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.     

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.     

Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.     

Zmpste24基因缺失与早老症

衰老是一种生理完整性丧失,功能受损,疾病和死亡风险增加的过程。早老症（HGPS）是一种加速化的衰老疾病,是研究人类正常衰老理想的疾病模型。由LMNA基因突变产生prelamin AΔ50在细胞内累积是造成早老症的主要原因,早老症病人表现出寿命急剧缩短,老化特征明显的现象,例如脱发、皮下脂肪减少、骨质疏松以及早逝。 锌金属蛋白酶Zmpste24 是prelamin A加工成为成熟lamin A蛋白的关键酶。敲除Zmpste24基因的小鼠表现出与早老症高度一致的衰老表型,同时也存在非常相似的发病机制,如染色质异常、DNA损伤和干细胞功能缺失等。Zmpste24缺失小鼠作为典型的早老模型小鼠因其衰老周期短,衰老特征明显而获得广泛应用。本文总结了以Zmpste24缺失早老小鼠为模型取得的早老相关分子机制的研究进展,以及抗衰老策略的最新发现。     

核层蛋白A前体的法尼基化与衰老

编码核层蛋白A(lamin A)的LMNA基因突变导致法尼基化的核层蛋白A前体(prelamin A)不能被进一步加工成成熟的核层蛋白A,从而导致一种Hutchinson-Gilford早老症综合征(Hutchinson-Gilford progeria syndrome,HGPS)。一种更严重的早老症——限制性皮肤病(restrictive dermopathy,RD),是由于缺失核层蛋白A前体加工过程中的剪切酶ZMPSTE24引起的。ZMPSTE24的缺失阻止了法尼基化的核层蛋白A前体不能正常加工成为成熟的核层蛋白A,同时导致法尼基化的核层蛋白A前体的堆积。在HGPS和RD病人的成纤维细胞中,发现法尼基化的核层蛋白A前体都定位在核膜,从而影响细胞核膜的完整性,并导致细胞核形的异常,进而导致衰老。最近研究表明经过法尼基酰转移酶抑制剂(farnesyltransferase inhibitor,FTI)处理后的细胞的核形异常减少。同时,FTI能够改善HGPS和RD小鼠的早老症状。本文就核层蛋白A前体的法尼基化对衰老的影响有关研究进展作一综述。     

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.     

Prelamin A farnesylation and progeroid syndromes

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation that leads to the synthesis of a mutant prelamin A that is farnesylated but cannot be further processed to mature lamin A. A more severe progeroid disorder, restrictive dermopathy (RD), is caused by the loss of the prelamin A-processing enzyme, ZMPSTE24. The absence of ZMPSTE24 prevents the endoproteolytic processing of farnesyl-prelamin A to mature lamin A and leads to the accumulation of farnesyl-prelamin A. In both HGPS and RD, the farnesyl-prelamin A is targeted to the nuclear envelope, where it interferes with the integrity of the nuclear envelope and causes misshapen cell nuclei. Recent studies have shown that the frequency of misshapen nuclei can be reduced by treating cells with a farnesyltransferase inhibitor (FTI). Also, administering an FTI to mouse models of HGPS and RD ameliorates the phenotypes of progeria. These studies have prompted interest in testing the efficacy of FTIs in children with HGPS.     

Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence

Lipodystrophic syndromes associated with mutations in LMNA, encoding A-type lamins, and with HIV antiretroviral treatments share several clinical characteristics. Nuclear alterations and prelamin A accumulation have been reported in fibroblasts from patients with LMNA mutations and adipocytes exposed to protease inhibitors (PI). As genetically altered lamin A maturation also results in premature ageing syndromes with lipodystrophy, we studied prelamin A expression and senescence markers in cultured human fibroblasts bearing six different LMNA mutations or treated with PIs. As compared to control cells, fibroblasts with LMNA mutations or treated with PIs had nuclear shape abnormalities and reduced proliferative activity that worsened with increasing cellular passages. They exhibited prelamin A accumulation, increased oxidative stress, decreased expression of mitochondrial respiratory chain proteins and premature cellular senescence. Inhibition of prelamin A farnesylation prevented cellular senescence and oxidative stress. Adipose tissue samples from patients with LMNA mutations or treated with PIs also showed retention of prelamin A, overexpression of the cell cycle checkpoint inhibitor p16 and altered mitochondrial markers. Thus, both LMNA mutations and PI treatment result in accumulation of farnesylated prelamin A and oxidative stress that trigger premature cellular senescence. These alterations could participate in the pathophysiology of lipodystrophic syndromes and lead to premature ageing complications.     

