Metabolic Control of Longevity.

Several metabolic alterations accumulate over time along with a reduction in biological fitness, suggesting the existence of a "metabolic clock" that controls aging. Multiple inborn defects in metabolic circuitries accelerate aging, whereas genetic loci linked to exceptional longevity influence metabolism. Each of the nine hallmarks of aging is connected to undesirable metabolic alterations. The main features of the "westernized" lifestyle, including hypercaloric nutrition and sedentariness, can accelerate aging as they have detrimental metabolic consequences. Conversely, lifespan-extending maneuvers including caloric restriction impose beneficial pleiotropic effects on metabolism. The introduction of strategies that promote metabolic fitness may extend healthspan in humans.

The microRNA-29/PGC1α regulatory axis is critical for metabolic control of cardiac function.

Different microRNAs (miRNAs), including miR-29 family, may play a role in the development of heart failure (HF), but the underlying molecular mechanisms in HF pathogenesis remain unclear. We aimed at characterizing mice deficient in miR-29 in order to address the functional relevance of this family of miRNAs in the cardiovascular system and its contribution to heart disease. In this work, we show that mice deficient in miR-29a/b1 develop vascular remodeling and systemic hypertension, as well as HF with preserved ejection fraction (HFpEF) characterized by myocardial fibrosis, diastolic dysfunction, and pulmonary congestion, and die prematurely. We also found evidence that the absence of miR-29 triggers the up-regulation of its target, the master metabolic regulator PGC1α, which in turn generates profound alterations in mitochondrial biogenesis, leading to a pathological accumulation of small mitochondria in mutant animals that contribute to cardiac disease. Notably, we demonstrate that systemic hypertension and HFpEF caused by miR-29 deficiency can be rescued by PGC1α haploinsufficiency, which reduces cardiac mitochondrial accumulation and extends longevity of miR-29-mutant mice. In addition, PGC1α is overexpressed in hearts from patients with HF. Collectively, our findings demonstrate the in vivo role of miR-29 in cardiovascular homeostasis and unveil a novel miR-29/PGC1α regulatory circuitry of functional relevance for cell metabolism under normal and pathological conditions.

Nuclear lamina defects cause ATM-dependent NF-κB activation and link accelerated aging to a systemic inflammatory response.

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 Lmna(G609G/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.

Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice.

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.

USP39 Deubiquitinase Is Essential for KRAS Oncogene-driven Cancer.

KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Loss of GLUT4 induces metabolic reprogramming and impairs viability of breast cancer cells.

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.

Functional analysis of sucrase-isomaltase mutations from chronic lymphocytic leukemia patients.

Next-generation sequencing techniques have emerged as powerful tools for the understanding of cancer genomes. In recent years, whole-exome and whole-genome sequencing strategies have enabled the annotation of a comprehensive mutation landscape of chronic lymphocytic leukemia (CLL), the most frequent leukemia in western countries. Several recurrently mutated genes have been identified, with a subset being validated as neoplastic drivers. Still, a main challenge remains for the differentiation between driver and passenger mutations among candidates as well as for the functional description of the newly discovered leukemogenic genes that could be utilized for personalized anti-tumor strategies. In this scenario, we have identified the metabolic enzyme sucrase-isomaltase (SI) as one of the most frequently mutated genes in a cohort of 105 CLL patients. Here, we demonstrate that these SI mutations result in loss of enzyme function by preventing the biosynthesis of catalytically competent SI at the cell surface. Transcriptome analyses of RNA from CLL patients with SI loss-of-function mutations have uncovered gene expression patterns that depict ample metabolic reprogramming, pinpointing SI as a putative player in the cancer-associated metabolic switch. These results highlight SI as a relevant target for clinical evaluation in future CLL studies.

Matrix metalloproteinase Mmp-1a is dispensable for normal growth and fertility in mice and promotes lung cancer progression by modulating inflammatory responses.

Human MMP-1 is a matrix metalloproteinase repeatedly associated with many pathological conditions, including cancer. Thus, MMP1 overexpression is a poor prognosis marker in a variety of advanced cancers, including colorectal, breast, and lung carcinomas. Moreover, MMP-1 plays a key role in the metastatic behavior of melanoma, breast, and prostate cancer cells. However, functional and mechanistic studies on the relevance of MMP-1 in cancer have been hampered by the absence of an in vivo model. In this work, we have generated mice deficient in Mmp1a, the murine ortholog of human MMP1. Mmp1a(-/-) mice are viable and fertile and do not exhibit obvious abnormalities, which has facilitated studies of cancer susceptibility. These studies have shown a decreased susceptibility to develop lung carcinomas induced by chemical carcinogens in Mmp1a(-/-) mice. Histopathological analysis indicated that tumors generated in Mmp1a(-/-) mice are smaller than those of wild-type mice, consistently with the idea that the absence of Mmp-1a hampers tumor progression. Proteomic analysis revealed decreased levels of chitinase-3-like 3 and accumulation of the receptor for advanced glycation end-products and its ligand S100A8 in lung samples from Mmp1a(-/-) mice compared with those from wild-type. These findings suggest that Mmp-1a could play a role in tumor progression by modulating the polarization of a Th1/Th2 inflammatory response to chemical carcinogens. On the basis of these results, we propose that Mmp1a knock-out mice provide an excellent in vivo model for the functional analysis of human MMP-1 in both physiological and pathological conditions.

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome.

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.

The hallmarks of aging as a conceptual framework for health and longevity research.

