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。     

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24^−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24^−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24^−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24^−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24^−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.     

HP1α在Zmpste24-缺陷早老小鼠胚胎成纤维细胞中表达和磷酸化水平升高

本实验旨在研究异染色质蛋白1（heterochromatin protein 1, HP1）在Zmpste24基因敲除早老小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)中的表达量和磷酸化水平,探索异染色质功能异常与早老发病机制的内在联系．首先取雄雌Zmpste24杂合子小鼠胚胎,原代培养MEFs;分别用PCR和Western 印迹检测MEFs基因型和A型核纤层蛋白（laminA）表达以区分野生型与Zmpste24-缺陷型早老细胞;用与衰老相关的β-半乳糖苷酶染色法 (senescence associated-β-galactosidase assay, SA-β-gal)确定早老细胞出现衰老表型的传代数．用Western 印迹和phos-tag Western 印迹分别检测HP1在Zmpste24+/+和Zmpste24-/- MEFs中表达量和磷酸化水平的差异．实验结果显示,Zmpste24-/- MEFs中存在异常的LaminA,且在传代培养第5代出现明显的细胞衰老现象．选用培养至第3代或第4代的MEFs细胞进行下述实验发现,Zmpste24-/-MEFs中HP1α表达量明显高于Zmpste24+/+ MEFs,而HP1β未发现明显升高;Zmpste24+/+ MEFs中HP1α以非磷酸化状态为主,但在Zmpste24-/- MEFs中磷酸化HP1α比例明显升高;2种细胞中均未检测到磷酸化HP1β. 本研究结果证明,HP1α在Zmpste24-缺陷型早老小鼠传代早期MEFs中的表达量和磷酸化水平均有升高,提示HP1α参与A型核纤层蛋白相关的早老小鼠发病机制．     

The R527H mutation in LMNA gene causes an increased sensitivity to ionizing radiation

Mandibuloacral dysplasia type A (MADA; OMIM # 248370) is a premature ageing disease caused by the homozygous R527H mutation in the LMNA gene. At the cellular level, MADA is characterized by unprocessed prelamin A accumulation, nuclear architecture alterations, chromatin defects and increased incidence of apoptosis. In some progeroid laminopathies (e.g. HGPS) it has been demonstrated that such biochemical and morphological alterations are strongly linked with genomic instability. To test this also in MADA fibroblasts, their response to the ionising radiation- induced damage was analysed. We observed that their ability to repair the damage was significantly impaired, as demonstrated by the increased chromosome damage and the higher percentage of residual γ-H2AX foci, corresponding to unrepaired DNA-damage sites. Moreover, MADA fibroblasts showed a markedly reduced phosphorylation of p53 at Ser15(S15) and a lower induction of p53 and CDKN1A proteins after irradiation, compared to the control cell line. Upon irradiation, we also detected differences in the expression of some p53 downstream target genes. In addition, MADA cells showed partial defects in the checkpoint response, particularly in G1/S transition. Our results indicate that accumulation of the lamin A precursor protein determines a defect in DNA damage response after X-ray exposure, supporting a crucial role of lamin A in regulating DNA repair process and cell cycle control.     

Lamin A/C, laminopathies and premature ageing

Lamin A/C belongs to type V intermediate filaments and constitutes the nuclear lamina and nuclear matrix, where a variety of nuclear activities occur. Lamin A/C protein is firstly synthesized as a precursor and is further proteolytically processed by the zinc metallo-proteinase Ste24 (Zmpste24). Lamin A/C mutations cause a series of human diseases, collectively called laminopathies, the most severe of which is Hutchinson Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) which arises due to an unsuccessful maturation of prelamin A. Although the exact underlying molecular mechanisms are still poorly understood, genomic instability, defective nuclear mechanics and mechanotransduction, have been hypothesized to be responsible for laminopathy-based premature ageing. Removal of unprocessed prelamin A (progerin) or rescue of defective DNA repair could be potential therapeutic strategies for the treatment of HGPS in future.     

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.     

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.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.     

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.     

Proteomic profiling of adipose tissue from Zmpste24-/- mice, a model of lipodystrophy and premature aging, reveals major changes in mitochondrial function and vimentin processing

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.     

