Single loss of a Trp53 allele triggers an increased oxidative, DNA damage and cytokine inflammatory responses through deregulation of IκBα expression.

Dose of Trp53, the main keeper of genome stability, influences tumorigenesis; however, the causes underlying and driving tumorigenesis over time by the loss of a single p53 allele are still poorly characterized. Here, we found that single p53 allele loss specifically impacted the oxidative, DNA damage and inflammatory status of hematopoietic lineages. In particular, single Trp53 allele loss in mice triggered oxidative stress in peripheral blood granulocytes and spleenocytes, whereas lack of two Trp53 alleles produced enhanced oxidative stress in thymus cells, resulting in a higher incidence of lymphomas in the Trp53 knockout (KO) mice compared with hemizygous (HEM). In addition, single or complete loss of Trp53 alleles, as well as p53 downregulation, led to a differential increase in basal, LPS- and UVB-induced expression of a plethora of pro-inflammatory cytokine, such as interleukin-12 (Il-12a), TNFα (Tnfa) and interleukin (Il-23a) in bone marrow-derived macrophage cells (BMDMs) compared to WT cells. Interestingly, p53-dependent increased inflammatory gene expression correlated with deregulated expression of the NF-κB pathway inhibitor IκBα. Chromatin immunoprecipitation data revealed decreased p65 binding to Nfkbia in the absence of p53 and p53 binding to Nfkbia promoter, uncovering a novel crosstalk mechanism between p53 and NF-κB transcription factors. Overall, our data suggest that single Trp53 allele loss can drive a sustained inflammatory, DNA damage and oxidative stress response that, over time, facilitate and support carcinogenesis.

EndoG Knockout Mice Show Increased Brown Adipocyte Recruitment in White Adipose Tissue and Improved Glucose Homeostasis.

Brown adipose tissue (BAT) plays a central role in the regulation of whole-body energy and glucose homeostasis owing to its elevated capacity for lipid and glucose oxidation. The BAT thermogenic function, which is essential for the defense of body temperature against exposure to low environmental temperatures, relies on the expression in the inner membrane of brown adipocyte's mitochondria of uncoupling protein-1, a protein that uncouples substrate oxidation from oxidative phosphorylation and leads to the production of heat instead of ATP. BAT thermogenesis depends on proper mitochondrial biogenesis during the differentiation of brown adipocytes. Despite the data that support a role for Endonuclease G (EndoG) in the process of mitochondrial biogenesis, its function in BAT has not been explored. Here, using an EndoG knockout mouse model, we demonstrate that EndoG is not essential for the expression of mitochondrial genes involved in substrate oxidation or for the induction of thermogenic genes in BAT in response to cold exposure. We also show that a lack of EndoG is associated with an increased expression of thermogenic genes (ie, uncoupling protein-1, peroxisome proliferator-activated receptor-γ coactivator-1α) in white adipose tissue (WAT) that correlates with the appearance of brown adipocyte-like cells interspersed among white adipocytes. Interestingly, the increased browning of WAT elicited by the lack of EndoG was associated with a better glucose tolerance and reduced fat mass. Our results suggest that the induction of browning in WAT by means of inhibiting EndoG activity appears as a potential therapeutic strategy to prevent obesity and ameliorate glucose intolerance.

Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart.

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

Multifaceted role of TREX2 in the skin defense against UV-induced skin carcinogenesis.

TREX2 is a 3'-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skin carcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skin carcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFNγ, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low-density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response.

Neurobehavioral characterization of Endonuclease G knockout mice reveals a new putative molecular player in the regulation of anxiety.

Endonuclease G (EndoG) has been largely related with a role in the modulation of a caspase-independent cell death pathway in many cellular systems. However, whether this protein plays a specific role in the brain remains to be elucidated. Here we have characterized the behavioral phenotype of EndoG(-/-) null mice and the expression of the nuclease among brain regions. EndoG(-/-) mice showed normal neurological function, learning, motor coordination and spontaneous behaviors. However, these animals displayed lower activity in a running wheel and, strikingly, they were consistently less anxious compared to EndoG(+/+) mice in different tests for anxiety such as plus maze and dark-light test. We next evaluated the expression of EndoG in different brain regions of wild type mice and found that it was expressed in all over but specially enriched in the striatum. Further, subcellular biochemical experiments in neocortical samples from wild type mice revealed that EndoG is localized in pre-synaptic compartments but not in post-synaptic compartments. Altogether these findings suggest that EndoG could play a highly specific role in the regulation of anxiety by modulating synaptic components.

D-Fagomine lowers postprandial blood glucose and modulates bacterial adhesion.

D-Fagomine is an iminosugar originally isolated from seeds of buckwheat (Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested D-fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, D-fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. D-Fagomine reduced the area under the curve (0-120 min) by 20 % (P < 0·01) and shifted the time to maximum blood glucose concentration (Tmax) by 15 min at doses of 1-2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, D-fagomine (0·14 mm) agglutinated 60 % of Enterobacteriaceae (Escherichia coli, Salmonella enterica serovar Typhimurium) populations (P < 0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, d-fagomine significantly (P < 0·001) inhibited the adhesion of Enterobacteriaceae (95-99 % cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56 % cells in the supernatant) to intestinal mucosa. D-Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, D-fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease.

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV(+)) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV(+) interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin-malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes.

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-x(L). These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.