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Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

Genome instability is a hallmark of both cancer and aging processes. Beyond cell-autonomous responses, it is known that DNA damage also elicits systemic mechanisms aimed at favoring survival and damaged cells clearance. Among these mechanisms, immune activation and NF-κB-mediated inflammation play central roles in organismal control of DNA damage. We focus herein on the different experimental evidences that have allowed gaining mechanistic insight about this relationship. We also describe the functional consequences of defective immune function in cancer development and age-related alterations. Finally, we discuss different intervention strategies based on enhancing immunity or on the modulation of the inflammatory response to improve organism homeostasis in cancer and aging.

Summary Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Particularly in the context of stem cell biology, analysis of single cell lineages in their native state has elucidated novel fates and highlighted heterogeneity of function. Coupling of such ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CARLIN (for CRISPR Array Repair LINeage tracing) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures based on in vivo stem cell function without a need for markers or cell sorting.

Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT)

The Instituto Universitario de Oncología del Principado de Asturias is supported by Fundación Bancaria Caja de Ahorros de Asturias. J.M.P.F. is supported by Ministerio de Economía y Competitividad (MINECO/FEDER: No. SAF2015-64157-R) and Gobierno del Principado de Asturias. C.L.-O. is supported by grants from the European Research Council (ERC-2016-ADG, DeAge), Ministerio de Economía y Competitividad (MINECO/FEDER: Nos. SAF2014-52413-R and SAF2017-87655-R), Instituto de Salud Carlos III (RTICC) and Progeria Research Foundation (No. PRF2016-66). O.S.-F. is recipient of an FPU fellowship. A.R.F. is recipient of a Ramón y Cajal fellowship. The generation of progerin antibody was funded by the Wellcome Trust (No. 098291/Z/12/Z to S.N.).

Myeloproliferative neoplasms (MPNs) represent a frequently occurring group of heterogeneous hematologic malignancies. In the last decade, the identification of JAK2-activating mutations in a significant proportion of MPN patients gave rise to the first molecularly driven therapy for BCR-ABL-negative patients. Nevertheless, current efforts are still focused on the identification of novel therapeutic targets to achieve permanent remission. In this perspective, we focus on the recent findings in this field and highlight new evidence linking proteostasis deregulation with myeloid transformation. We recently reported that the proteostasis regulator AIRAPL acts as a tumor suppressor in MPNs through the modulation of insulin-like growth factor receptor levels at the endoplasmic reticulum. This finding paves the way for new therapeutic approaches to these neoplasms and indicates the importance of protein homeostasis maintenance for normal hematopoiesis.

The authors uncover a role for the proteostasis modulator AIRAPL as a tumor suppressor in myeloproliferative malignancies, through its regulation of IGFR stability. The results ascribe a biological function to AIRAPL, and they implicate prosteostatic deregulation as an oncogenic mechanism in myeloid transformation, thus suggesting potential novel therapeutic strategies. AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown1,2,3. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2V617F mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms4,5,6. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

Summary Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age‐dependent metabolic changes, but the mechanism is unknown. Here, we profile lamina‐associated domains (LADs) using lamin B1 ChIP‐Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1‐associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24‐deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de‐repression of PPAR‐ and LXR‐dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de‐repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.

Summary Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis., Graphical Abstract, Highlights • Base substitution rate is similar among human HSCs and MPPs • Mutations accumulate with 14 mutations per year per cell • Three signatures explain mutation spectra in HSC/MPPs and are also present in AML • Shared mutations allow construction of a developmental lineage tree, Osorio et al. report lifelong mutation accumulation in human hematopoietic stem and progenitor cells, which is explained by three distinct mutational signatures. Shared somatic mutations between cells of the same donor enable the construction of a developmental lineage tree and quantification of each branch to mature blood cell populations.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

MT1‐MMP ( MMP14 ) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14 −/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1‐MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1‐MMP induces a senescent phenotype characterized by up‐regulation of p16 INK 4a and p21 CIP 1/ WAF 1 , increased activity of senescence‐associated β‐galactosidase, generation of a senescence‐associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14 −/− mice with all‐ trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.

UNLABELLED: Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental premature aging disorder that recapitulates some biological and physical aspects of physiological aging. The disease is caused by a sporadic dominant mutation in the LMNA gene that leads to the expression of progerin, a mutant form of lamin A that lacks 50 amino acids and retains a toxic farnesyl modification in its carboxy-terminus. However, the mechanisms underlying cellular damage and senescence and accelerated aging in HGPS are incompletely understood. Here, we analyzed fibroblasts from healthy subjects and HGPS patients using SILAC (stable isotope labeling with amino acids in cell culture). We found in HGPS cells a marked downregulation of mitochondrial oxidative phosphorylation proteins accompanied by mitochondrial dysfunction, a process thought to provoke broad organ decline during normal aging. We also found mitochondrial dysfunction in fibroblasts from adult progeroid mice expressing progerin (Lmna(G609G/G609G) knock-in mice) or prelamin A (Zmpste24-null mice). Analysis of tissues from these mouse models revealed that the damaging effect of these proteins on mitochondrial function is time- and dose-dependent. Mitochondrial alterations were not observed in the brain, a tissue with extremely low progerin expression that seems to be unaffected in HGPS. Remarkably, mitochondrial function was restored in progeroid mouse fibroblasts treated with the isoprenylation inhibitors FTI-277 or pravastatin plus zoledronate, which are being tested in HGPS clinical trials. Our results suggest that mitochondrial dysfunction contributes to premature organ decline and aging in HGPS. Beyond its effects on progeria, prelamin A and progerin may also contribute to mitochondrial dysfunction and organ damage during normal aging, since these proteins are expressed in cells and tissues from non-HGPS individuals, most prominently at advanced ages. BIOLOGICAL SIGNIFICANCE: Mutations in LMNA or defective processing of prelamin A causes premature aging disorders, including Hutchinson-Gilford progeria syndrome (HGPS). Most HGPS patients carry in heterozygosis a de-novo point mutation (c.1824C>T: GGC>GGT; p.G608G) which causes the expression of the lamin A mutant protein called progerin. Despite the importance of progerin and prelamin A in accelerated aging, the underlying molecular mechanisms remain largely unknown. To tackle this question, we compared the proteome of skin-derived dermal fibroblast from HGPS patients and age-matched controls using quantitative stable isotope labeling with amino acids in cell culture (SILAC). Our results show a pronounced down-regulation of several components of the mitochondrial ATPase complex accompanied by up-regulation of some glycolytic enzymes. Accordingly, functional studies demonstrated mitochondrial dysfunction in HGPS fibroblasts. Moreover, our expression and functional studies using cellular and animal models confirmed that mitochondrial dysfunction is a feature of progeria which develops in a time- and dose-dependent manner. Finally, we demonstrate improved mitochondrial function in progeroid mouse cells treated with a combination of statins and aminobisphosphonates, two drugs that are being evaluated in ongoing HGPS clinical trials. Although further studies are needed to unravel the mechanisms through which progerin and prelamin A provoke mitochondrial abnormalities, our findings may pave the way to improved treatments of HGPS. These studies may also improve our knowledge of the mechanisms leading to mitochondrial dysfunction during normal aging, since both progerin and prelamin A have been found to accumulate during normal aging. Work in the author's laboratories is supported by grants from the Spanish Ministry of Economy and Competiveness (MINECO) (SAF2010-16044; SAF2011-23089, SAF2009-08007, CSD2007-00020, CTQ2010-18644), Instituto de Salud Carlos III (ISCIII) (RD06/0014/ 0021, RD12/0042/0028), the Progeria Research Foundation (Innovator Award PRF 2012-42), and Comunidad de Madrid (S2011/BMD-2402). P.C.-S. was financially supported by an FPU scholarship from the Spanish Ministry of Education. The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by MINECO and Pro-CNIC Foundation, and the Instituto Universitario de Oncología by Obra Social Cajastur. Sí

Background— Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS. Methods and Results— We performed expression and functional studies in wild-type mice and knock-in Lmna G609G/+ mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna G609G/+ mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna G609G/+ vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna G609G/+ mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice. Conclusions— Excessive vascular calcification in Lmna G609G mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases.

