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.     

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.     

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.     

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.     

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．     

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.     

Integrating the contributions of mitochondrial oxidative metabolism to lipotoxicity and inflammation in NAFLD pathogenesis

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.     

Altered mitochondrial function and metabolic inflexibility associated with loss of caveolin-1

Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility.     

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.     

Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice

Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, β-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and PGC-1β, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.     

Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

Peroxisome-generated succinate induces lipid accumulation and oxidative stress in the kidneys of diabetic mice

Diabetes normally causes lipid accumulation and oxidative stress in the kidneys, which plays a critical role in the onset of diabetic nephropathy; however, the mechanism by which dysregulated fatty acid metabolism increases lipid and reactive oxygen species (ROS) formation in the diabetic kidney is not clear. As succinate is remarkably increased in the diabetic kidney, and accumulation of succinate suppresses mitochondrial fatty acid oxidation and increases ROS formation, we hypothesized that succinate might play a role in inducing lipid and ROS accumulation in the diabetic kidney. Here we demonstrate a novel mechanism by which diabetes induces lipid and ROS accumulation in the kidney of diabetic animals. We show that enhanced oxidation of dicarboxylic acids by peroxisomes leads to lipid and ROS accumulation in the kidney of diabetic mice via the metabolite succinate. Furthermore, specific suppression of peroxisomal β-oxidation improved diabetes-induced nephropathy by reducing succinate generation and attenuating lipid and ROS accumulation in the kidneys of the diabetic mice. We suggest that peroxisome-generated succinate acts as a pathological molecule inducing lipid and ROS accumulation in kidney, and that specifically targeting peroxisomal β-oxidation might be an effective strategy in treating diabetic nephropathy and related metabolic disorders.     

Berardinelli-Seip congenital lipodystrophy 2 regulates adipocyte lipolysis,browning, and energy balance in adult animals

Mutations in BSCL2/SEIPIN cause Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), but the mechanisms whereby Bscl2 regulates adipose tissue function are unclear. Here, we generated adipose tissue (mature) Bscl2 knockout (Ad-mKO) mice, in which Bscl2 was specifically ablated in adipocytes of adult animals, to investigate the impact of acquired Bscl2 deletion on adipose tissue function and energy balance. Ad-mKO mice displayed reduced adiposity and were protected against high fat diet-induced obesity, but not insulin resistance or hepatic steatosis. Gene expression profiling and biochemical assays revealed increased lipolysis and fatty acid oxidation in white adipose tissue (WAT) and brown adipose tissue , as well as browning of WAT, owing to induction of cAMP/protein kinase A signaling upon Bscl2 deletion. Interestingly, Bscl2 deletion reduced food intake and downregulated adipose β3-adrenergic receptor (ADRB3) expression. Impaired ADRB3 signaling partially offsets upregulated browning-induced energy expenditure and thermogenesis in Ad-mKO mice housed at ambient temperature. However, this counter-regulatory response was abrogated under thermoneutral conditions, resulting in even greater body mass loss in Ad-mKO mice. These findings suggest that Bscl2 regulates adipocyte lipolysis and β-adrenergic signaling to produce complex effects on adipose tissues and whole-body energy balance.     

Lipotoxicity,fatty acid uncoupling and mitochondrial carrier function

Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis.