Failure of metabolic checkpoint control during late-stage granulopoiesis drives neutropenia in reticular dysgenesis.

Cellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Here, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in reticular dysgenesis patient samples with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated AMP and IMP levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis.

Gene replacement therapy provides benefit in an adult mouse model of Leigh syndrome.

Mutations in nuclear-encoded mitochondrial genes are responsible for a broad spectrum of disorders among which Leigh syndrome is the most common in infancy. No effective therapies are available for this severe disease mainly because of the limited capabilities of the standard adeno-associated viral (AAV) vectors to transduce both peripheral organs and the CNS when injected systemically in adults. Here, we used the brain-penetrating AAV-PHP.B vector to reinstate gene expression in the Ndufs4 knockout mouse model of Leigh syndrome. Intravenous delivery of an AAV.PHP.B-Ndufs4 vector in 1-month-old knockout mice restored mitochondrial complex I activity in several organs including the CNS. This gene replacement strategy extended lifespan, rescued metabolic parameters, provided behavioural improvement, and corrected the pathological phenotype in the brain, retina, and heart of Ndufs4 knockout mice. These results provide a robust proof that gene therapy strategies targeting multiple organs can rescue fatal neurometabolic disorders with CNS involvement.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

RNA Dynamics in the Control of Circadian Rhythm.

The circadian oscillator is based on transcription-translation feedback loops that generate 24 h oscillations in gene expression. Although circadian regulation of mRNA expression at the transcriptional level is one of the most important steps for the generation of circadian rhythms within the cell, multiple lines of evidence point to a disconnect between transcript oscillation and protein oscillation. This can be explained by regulatory RNA-binding proteins acting on the nascent transcripts to modulate their processing, export, translation and degradation rates. In this chapter we will review what is known about the different steps involved in circadian gene expression from transcription initiation to mRNA stability and translation efficiency. The role of ribonucleoprotein particles in the generation of rhythmic gene expression is only starting to be elucidated, but it is likely that they cooperate with the basal transcriptional machinery to help to maintain the precision of the clock under diverse cellular and environmental conditions.

Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis.

INTRODUCTION: We previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention. METHODS: HER2/neu transgenic mice with homozygous knockout of IL-15 (here referred to as IL15KO/NeuT mice) were compared to IL-15 wild-type HER2/neu transgenic mice (NeuT) regarding mammary carcinogenesis, profile of peripheral blood lymphocytes and splenocytes and humoral and cellular responses induced by the vaccine. RESULTS: IL15KO/NeuT mice showed a significantly earlier mammary cancer onset than NeuT mice, with median latency times of 16 and 20 weeks respectively, suggesting a role for IL-15 in cancer immunosurveillance. Natural killer (NK) and CD8+ lymphocytes were significantly lower in IL15KO/NeuT mice compared to mice with wild-type IL-15. The IL-12-adjuvanted allogeneic HER2/neu-expressing cell vaccine was still able to delay mammary cancer onset but efficacy in IL-15-lacking mice vanished earlier: all vaccinated IL15KO/NeuT mice developed tumors within 80 weeks of age (median latency of 53 weeks), whereas more than 70 % of vaccinated NeuT mice remained tumor-free up to 80 weeks of age. Vaccinated IL15KO/NeuT mice showed less necrotic tumors with fewer CD3+ lymphocyes and lacked perforin-positive infiltrating cells compared to NeuT mice. Concerning the anti-vaccine antibody response, antibody titer was unaffected by the lack of IL-15, but less antibodies of IgM and IgG1 isotypes were found in IL15KO/NeuT mice. A lower induction by vaccine of systemic interferon-gamma (IFN-γ) and interleukin-5 (IL-5) was also observed in IL15KO/NeuT mice when compared to NeuT mice. Finally, we found a lower level of CD8+ memory cells in the peripheral blood of vaccinated IL15KO/NeuT mice compared to NeuT mice. CONCLUSIONS: We demonstrated that IL-15 has a role in mammary cancer immunosurveillance and that IL-15-regulated NK and CD8+ memory cells play a role in long-lasting immunoprevention, further supporting the potential use of IL-15 as adjuvant in immunological strategies against tumors.

Interplay between SOX9, ß-catenin and PPARγ activation in colorectal cancer.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and ß-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, ß-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, ß-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and ß-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, ß-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and ß-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, ß-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and ß-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

Systems genetics of metabolic health in the BXD mouse genetic reference population.

Susceptibility to metabolic syndrome (MetS) is dependent on genetics, environment, and gene-by-environment interactions, rendering the study of underlying mechanisms challenging. The majority of experiments in model organisms do not incorporate genetic variation and lack specific evaluation criteria for MetS. Here, we derived a continuous metric, the metabolic health score (MHS), based on standard clinical parameters and defined its molecular signatures in the liver and circulation. In human UK Biobank, the MHS associated with MetS status and was predictive of future disease incidence, even in individuals without MetS. Using quantitative trait locus analyses in mice, we found two MHS-associated genetic loci and replicated them in unrelated mouse populations. Through a prioritization scheme in mice and human genetic data, we identified TNKS and MCPH1 as candidates mediating differences in the MHS. Our findings provide insights into the molecular mechanisms sustaining metabolic health across species and uncover likely regulators. A record of this paper's transparent peer review process is included in the supplemental information.