This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grants SAF2010-16044 and SAF2013-46663-R (to V.A.), SAF2011-30312 and SAF2014-58286-C2-1-R (to L.H.-M.), SAF2011-30088 (to E.D.), and SAF2014-52413-R (to C.L.-O.) and Fondo de Investigación Sanitaria del Instituto de Salud Carlos III Grants RD12/0042/0028 (to V.A.), RD12/0042/0011 (to J.T.), and RD12/0042/0002 (to L.H.-M.), with cofunding from the Fondo Europeo de Desarrollo Regional and the Progeria Research Foundation. J.A.G. is the recipient of a U-Mobility Grant from the Marie Curie cofunding of Regional, National and International Programme (Grant 246550). The Instituto Universitario de Oncología is supported by Obra Social Cajastur. The CNIC is supported by the MINECO and the Pro CNIC Foundation, and it is a Severo Ochoa Center of Excellence (MINECO Award SEV-2015-0505)., Rivera-Torres, J., Calvo, C.J., Llach, A., Guzmán-Martínez, G., Caballero, R., González-Gómez, C., Jiménez-Borreguero, L.J., Guadix, J.A., Osorio, F.G., López-Otín, C., Herraiz-Martínez, A., Cabello, N., Vallmitjana, A., Benítez, R., Gordon, L.B., Jalife, J., Pérez-Pomares, J.M., Tamargo, J., Delpón, E., Hove-Madsen, L., Filgueiras-Rama, D., Andrés, V.

NF-κB signaling exerts essential roles in immunity and cellular stress responses, regulating many functions related with organism innate defense. Besides, NF-κB altered signaling has been causally linked to ageing and diverse pathological conditions. We discuss herein the functional involvement of this signaling pathway in ageing, visiting recent experimental evidence about NF-κB activation in this complex process, its functional consequences and the novel biological functions raised from these works. Moreover, we discuss ageing intervention strategies based on NF-κB inhibition, which have demonstrated to be effective at delaying and even reverting different ageing manifestations in human and mouse models of both normal and accelerated ageing. Altogether, the current evidence supports that NF-κB activation constitutes a driving force of the ageing process and a preferential target for rejuvenation-aimed approaches.

International audience; Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C\textgreaterT; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C\textgreaterT mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C\textgreaterT) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.

Mutations in the nuclear envelope protein lamin A or in its processing protease ZMPSTE24 cause human accelerated aging syndromes, including Hutchinson-Gilford progeria syndrome. Similarly, Zmpste24-deficient mice accumulate unprocessed prelamin A and develop multiple progeroid symptoms, thus representing a valuable animal model for the study of these syndromes. Zmpste24-deficient mice also show marked transcriptional alterations associated with chromatin disorganization, but the molecular links between both processes are unknown. We report herein that Zmpste24-deficient mice show a hypermethylation of rDNA that reduces the transcription of ribosomal genes, being this reduction reversible upon treatment with DNA methyltransferase inhibitors. This alteration has been previously described during physiological aging in rodents, suggesting its potential role in the development of the progeroid phenotypes. We also show that Zmpste24-deficient mice present global hypoacetylation of histones H2B and H4. By using a combination of RNA sequencing and chromatin immunoprecipitation assays, we demonstrate that these histone modifications are associated with changes in the expression of several genes involved in the control of cell proliferation and metabolic processes, which may contribute to the plethora of progeroid symptoms exhibited by Zmpste24-deficient mice. The identification of these altered genes may help to clarify the molecular mechanisms underlying aging and progeroid syndromes as well as to define new targets for the treatment of these dramatic diseases.

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A-dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response. Spanish Ministerio de Economia y Competitividad (MINECO) [SAF2011-25834, SAF2010-16044]; Comunidad de Madrid [INDISNET-S2011/BMD-2332]; Instituto de Salud Carlos III (ISCIII) [RD12/0042/0028, RD12/0042/0056, CP11/00145]; Progeria Research Foundation [PRF 2012-42]; European Commission [ERC-2011AdG 294340-GENTRIS, 317916-Liphos]; Sara Borrell ISCIII program [CP11/00145]; Miguel Servet ISCIII program [CP11/00145]; Fundacion Mario Losantos del Campo; Fundacion Ferrer para la Investigacion; Obra Social Cajastur; Spanish MINECO; Pro-CNIC Foundation Sí