Targeting RAS‐converting enzyme 1 overcomes senescence and improves progeria‐like phenotypes of ZMPSTE24 deficiency

Several progeroid disorders are caused by deficiency in the endoprotease ZMPSTE24 which leads to accumulation of prelamin A at the nuclear envelope. ZMPSTE24 cleaves prelamin A twice: at the third carboxyl‐terminal amino acid following farnesylation of a –CSIM motif; and 15 residues upstream to produce mature lamin A. The carboxyl‐terminal cleavage can also be performed by RAS‐converting enzyme 1 (RCE1) but little is known about the importance of this cleavage for the ability of prelamin A to cause disease. Here, we found that knockout of RCE1 delayed senescence and increased proliferation of ZMPSTE24‐deficient fibroblasts from a patient with non‐classical Hutchinson‐Gilford progeria syndrome (HGPS), but did not influence proliferation of classical LMNA‐mutant HGPS cells. Knockout of Rce1 in Zmpste24‐deficient mice at postnatal week 4–5 increased body weight and doubled the median survival time. The absence of Rce1 in Zmpste24‐deficient fibroblasts did not influence nuclear shape but reduced an interaction between prelamin A and AKT which activated AKT‐mTOR signaling and was required for the increased proliferation. Prelamin A levels increased in Rce1‐deficient cells due to a slower turnover rate but its localization at the nuclear rim was unaffected. These results strengthen the idea that the presence of misshapen nuclei does not prevent phenotype improvement and suggest that targeting RCE1 might be useful for treating the rare progeroid disorders associated with ZMPSTE24 deficiency.     

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.     

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.     

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.     

Analysis of prelamin A biogenesis reveals the nucleus to be a CaaX processing compartment

Proteins establish and maintain a distinct intracellular localization by means of targeting, retention, and retrieval signals, ensuring most proteins reside predominantly in one cellular location. The enzymes involved in the maturation of lamin A present a challenge to this paradigm. Lamin A is first synthesized as a 74-kDa precursor, prelamin A, with a C-terminal CaaX motif and undergoes a series of posttranslational modifications including CaaX processing (farnesylation, aaX cleavage and carboxylmethylation), followed by endoproteolytic cleavage by Zmpste24. Failure to cleave prelamin A results in progeria and related premature aging disorders. Evidence suggests prelamin A is imported directly into the nucleus where it is processed. Paradoxically, the processing enzymes have been shown to reside in the cytosol (farnesyltransferase), or are ER membrane proteins (Zmpste24, Rce1, and Icmt) with their active sites facing the cytosol. Here we have reexamined the cellular site of prelamin A processing, and show that the mammalian and yeast processing enzymes Zmpste24 and Icmt exhibit a dual localization to the inner nuclear membrane, as well as the ER membrane. Our findings reveal the nucleus to be a physiologically relevant location for CaaX processing, and provide insight into the biology of a protein at the center of devastating progeroid diseases.     

Lamin A, farnesylation and aging

Lamin A is a component of the nuclear envelope that is synthesized as a precursor prelamin A molecule and then processed into mature lamin A through sequential steps of posttranslational modifications and proteolytic cleavages. Remarkably, over 400 distinct point mutations have been so far identified throughout the LMNA gene, which result in the development of at least ten distinct human disorders, collectively known as laminopathies, among which is the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). The majority of HGPS cases are associated with a single point mutation in the LMNA gene that causes the production of a permanently farnesylated mutant lamin A protein termed progerin. The mechanism by which progerin leads to premature aging and the classical HGPS disease phenotype as well as the relationship between this disorder and the onset of analogous symptoms during the lifespan of a normal individual are not well understood. Yet, recent studies have provided critical insights on the cellular processes that are affected by accumulation of progerin and have suggested that cellular alterations in the lamin A processing pathway leading to the accumulation of farnesylated prelamin A intermediates may play a role in the aging process in the general population. In this review we provide a short background on lamin A and its maturation pathway and discuss the current knowledge of how progerin or alterations in the prelamin A processing pathway are thought to influence cell function and contribute to human aging.     

Prelamin A impairs 53BP1 nuclear entry by mislocalizing NUP153 and disrupting the Ran gradient

The nuclear lamina is essential for the proper structure and organization of the nucleus. Deregulation of A‐type lamins can compromise genomic stability, alter chromatin organization and cause premature vascular aging. Here, we show that accumulation of the lamin A precursor, prelamin A, inhibits 53BP1 recruitment to sites of DNA damage and increases basal levels of DNA damage in aged vascular smooth muscle cells. We identify that this genome instability arises through defective nuclear import of 53BP1 as a consequence of abnormal topological arrangement of nucleoporin NUP153. We show for the first time that this nucleoporin is important for the nuclear localization of Ran and that the deregulated Ran gradient is likely to be compromising the nuclear import of 53BP1. Importantly, many of the defects associated with prelamin A expression were significantly reduced upon treatment with Remodelin, a small molecule recently reported to reverse deficiencies associated with abnormal nuclear lamina.