS disease mechanism.

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.

Understanding the interplay between reversible epigenetic changes and potentially more difficult to reverse accumulation of damaged macromolecules is a central challenge in developing treatments for aging-associated dysfunction. One hypothesis is that epigenetic drift leads to subtle losses of homeostatic maintenance mechanisms, that in turn, lead to the accumulation of damaged macromolecules, which then further degrade homeostasis. A key mechanism of maintaining optimal cell function is asymmetrical division, whereby cellular damage is segregated away from cells that need to undergo further proliferation, such as stem cells. Such asymmetrical distribution of damaged macromolecules has been observed during cell division in many organisms, from yeast to human embryonic stem cells, and depends on diffusion barriers (DBs) in the membrane of the endoplasmic reticulum (ER). In a recent study, these results have been extended to neural stem cells (NSCs), in which the ability of the ER DB to promote asymmetrical distribution of damaged proteins deteriorates with age. NSC function declines with age as proliferative capacity is reduced. The loss of asymmetric protein distribution correlates with the loss of NSC proliferative capacity. Ectopic expression of progerin, an altered form of lamin A, is associated with the premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS). Progerin's expression also increases with normal aging due to mis-splicing, weakening the ER DB. Recent work suggests that many cell signaling pathway changes associated with HGPS are replicated during normal aging in cultured cells. Moreover, the detrimental changes associated with progerin expression in HGPS are partially reversible experimentally after treatment with statins, a farnesyltransferase inhibitor, a isoprenylcysteine carboxyl methyltransferase inhibitor, or sulforaphane. It will be of great interest if these compounds can also reverse the aging-associated permeability of the ER DB and restore stem cell function.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disorder that causes premature aging, affecting approximately one in 4-8 million births. Most cases result from a mutation in the lamin A/C (LMNA) gene, leading to the production of progerin, an aberrant lamin A variant that disrupts nuclear architecture and alters gene expression, including microRNA (miRNA) deregulation. This study aimed to investigate the molecular mechanisms underlying HGPS and aging using global miRNA sequencing to identify key deregulated miRNAs. Both miR-145 and miR-27b were significantly altered in HGPS. Functional experiments further revealed their crucial role in adipogenesis. Downregulation of these miRNAs in HGPS cells enhanced adipocyte differentiation, whereas their upregulation in control cells suppressed this process. These findings indicate that elevated levels of miR-145-5p and miR-27b-3p impair adipogenesis, providing mechanistic insights into HGPS pathophysiology and highlight new potential therapeutic avenues for both HGPS and metabolic disorders.

Hutchinson-Gilford progeria (HGPS) is a rare, genetic progeroid disorder that causes premature ageing, nuclear lamina shape abnormalities, growth impairment, and early death at ~13 year of age (Gordon et al., 2013). The disorder is a result of a spontaneous point mutation in the gene lamin A/C (LMNA) which encodes for the nuclear lamina scaffold protein, prelamin A. The most common point mutation occurs within exon 11 and results in a silent Gly-to-Gly mutation that causes increased usage of an internal cryptic splice site. This cryptic splice site produces a truncated form of prelamin A known as progerin (Eriksson et al., 2003). In non-mutated cells, prelamin A undergoes a series of modifications to produce lamin A, a vital nuclear lamina structural protein. Prelamin A contains a carboxyterminal CAAX motif that is farnesylated on the CAAX motif cysteine by farnesyltransferase (FTase). Farnesylation then targets prelamin A to the inner nuclear membrane where the last three amino acids are cleaved by zinc metallopeptidase STE24 (ZMPSTE24). This is followed by immediate methylation of the farnesylcysteine by isoprenylcysteine carboxyl methyltransferase (ICMT) and subsequent cleavage by ZMPSTE24 to produce lamin A. Following cleavage from the nuclear membrane, lamin A is capable of migrating to the nucleoplasm. In HGPS, progerin lacks a vital cleavage site utilized by ZMPSTE24. This results in progerin remaining permanently attached to the inner nuclear membrane and is suspected to contribute the HGPS phenotype (Fantle et al., 1994; Davies et al., 2009). Although previous studies have been dedicated to treating HGPS by halting farnesylation via FTase inhibitors, Ibrahim et al. targeted Icmt expression as a means to reverse progeria-like symptoms in a mouse model of HGPS (Ibrahim et al., 2013). Methylation of other CAAX protein motifs has been shown to play a role in protein membrane targeting (Bergo et al., 2002; Michaelson et al., 2005). To explore the role of CAAX methylation in HGPS, Ibrahim et al. introduced a hypomorphic allele of Icmt into a mouse model of HGPS that utilizes Zmpste24 deficient mice. It was found that the mice hypomorphic for Icmt had increased body weight, normalized grip strength, decreased bone fractures and decreased death compared to non-hypomorphic mice. Analysis of primary mouse embryonic fibroblasts obtained from sacrificed mice indicate that reduced ICMT activity does not affect levels of prelamin A, but instead causes mislocalization of prelamin A away from the nuclear rim. Interestingly, there is not a subsequent reduction in the number of misshapen nuclei in Icmt hypomorphic mice fibroblasts. This indicates that misshapen nuclei play a lesser role in the HGPS phenotype than previously thought. Icmt hypomorphic mice fibroblasts also restored cell proliferation to rates similar to wild type fibroblasts. To further elucidate the role of ICMT within cell proliferation, Ibrahim et al. evaluated the impact of ICMT on the on AKT-mTOR cell growth, proliferation and survival pathway. It was found that reduced ICMT activity triggers prelamin A dependent activation of AKT-mTOR, which decreases premature senescence of Zmpste24 deficient fibroblasts. Furthermore, ICMT activates the pathway through AKT and not mTOR. The precise mechanism of interaction was not determined. The findings of the Ibrahim et al. study are interesting not only to the field of progeroid disorders, but to the field of ageing studies at large. Scaffidi and Misteli found that dermal fibroblasts with wild type LMNA are capable of utilizing the cryptic spice site seen in HGPS cells. The truncated protein was not found to accumulate with age, but the localization of the protein shifted from the nucleoplasm to the nuclear rim with increasing age (Scaffidi and Misteli, 2006). Other studies have found mixed results with some reporting a direct correlation between progerin accumulation and age and others finding no association (McClintock et al., 2007; Cao et al., 2011). Progerin accumulation has also been linked to telomere dysfunction in normal human fibroblasts. Cells utilizing the LMNA cryptic splice site have shorter telomeres and high senescence-associated β-gal activity (Cao et al., 2011). Thus, the LMNA splice site plays a critical role not only in HGPS but also in normal ageing and cellular senescence. The Ibrahim et al. study has provided promising results for preventing progerin accumulation at the nuclear rim by reducing ICMT activity. Finding endogenous and exogenous mediators that can control the ICMT activity seen in aging conditions is an important step to discover pharmaceutical intervention and even possible reverse aging. Understanding the influence of ICMT in aging is likely to provide important insights that will not only guide investigation of the molecular and cellular basis of aging, but may also help to identify novel treatment strategies targeting these pathways.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. The majority of HGPS cases are caused by a de novo point mutation in the LMNA gene (c.1824C>T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes normal angiogenic function in HGPS. Here, we use human iPSC‐derived endothelial cell (iPSC‐EC) models to study angiogenesis in HGPS. We cultured normal and HGPS iPSC‐ECs under both static and fluidic culture conditions. HGPS iPSC‐ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared with normal controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary‐like microvascular network formation. Furthermore, the expression of matrix metalloproteinase 9 (MMP‐9) was reduced in HGPS iPSC‐ECs, while the expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) was upregulated relative to healthy controls. Finally, we used an adenine base editor (ABE7.10max‐VRQR) to correct the pathogenic c.1824C>T allele in HGPS iPSC‐ECs. Remarkably, ABE7.10max‐VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized the misregulated TIMPs, and restored angiogenic competence in HGPS iPSC‐ECs. Together, these results provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE could be a promising therapeutic approach for correcting HGPS‐related cardiovascular phenotypes.

