Isabel C. Lopez-Mejia

Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging

Antisense oligonucleotides reverse premature aging and extend life span in mutant mice that mimic aberrant splicing in progeria patients. Countering Careless Cutting Carpenters warn that one should “measure twice, cut once” to avoid unfixable assaults on building materials. Indeed, careless cutting lies at the heart of Hutchinson-Gilford progeria syndrome (HGPS). This premature aging disease is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site in LMNA RNA; aberrant cutting and splicing results in the production of an mRNA that encodes progerin, a truncated form of the lamin A protein that is also produced in small amounts during normal aging. Until now, no model system has recapitulated the pathogenic LMNA splicing that occurs in HGPS patients. Here, Osorio et al. characterize such HGPS mutant mice mimics—called LmnaG609G/G609G mice—and show that antisense oligonucleotide–based therapy reverses various premature aging phenotypes and extends life span. Encoded by the LMNA gene, lamin A is a nuclear envelope protein that is important for nuclear stability, chromatin structure, and regulation of gene expression. Osorio et al. showed that the LmnaG609G/G609G mice produced reduced amounts of intact lamin A, accumulated progerin, displayed the nuclear abnormalities and transcriptional alterations seen in other progeroid models, and sported the key clinical features of human HGPS, such as a shortened life span, reduced size, disrupted metabolism, and enhanced bone and cardiovascular maladies relative to wild-type animals. The authors then used their newly characterized HGPS animal model to test the effects of antisense morpholino oligonucleotides that bound to and blocked the aberrant splice donor site in Lmna RNA. These reagents reduced progerin accumulation and corrected the nuclear abnormalities in both cultured mutant mouse and human HGPS fibroblasts. Furthermore, LmnaG609G/G609G mice that were treated with a combination of two antisense oligonucleotides that blocked aberrant splicing displayed reduced amounts of accumulated progerin, enhanced life expectancy, and a reversal of the phenotypical and molecular alterations associated with HGPS, including the righting of gene expression aberrations and normalization of blood glucose levels. Together, these findings provide preclinical proof of concept for the use of antisense oligonucleotide–based therapies in the treatment of HGPS. Furthermore, because progerin also accumulates during normal aging, the LmnaG609G/G609G mutant mice may be useful for preclinical testing of therapies designed to slow the human aging process and prevent age-related diseases. As the poet Ralph Waldo Emerson noted, “All diseases run into one—old age.” 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.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

Cell cycle regulation of mitochondrial function

Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response.

E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis

E2F transcription factors are known regulators of the cell cycle, proliferation, apoptosis, and differentiation. Here, we reveal that E2F1 plays an essential role in liver physiopathology through the regulation of glycolysis and lipogenesis. We demonstrate that E2F1 deficiency leads to a decrease in glycolysis and de novo synthesis of fatty acids in hepatocytes. We further demonstrate that E2F1 directly binds to the promoters of key lipogenic genes, including Fasn, but does not bind directly to genes encoding glycolysis pathway components, suggesting an indirect effect. In murine models, E2F1 expression and activity increased in response to feeding and upon insulin stimulation through canonical activation of the CDK4/pRB pathway. Moreover, E2F1 expression was increased in liver biopsies from obese, glucose-intolerant humans compared with biopsies from lean subjects. Finally, E2f1 deletion completely abrogated hepatic steatosis in different murine models of nonalcoholic fatty liver disease (NAFLD). In conclusion, our data demonstrate that E2F1 regulates lipid synthesis and glycolysis and thus contributes to the development of liver pathology.

Modulation of mTOR Signalling Triggers the Formation of Stem Cell-like Memory T Cells

Robust, long-lasting immune responses are elicited by memory T cells that possess properties of stem cells, enabling them to persist long-term and to permanently replenish the effector pools. Thus, stem cell-like memory T (TSCM) cells are of key therapeutic value and efforts are underway to characterize TSCM cells and to identify means for their targeted induction. Here, we show that inhibition of mechanistic/mammalian Target of Rapamycin (mTOR) complex 1 (mTORC1) by rapamycin or the Wnt-β-catenin signalling activator TWS119 in activated human naive T cells leads to the induction of TSCM cells. We show that these compounds switch T cell metabolism to fatty acid oxidation as favoured metabolic programme for TSCM cell generation. Of note, pharmacologically induced TSCM cells possess superior functional features as a long-term repopulation capacity after adoptive transfer. Furthermore, we provide insights into the transcriptome of TSCM cells. Our data identify a mechanism of pharmacological mTORC1 inhibitors, allowing us to confer stemness to human naive T cells which may be significantly relevant for the design of innovative T cell-based cancer immunotherapies.

CDK4 is an essential insulin effector in adipocytes

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan

Alternative RNA processing of LMNA pre‐mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson‐Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin‐expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS‐like mice, whereas lamin C‐only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C‐only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

Dietary Fiber Confers Protection against Flu by Shaping Ly6c− Patrolling Monocyte Hematopoiesis and CD8+ T Cell Metabolism

SUMMARY Dietary fiber protects against chronic inflammatory diseases by dampening immune responses through short‐chain fatty acids (SCFAs). Here we examined the effect of dietary fiber in viral infection, where the anti‐inflammatory properties of SCFAs in principle could prevent protective immunity. Instead, we found that fermentable dietary fiber increased survival of influenza‐infected mice through two complementary mechanisms. High‐fiber diet (HFD)‐fed mice exhibited altered bone marrow hematopoiesis, characterized by enhanced generation of Ly6c− patrolling monocytes, which led to increased numbers of alternatively activated macrophages with a limited capacity to produce the chemokine CXCL1 in the airways. Blunted CXCL1 production reduced neutrophil recruitment to the airways, thus limiting tissue immunopathology during infection. In parallel, diet‐derived SCFAs boosted CD8+ T cell effector function by enhancing cellular metabolism. Hence, dietary fermentable fiber and SCFAs set an immune equilibrium, balancing innate and adaptive immunity so as to promote the resolution of influenza infection while preventing immune‐associated pathology. Graphical Abstract Figure. No Caption available. HighlightsThe fermentable fiber inulin and SCFAs protect against influenza‐induced pathologySCFAs alter hematopoiesis by increasing macrophage precursors in the bone marrowSCFAs shape macrophage functionality to alleviate neutrophil‐mediated tissue damageSCFAs enhance CD8+ T cell functionality by altering their metabolism &NA; Trompette et al. report that a diet rich in the fermentable fiber inulin and the associated metabolites—short‐chain fatty acids—improve the response of mice to influenza infection by dampening deleterious immunopathology caused by neutrophils while enhancing anti‐viral CD8+ T cell responses through a boost in T cell metabolism.

Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose and Fatty Acid Disposal in Muscle Cells

Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non‐targeting RNAs, myotubes silenced for Rb (by 80–90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5‐fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up‐regulated levels of glucose transporter type 4 expression (∼5‐fold increase), increased basal glucose uptake, and enhanced insulin‐induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. J. Cell. Physiol. 231: 708–718, 2016.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

Matching sets of human primary fibroblasts cocultured with placenta explants are used to compare tissue capacities to support trophoblast invasion. Substituting endometrium with dermis dramatically reduces EVCT interstitial invasion, a phenomenon related to the ECM fibronectin content, FN alternative splicing, and expression of the SR protein SRSF1.