Background: Monitoring activity of specific signaling pathways in vivo is challenging and requires highly sensitive methods to detect dynamic perturbations in whole organisms. Results: In vivo gene delivery of a luciferase reporter followed by bioluminiscence imaging allows measuring NF-κB activity in mice liver and lungs. Conclusions: This protocol allows a direct measure of NF-κB activity through quantification of bioluminescence signal, demonstrating its accuracy and sensitivity in different animal models and experimental conditions. Variants could be also applied for the analysis of NF-κB activity in different tissues or for studying other signaling pathways in vivo. Background Monitoring biological processes in vivo remains experimentally challenging. Visualization of biochemical perturbations in whole organisms requires highly sensitive methods that allow detection in a quantitative and reproducible manner [1-3]. Reporter gene approaches are based on the use of specific regulatory sequences attached to genes that confer organisms a property that could be easily detected and quantified, typically light or fluorescence emission [4]. Different luciferase and fluorescent proteins-based strategies have been developed for monitoring signaling pathways involved in cancer and aging [5-11]. Most of the available strategies rely on the generation of transgenic mice that stably express reporter genes. These mutant animals allow dynamic studies of complex processes, as the activity of the monitored signaling pathways could be recorded at different time points during lifetime. The main limitation of these approaches is that they imply the generation of transgenic strains, involving a long and expensive process that requires the use of a large number of experimentation animals. An alternative approach is described herein, based on the use of in vivo gene delivery strategies that potentially overcome previous limitations, allowing a fast and reliable quantification of the activity of signaling pathways in a wide variety of experimental models and physiological situations. Here we specifically describe a luciferase-based approach to measure in vivo NF-κB activity in different mice tissues. NF-κB is involved in the response against a large variety of external and internal stress signals, having essential roles in inflammation, immune response, cell proliferation and protection against apoptosis [12-14]. Consequently with its key role in cellular stress response, NF-κB aberrant activation has been linked with several pathophysiological conditions including cancer and accelerated aging [15,16]. In this regard, it has been described that not only acute NF-κB activation is related with disease but also subtle changes in NF-κB activity that persist in time have important biological consequences, giving rise to chronic inflammatory conditions. The monitoring of these subtle changes in NF-κB activity has been especially challenging.

Gene mutations that cause defects in the nuclear envelope are responsible for progeroid syndromes, characterized by exacerbated cell senescence and accelerated aging. Consequently, morphological abnormalities of the nucleus represent a cellular phenotype whose analysis allows for both the characterization of the consequences of particular mutations and the assessment of the impact of approaches aimed at reversing their pathological effects. To obtain reliable results, systematic and reproducible procedures are required. Here, we describe a simple fluorescence microscopy-based protocol to detect nuclear envelope alterations in the study of cellular senescence.

Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A-accumulating) and Zmpste24-proficient (mature lamin A-containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.

Gene mutations that cause defects in the nuclear envelope are responsible for progeroid syndromes, characterized by exacerbated cell senescence and accelerated aging. Consequently, morphological abnormalities of the nucleus represent a cellular phenotype whose analysis allows for both the characterization of the consequences of particular mutations and the assessment of the impact of approaches aimed at reversing their pathological effects. To obtain reliable results, systematic and reproducible procedures are required. Here, we describe a simple fluorescence microscopy-based protocol to detect nuclear envelope alterations in the study of cellular senescence.

Alterations in the architecture and dynamics of the nuclear lamina have a causal role in normal and accelerated aging through both cell-autonomous and systemic mechanisms. However, the precise nature of the molecular cues involved in this process remains incompletely defined. Here we report that the accumulation of prelamin A isoforms at the nuclear lamina triggers an ATM- and NEMO-dependent signaling pathway that leads to NF-κB activation and secretion of high levels of proinflammatory cytokines in two different mouse models of accelerated aging (Zmpste24−/− and LmnaG609G/G609G mice). Causal involvement of NF-κB in accelerated aging was demonstrated by the fact that both genetic and pharmacological inhibition of NF-κB signaling prevents age-associated features in these animal models, significantly extending their longevity. Our findings provide in vivo proof of principle for the feasibility of pharmacological modulation of the NF-κB pathway to slow down the progression of physiological and pathological aging.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino¿based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide¿based therapies for treating human diseases of accelerated aging.