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

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

Background:Isoprenylcysteine carboxyl methyltransferase (ICMT) is an enzyme crucial for the post-translational processing of Ras oncoproteins. Pharmacological inhibition of ICMT can mislocalize Ras and disrupt oncogenic signaling, exhibiting anticancer effects. Radicicol (RAD) is a 14-membered resorcylic acid lactone biosynthesized by fungi and possesses diverse bioactivities.Objective:The current work explored RAD’s interaction with ICMT through molecular docking and molecular dynamics simulations, comparing Radicicol’s binding mode to that of the native ICMT ligand.Method:Docking simulations were performed using Glide XP mode, and binding energies were refined by MM-GBSA calculations. Molecular dynamics simulation of 100 ns was conducted to assess the binding stability of the Radicicol–ICMT complex using Desmond, with analysis of RMSD and protein–ligand interactions.Results:Molecular docking and molecular dynamics simulations revealed that RAD stably bound within the ICMT active site by bridging both the S-adenosylmethionine cofactor pocket and the hydrophobic prenyl substrate tunnel. Its key interactions included a persistent hydrogen bond with Val116 and an induced-fit engagement of Arg125, supporting a snug and stable RAD–ICMT complex.Conclusion:These computational insights suggested that RAD inhibited ICMT by dual-site binding, simultaneously occupying the cofactor and substrate pockets. Such a dual engagement could mislocalize prenylated proteins (like Ras) and represent a novel mechanism of action for RAD.

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.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to a severe premature ageing phenotype, caused by mutations in the LMNA gene. The LMNA gene codes for lamin-A and lamin-C proteins, which are structural components of the nuclear lamina. HGPS is usually caused by a de novo C1824T mutation that leads to the accumulation of a dominant negative form of lamin-A called progerin. Progerin also accumulates physiologically in normal ageing cells as a rare splicing form of lamin-A transcripts. From this perspective, HGPS cells seem to be good candidates for the study of the physiological mechanisms of ageing. Progerin accumulation leads to faster cellular senescence, stem cell depletion and the progeroid phenotype. Tissues of mesodermic origin are especially affected by HGPS. HGPS patients usually have a bad quality of life and, with current treatments, their life expectancy does not exceed their second decade at best. Though progerin can be expressed in almost any tissue, when death occurs, it is usually due to cardiovascular complications. In HGPS, severe epigenetic alterations have been reported. Histone-covalent modifications are radically different from control specimens, with the tendency to lose the bipartition into euchromatin and heterochromatin. This is reflected in an altered spatial compartmentalization and conformation of chromatin within the nucleus. Moreover, it seems that microRNAs and microRNA biosynthesis might play a role in HGPS. Exemplary in this connection is the suggested protective effect of miR-9 on the central nervous system of affected individuals. This mini-review will report on the state of the art of HGPS epigenetics, and there will be a discussion of how epigenetic alterations in HGPS cells can alter the cellular metabolism and lead to the systemic syndrome.