Cell autonomous and systemic factors in progeria development

Progeroid laminopathies are accelerated aging syndromes caused by defects in nuclear envelope proteins. Accordingly, mutations in the LMNA gene and functionally related genes have been described to cause HGPS (Hutchinson-Gilford progeria syndrome), MAD (mandibuloacral dysplasia) or RD (restrictive dermopathy). Functional studies with animal and cellular models of these syndromes have facilitated the identification of the molecular alterations and regulatory pathways involved in progeria development. We have recently described a novel regulatory pathway involving miR-29 and p53 tumour suppressor which has provided valuable information on the molecular components orchestrating the response to nuclear damage stress. Furthermore, by using progeroid mice deficient in ZMPSTE24 (zinc metalloprotease STE24 homologue) involved in lamin A maturation, we have demonstrated that, besides these abnormal cellular responses to stress, dysregulation of the somatotropic axis is responsible for some of the alterations associated with progeria. Consistent with these observations, pharmacological restoration of the somatotroph axis in these mice delays the onset of their progeroid features, significantly extending their lifespan and supporting the importance of systemic alterations in progeria progression. Finally, we have very recently identified a novel progeroid syndrome with distinctive features from HGPS and MAD, which we have designated NGPS (Néstor-Guillermo progeria syndrome) (OMIM #614008). This disorder is caused by a mutation in BANF1, a gene encoding a protein with essential functions in the assembly of the nuclear envelope, further illustrating the importance of the nuclear lamina integrity for human health and providing additional support to the study of progeroid syndromes as a valuable source of information on human aging.     

Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs as novel SIRT1 activators

SIRT1 is an NAD⁺-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.     

白藜芦醇通过促进SIRT1表达，抑制NF-κB和TNF-α表达抵抗牛磺胆酸诱导的胎盘合体滋养细胞HTR-8的炎症损伤

摘要 已有研究证明,沉默信息调节子1(silent information regulator , SIRT1）和核因子κB(nuclear factor-κB, NF-κB）参与多种炎性反应调节;SIRT1激活剂——白藜芦醇（resveratrol）可通过依赖或不依赖于SIRT1的途径抑制炎症反应。然而,SIRT1及白藜芦醇在妊娠期肝内胆汁瘀积症（intrahepatic cholestasis of pregnancy, ICP）炎性反应中的作用在国内外尚未见报道。本研究证明,白藜芦醇可通过上调SIRT1表达,下调NF-κB及肿瘤坏死因子α（tumor necrosis factor-alpha, TNF-α）表达保护牛磺胆酸 （taurocholic acid, TCA）引起的胎盘合体滋养细胞HTR-8炎性损伤。免疫组化及Western印迹显示,SIRT1蛋白在正常胎盘组织的表达水平明显高于妊娠期ICP胎盘组织,而NF-κB蛋白表达在正常胎盘组织的表达低于ICP胎盘组织。Western印迹揭示,SIRT1蛋白在HTR-8细胞中的表达水平随暴露的TCA浓度增高而降低;相反,NF-κB和TNF-α蛋白质水平随TCA浓度增高而升高。采用白藜芦醇处理HTR-8细胞,该差异可被逆转;且白藜芦醇这一作用可被Ex-527（一种SIRT1 抑制剂）阻断。上述结果证明,在ICP胎盘合体滋养细胞炎性反应中SIRT1表达下调,而NF-κB表达上调;其可能的机制是ICP高浓度胆汁酸干扰胎盘合体滋养细胞SIRT1-NF-κB通路,诱导炎症反应。上述结果还提示,SIRT1激动剂——白藜芦醇可通过诱导SIRT1表达,抑制NF-κB及TNF-α表达,保护牛磺胆酸诱导的胎盘合体滋养细胞的炎性损伤。本研究为白藜芦醇临床治疗（妊娠期）肝内胆汁瘀积症提供了新的证据。至于白藜芦醇是否可直接抑制NF-κB的表达还有待进一步探讨。     

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.     

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.     

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。     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

Red wine triggers cell death and thioredoxin reductase inhibition: Effects beyond resveratrol and SIRT1

Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 μM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (≥ 10 μM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v) - but not white wine - induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

Characterization of the transactivation domain in the peroxisome-proliferator-activated receptor gamma co-activator (PGC-1)

Resveratrol, a polyphenol found in fruits, possesses chemopreventive and chemotherapeutic properties and has been shown to increase lifespan in yeast and metazoans, including mice. Genetic evidence and in vitro enzymatic measurements indicate that the deacetylase Sir2/SIRT1, an enzyme promoting stress resistance and aging, is the target of resveratrol. Similarly, down-regulation of insulin-like pathways, of which PI3K (phosphoinositide 3-kinase) is a key mediator, promotes longevity and is an attractive strategy to fight cancer. We show here that resveratrol inhibits, in vitro and in cultured muscle cell lines, class IA PI3K and its downstream signalling at the same concentration range at which it activates sirtuins. Our observations define class IA PI3K as a target of resveratrol that may contribute to the longevity-promoting and anticancer properties and identify resveratrol as a natural class-specific PI3K inhibitor.     

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.     

Perturbation of wild-type lamin A metabolism results in a progeroid phenotype

Mutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of disease-linked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

SIRT1 participates in the response to methylglyoxal-dependent glycative stress in mouse oocytes and ovary

Methylglyoxal (MG), a highly reactive dicarbonyl derived from metabolic processes, is the most powerful precursor of advanced glycation end products (AGEs). Glycative stress has been recently associated with ovarian dysfunctions in aging and PCOS syndrome. We have investigated the role of the NAD⁺-dependent Class III deacetylase SIRT1 in the adaptive response to MG in mouse oocytes and ovary. In mouse oocytes, MG induced up-expression of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) genes, components of the main MG detoxification system, whereas inhibition of SIRT1 by Ex527 or sirtinol reduced this response. In addition, the inhibition of SIRT1 worsened the effects of MG on oocyte maturation rates, while SIRT1 activation by resveratrol counteracted MG insult. Ovaries from female mice receiving 100 mg/kg MG by gastric administration for 28 days (MG mice) exhibited increased levels of SIRT1 along with over-expression of catalase, superoxide dismutase 2, SIRT3, PGC1α and mtTFA. Similar levels of MG-derived AGEs were observed in the ovaries from MG and control groups, along with enhanced protein expression of glyoxalase 1 in MG mice. Oocytes ovulated by MG mice exhibited atypical meiotic spindles, a condition predisposing to embryo aneuploidy. Our results from mouse oocytes revealed for the first time that SIRT1 could modulate MG scavenging by promoting expression of glyoxalases. The finding that up-regulation of glyoxalase 1 is associated with that of components of a SIRT1 functional network in the ovaries of MG mice provides strong evidence that SIRT1 participates in the response to methylglyoxal-dependent glycative stress in the female gonad.