Background A subset of lung cancer patients harbour EGFR somatic mutations in their tumours and are candidates for treatment with EGFR tyrosine kinase inhibitors. In a few cases EGFR mutations have also been found in the germ line, suggesting a role in lung carcinogenesis. Objetives of this study were: 1) To analyze the EGFR gene mutations in a population diagnosed with lung adenocarcinoma from Northern Spain. 2) To determine the frequency of a new germ-line mutation found in our laboratory as well as the frequency in our population of three other EGFR germ-line mutations detected by other authors. 3) To determine whether the novel mutation detected may have a functional effect on the EGFR protein. Methods Tumour DNA samples were obtained from frozen or paraffin embedded tumour tissues. Samples of DNA from peripheral blood cells were obtained from 912 individuals with lung cancer recruited from the CAPUA study [1, 2], 477 unrelated healthy donor individuals and 32 individuals with other types of cancer. EGFR gene exons 18 to 21 were studied by direct standard dideoxy sequencing. Specific mutations were determined either by direct sequencing or by specific RFLP analysis. Cell lines were transfected with EGFR-mutant plasmids and analysed by western blot with antibodies specific for total or phosphorylated-EGFR. Results We found EGFR mutation in 12 of the 71 tumour samples (17%). One tumour contained two mutations. One mutation (p.R776G) was present as a germ line. Using an RFLP analysis, this mutation was not found in 954 alleles from healthy individuals studied, concluding that it is not a polymorphism. The mutation was not found either in genomic DNA from 912 lung cancer patients. Three additional EGFR germ-line mutations that were already described were not found in any of the studied samples. These observations show that EGFR mutated alleles are rare in the population. In vitro studies revealed that tyrosine autophosphorylation is enhanced in p.R776G-mutant EGFR when compared with wild-type EGFR. This enhanced autophosphorylation in the absence of ligand may be associated with a proliferative advantage. Conclusions Germ-line mutations in EGFR are rare but may contribute to oncogenesis

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.

Mutations in the nuclear envelope protein lamin A or in its processing protease ZMPSTE24 cause human accelerated aging syndromes, including Hutchinson-Gilford progeria syndrome. Similarly, Zmpste24-deficient mice accumulate unprocessed prelamin A and develop multiple progeroid symptoms, thus representing a valuable animal model for the study of these syndromes. Zmpste24-deficient mice also show marked transcriptional alterations associated with chromatin disorganization, but the molecular links between both processes are unknown. We report herein that Zmpste24-deficient mice show a hypermethylation of rDNA that reduces the transcription of ribosomal genes, being this reduction reversible upon treatment with DNA methyltransferase inhibitors. This alteration has been previously described during physiological aging in rodents, suggesting its potential role in the development of the progeroid phenotypes. We also show that Zmpste24-deficient mice present global hypoacetylation of histones H2B and H4. By using a combination of RNA sequencing and chromatin immunoprecipitation assays, we demonstrate that these histone modifications are associated with changes in the expression of several genes involved in the control of cell proliferation and metabolic processes, which may contribute to the plethora of progeroid symptoms exhibited by Zmpste24-deficient mice. The identification of these altered genes may help to clarify the molecular mechanisms underlying aging and progeroid syndromes as well as to define new targets for the treatment of these dramatic diseases.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A, an essential component of the nuclear envelope. Zmpste24-deficient mice exhibit multiple defects that phenocopy human accelerated aging processes such as Hutchinson–Gilford progeria syndrome. In this work, we report that progeroid Zmpste24 -/ − mice present profound transcriptional alterations in genes that regulate the somatotroph axis, together with extremely high circulating levels of growth hormone (GH) and a drastic reduction in plasma insulin-like growth factor 1 (IGF-1). We also show that recombinant IGF-1 treatment restores the proper balance between IGF-1 and GH in Zmpste24 -/ − mice, delays the onset of many progeroid features, and significantly extends the lifespan of these progeroid animals. Our findings highlight the importance of IGF/GH balance in longevity and may be of therapeutic interest for devastating human progeroid syndromes associated with nuclear envelope abnormalities.

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.

International audience; 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.