Accelerated ageing: from mechanism to therapy through animal models

Ageing research benefits from the study of accelerated ageing syndromes such as Hutchinson-Gilford progeria syndrome (HGPS), characterized by the early appearance of symptoms normally associated with advanced age. Most HGPS cases are caused by a mutation in the gene LMNA, which leads to the synthesis of a truncated precursor of lamin A known as progerin that lacks the target sequence for the metallopotease FACE-1/ZMPSTE24 and remains constitutively farnesylated. The use of Face-1/Zmpste24-deficient mice allowed us to demonstrate that accumulation of farnesylated prelamin A causes severe abnormalities of the nuclear envelope, hyper-activation of p53 signalling, cellular senescence, stem cell dysfunction and the development of a progeroid phenotype. The reduction of prenylated prelamin A levels in genetically modified mice leads to a complete reversal of the progeroid phenotype, suggesting that inhibition of protein farnesylation could represent a therapeutic option for the treatment of progeria. However, we found that both prelamin A and its truncated form progerin can undergo either farnesylation or geranylgeranylation, revealing the need of targeting both activities for an efficient treatment of HGPS. Using Face-1/Zmpste24-deficient mice as model, we found that a combination of statins and aminobisphosphonates inhibits both types of modifications of prelamin A and progerin, improves the ageing-like symptoms of these mice and extends substantially their longevity, opening a new therapeutic possibility for human progeroid syndromes associated with nuclear-envelope defects. We discuss here the use of this and other animal models to investigate the molecular mechanisms underlying accelerated ageing and to test strategies for its treatment.     

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation.In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.     

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.     

Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging

Several human progerias, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by the accumulation at the nuclear envelope of farnesylated forms of truncated prelamin A, a protein that is also altered during normal aging. Previous studies in cells from individuals with HGPS have shown that farnesyltransferase inhibitors (FTIs) improve nuclear abnormalities associated with prelamin A accumulation, suggesting that these compounds could represent a therapeutic approach for this devastating progeroid syndrome. We show herein that both prelamin A and its truncated form progerin/LADelta50 undergo alternative prenylation by geranylgeranyltransferase in the setting of farnesyltransferase inhibition, which could explain the low efficiency of FTIs in ameliorating the phenotypes of progeroid mouse models. We also show that a combination of statins and aminobisphosphonates efficiently inhibits both farnesylation and geranylgeranylation of progerin and prelamin A and markedly improves the aging-like phenotypes of mice deficient in the metalloproteinase Zmpste24, including growth retardation, loss of weight, lipodystrophy, hair loss and bone defects. Likewise, the longevity of these mice is substantially extended. These findings open a new therapeutic approach for human progeroid syndromes associated with nuclear-envelope abnormalities.     

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.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.     

DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.     

Prelamin A-mediated nuclear envelope dynamics in normal and laminopathic cells

Prelamin A is the precursor protein of lamin A, a major constituent of the nuclear lamina in higher eukaryotes. Increasing attention to prelamin A processing and function has been given after the discovery, from 2002 to 2004, of diseases caused by prelamin A accumulation. These diseases, belonging to the group of laminopathies and mostly featuring LMNA mutations, are characterized, at the clinical level, by different degrees of accelerated aging, and adipose tissue, skin and bone abnormalities. The outcome of studies conducted in the last few years consists of three major findings. First, prelamin A is processed at different rates under physiological conditions depending on the differentiation state of the cell. This means that, for instance, in muscle cells, prelamin A itself plays a biological role, besides production of mature lamin A. Secondly, prelamin A post-translational modifications give rise to different processing intermediates, which elicit different effects in the nucleus, mostly by modification of the chromatin arrangement. Thirdly, there is a threshold of toxicity, especially of the farnesylated form of prelamin A, whose accumulation is obviously linked to cell and organism senescence. The present review is focused on prelamin A-mediated nuclear envelope modifications that are upstream of chromatin dynamics and gene expression mechanisms regulated by the lamin A precursor.     

Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein.

Results

We have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells.

Conclusions

This study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.     

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.