The inexorability of the aging process has sparked the curiosity of human beings since ancient times. However, despite this interest and the extraordinary scientific advances in the field, the complexity of the process has hampered its comprehension. In this context, The Hallmarks of Aging were defined in 2013 with the aim of establishing an organized, systematic and integrative view of this topic, which would serve as a conceptual framework for aging research. Ten years later and promoted by the progress in the area, an updated version included three new hallmarks while maintaining the original scope. The aim of this review is to determine to what extent The Hallmarks of Aging achieved the purpose that gave rise to them. For this aim, we have reviewed the literature citing any of the two versions of The Hallmarks of Aging and conclude that they have served as a conceptual framework not only for aging research but also for related areas of knowledge. Finally, this review discusses the new candidates to become part of the Hallmarks list, analyzing the evidence that supports whether they should or should not be incorporated.

Assessing microbiota composition in the context of aging.

The gut microbiota is a complex community of different microbial species that influence many aspects of health. Consequently, shifts in the composition of gut microbiome have been proposed to exert negative effects on the host physiology, leading to the pathogenesis of various age-related disorders, including cardiovascular and neurological diseases, type 2 diabetes, obesity, non-alcoholic liver disease, and other pathological conditions. Thus, understanding how the gut microbiota influences the aging-related decline is particularly topical. Advances in next-generation sequencing techniques, together with mechanistic experiments in animal models, have provided substantial improvements in microbiome analysis. However, standardization and best practices are needed to limit experimental variation between different studies. Here, we detail a simple method for microbiota composition analysis in mouse fecal samples using 16S rRNA next-generation sequencing.

Loss of ADAM29 does not affect viability and fertility in mice but improves wound healing.

ADAM29 (a disintegrin and metalloprotease domain 29) is a member of the membrane-anchored ADAM family of proteins, which is highly expressed in testis and may mediate different physiological and pathological processes. Although the functions of many ADAM family members have been well characterized, the biological relevance of ADAM29 has remained largely unknown. Here, we report the generation of an Adam29-deficient mouse model to delve deeper into the in vivo functions of this ADAM family member. We show that ADAM29 depletion does not affect mice viability, development, or fertility, but somehow impinges on metabolism and energy expenditure. We also report herein that ADAM29 deficiency leads to an accelerated wound healing process, without affecting cell reprogramming in mouse-derived fibroblasts. Collectively, our findings provide new insights into ADAM29 biological functions, highlighting the importance of non-catalytic ADAM proteases.

Noncoding RNA Contribution to Aging and Lifespan.

Aging is a multifactorial process characterized by an age-related decline in organismal fitness. This deterioration is the major risk factor for chronic diseases such as cardiovascular pathologies, neurodegeneration, or cancer, and it represents one of the main challenges of modern society. Therefore, understanding why and how we age would be a fundamental pillar to design strategies to promote a healthy aging. In the last decades, the study of the molecular bases of disease has been revolutionized by the discovery of different types of noncoding RNAs (ncRNAs) with regulatory potential. In this work, we will review the implication of ncRNAs in aging, with the aim to provide a first approach to the different aging-associated ncRNAs, their mechanism of action, and their potential relevance as therapeutic targets and disease biomarkers.

A conserved splicing mechanism of the LMNA gene controls premature aging.

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>T; 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>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) 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.

Mutational analysis of BRCA1 and BRCA2 in hereditary breast and ovarian cancer families from Asturias (Northern Spain).

BACKGROUND: The prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies according to geographical origin of studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian populations (Northern Spain). METHODS: In the present work, 256 unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analyzed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to May 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened both by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A total of 59 families (23%) were found to carry a pathogenic germ line mutation, 39 in BRCA1 and 20 in BRCA2. Twenty nine additional families (12%) carried an unknown significance variant. We detected 28 distinct pathogenic mutations (16 in BRCA1 and 12 in BRCA2), of which 3 mutations in BRCA1 (c.1674delA, c.1965C>A and c.2900_2901dupCT) and 5 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.8042_8043delCA) had not been previously described.The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 8 and 6 families respectively and clustered in two separated small geographically isolated areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.211A>G (9 families), and the common BRCA1 mutation c.3331_3334delCAAG (6 families), account for approximately 50% of all affected families. By contrast, very frequent mutations in other Spanish series such as the BRCA1 Ashkenazi founder mutation c.68_69delAG, was found in only one family. CONCLUSIONS: In this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differ from other areas of Spain. Our findings may help design a first step recurrent mutation panel for screening high-risk breast and/or ovarian cancer families from this specific area.

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

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

Insulin-like growth factor 1 treatment extends longevity in a mouse model of human premature aging by restoring somatotroph axis function.

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.

Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.

The mouse ortholog of human FACE-1, Zmpste24, is a multispanning membrane protein widely distributed in mammalian tissues and structurally related to Afc1p/ste24p, a yeast metalloproteinase involved in the maturation of fungal pheromones. Disruption of the gene Zmpste24 caused severe growth retardation and premature death in homozygous-null mice. Histopathological analysis of the mutant mice revealed several abnormalities, including dilated cardiomyopathy, muscular dystrophy and lipodystrophy. These alterations are similar to those developed by mice deficient in A-type lamin, a major component of the nuclear lamina, and phenocopy most defects observed in humans with diverse congenital laminopathies. In agreement with this finding, Zmpste24-null mice are defective in the proteolytic processing of prelamin A. This deficiency in prelamin A maturation leads to the generation of abnormalities in nuclear architecture that probably underlie the many phenotypes observed in both mice and humans with mutations in the lamin A gene. These results indicate that prelamin A is a specific substrate for Zmpste24 and demonstrate the usefulness of genetic approaches for identifying the in vivo substrates of proteolytic enzymes.