During decades, aging has been regarded as the consequence of a stochastic process caused by the accumulative effect of damaged molecules. However, recent experimental evidences have extended this view and suggested that aging also requires active signaling programs for the maintenance of the aged state [1]. Beyond cell-autonomous alterations, age signals get systemic through changes in intercellular communication pathways [2]. The identification of the precise nature of these mechanisms and signals could provide valuable information, uncovering potential targets for rejuvenation-aimed approaches [3]. Aging research has greatly benefited from the study of progeroid syndromes, accelerated aging conditions caused by an excessive accumulation of cellular damage or by an inefficient response of the repair mechanisms. Progeroid laminopathies are accelerated aging syndromes caused by defects of the nuclear lamina. Among them, Hutchinson-Gilford Progeria Syndrome (HGPS) is one the most intensely studied. This syndrome is caused by a point mutation in the LMNA gene, leading to the accumulation of a truncated form of lamin A called progerin which induces important alterations in the cell nucleus. Interestingly, progerin accumulation has also been reported during normal aging, adding a new layer of interest to the study of this syndrome. NF-κB transcription factors respond to a large variety of external and internal stress signals, having essential roles in development and tissue homeostasis maintenance. Through the study of two related mouse models of progeroid laminopathies (Zmpste24-deficient and LmnaG609G knock-in mice), we have recently found that aberrant activation of NF-κB is involved in the pathogenesis of accelerated aging syndromes, providing new insights into the mechanisms that allow the integration of cellular and systemic alterations in the aging process [4]. The in vivo monitoring of NF-κB activity by using a reporter-based assay revealed that this pathway was constitutively hyperactivated in progeroid mice. Further experiments allowed us to unveil the molecular pathway involved in this aberrant activation. Thus, in response to nuclear envelope alterations some important DNA damage sensors such as p53 or ATM were activated. In this context, we provide evidence that active ATM kinase cooperates with nuclear NEMO, an NF-κB regulatory subunit, resulting in the activation of NF-κB. We also found that, in response to NF-κB activation, several pro-inflammatory cytokines were significantly up-regulated. Among them, secretion of IL-6, CXCL-1 and TNF-α could have a causal role in the premature aging syndrome by establishing a chronic inflammatory situation through feed-forward regulatory signaling, affecting distant cells and tissues. Aimed at dissecting the precise contribution of NF-κB hyperactivation to the progeroid phenotype, we used an anti-inflammatory genetic strategy based on crossing Zmpste24-deficient mice with RelA haploinsufficient mice [5]. Interestingly, double mutant Zmpste24−/− RelA+/− mice displayed a retardation in the aging process, showing an extended longevity as compared with Zmpste24−/− RelA+/+ mice. Furthermore, double mutant mice showed a remarkable recovery of skin and immunological alterations, which is consistent with the proposed relevance of NF-κB activity in tissue homeostasis maintenance. These results prompted us to test a pharmacological approach to target NF-κB activation in progeroid mice. Thus, sodium salicylate treatment of both Zmpste24−/− and LmnaG609G/G609G mice extended longevity and led to a significant prevention of skin and immune alterations, demonstrating the feasibility of targeting this pathway for slowing down the progression of accelerated aging. Finally, our results indicate that these findings can be extended to normal aging, suggesting that a common accumulation of genetic damage and nuclear envelope alterations with age could be responsible, at least in part, of the abnormal NF-κB activity reported in tissues from advanced aged donors. The accumulation of senescent cells together with the decline in adult stem cell function is a primary cause of the compromise of tissue homeostasis during aging. The primary function of NF-κB activation in this context could be related to the prevention of apoptosis of damaged cells, so that chronic activation of this pathway with the subsequent immunological decline could preclude a proper clearance of senescent and damaged cells. In this regard, a recent report has described the causal involvement of inflammatory pathways in the age-related decline in stem cell function [6]. Globally, the data discussed herein clarify three important aspects that define NF-κB role during aging. Thus, experimental data confirm that NF-κB signaling is active during normal aging [7], its hyperactivation is associated with the development of accelerated aging and its amelioration retards the aging process [4-5, 8]. These characteristics support the use of strategies aimed at controlling NF-κB related inflammation as putative rejuvenation strategies during both normal and pathological aging. Over the last years, aging research has composed a complex picture where both cell autonomous and systemic alterations cooperate for establishing the aged state in organisms. Rational design of new interventions aimed to slow down this process should act in a coordinate way, targeting pro-aging signals as well as altered cellular communication pathways for the effective prevention of aging-related disorders. Figure 1 Exacerbated NF- κB signaling leads to age-related loss of tissue homeostasis, which takes place at an accelerated rate in progeroid syndromes. This phenomenon can be alleviated through NF- κB inhibition.