Genetic and dietary modulators of the inflammatory response in the gastrointestinal tract of the BXD mouse genetic reference population.

Inflammatory gut disorders, including inflammatory bowel disease (IBD), can be impacted by dietary, environmental, and genetic factors. While the incidence of IBD is increasing worldwide, we still lack a complete understanding of the gene-by-environment interactions underlying inflammation and IBD. Here, we profiled the colon transcriptome of 52 BXD mouse strains fed with a chow or high-fat diet (HFD) and identified a subset of BXD strains that exhibit an IBD-like transcriptome signature on HFD, indicating that an interplay of genetics and diet can significantly affect intestinal inflammation. Using gene co-expression analyses, we identified modules that are enriched for IBD-dysregulated genes and found that these IBD-related modules share cis-regulatory elements that are responsive to the STAT2, SMAD3, and REL transcription factors. We used module quantitative trait locus analyses to identify genetic loci associated with the expression of these modules. Through a prioritization scheme involving systems genetics in the mouse and integration with external human datasets, we identified Muc4 and Epha6 as the top candidates mediating differences in HFD-driven intestinal inflammation. This work provides insights into the contribution of genetics and diet to IBD risk and identifies two candidate genes, MUC4 and EPHA6, that may mediate IBD susceptibility in humans.

Sodium ferrous citrate and 5-aminolevulinic acid improve type 2 diabetes by maintaining muscle and mitochondrial health.

OBJECTIVE: Improving mitochondrial function is a promising strategy for intervention in type 2 diabetes mellitus. This study investigated the preventive effects of sodium ferrous citrate (SFC) and 5-aminolevulinic acid phosphate (ALA) on several metabolic dysfunctions associated with obesity because they have been shown to alleviate abnormal glucose metabolism in humans. METHODS: Six-week-old male C57BL/6J mice were fed with a normal diet, a high-fat diet, or a high-fat diet supplemented with SFC and ALA for 15 weeks. RESULTS: The simultaneous supplementation of SFC + ALA to high-fat diet-fed mice prevented loss of muscle mass, improved muscle strength, and reduced obesity and insulin resistance. SFC + ALA prevented abnormalities in mitochondrial morphology and reverted the diet effect on the skeletal muscle transcriptome, including the expression of glucose uptake and mitochondrial oxidative phosphorylation-related genes. In addition, SFC + ALA prevented the decline in mitochondrial DNA copy number by enhancing mitochondrial DNA maintenance and antioxidant transcription activity, both of which are impaired in high-fat diet-fed mice during long-term fasting. CONCLUSIONS: These findings suggest that SFC + ALA supplementation exerts its preventive effects in type 2 diabetes mellitus via improved skeletal muscle and mitochondrial health, further validating its application as a promising strategy for the prevention of obesity-induced metabolic disorders.

The genetic background shapes the susceptibility to mitochondrial dysfunction and NASH progression.

Non-alcoholic steatohepatitis (NASH) is a global health concern without treatment. The challenge in finding effective therapies is due to the lack of good mouse models and the complexity of the disease, characterized by gene-environment interactions. We tested the susceptibility of seven mouse strains to develop NASH. The severity of the clinical phenotypes observed varied widely across strains. PWK/PhJ mice were the most prone to develop hepatic inflammation and the only strain to progress to NASH with extensive fibrosis, while CAST/EiJ mice were completely resistant. Levels of mitochondrial transcripts and proteins as well as mitochondrial function were robustly reduced specifically in the liver of PWK/PhJ mice, suggesting a central role of mitochondrial dysfunction in NASH progression. Importantly, the NASH gene expression profile of PWK/PhJ mice had the highest overlap with the human NASH signature. Our study exposes the limitations of using a single mouse genetic background in metabolic studies and describes a novel NASH mouse model with features of the human NASH.

CASR gene activating mutations in two families with autosomal dominant hypocalcemia.

BACKGROUND: Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. AIM: To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. RESULTS: We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. CONCLUSIONS: We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.

SIRT1 and the clock gene machinery in colorectal cancer.

SIRT1 and the clock genes are involved in carcinogenesis. We evaluated SIRT1 expression in 19 human colorectal cancer (CRC) specimens and clock gene expression in SIRT1-overexpressing CaCo2 and SW480 cells. In CRC, SIRT1 mean expression level was decreased. Compared to CaCo2 cells, SW480 cells displayed lower levels of SIRT1 and PER3 and higher levels of ARNTL1, CLOCK, PER1, PER2, CRY1, TIPIN, and CSNKIE. SIRT1 overexpression induced PER1 upregulation in CaCo2 and downregulation in SW480 cells. SIRT1 expression was heterogeneous in human CRC and in CRC cell lines. These results might have relevant implications for a better understanding of colorectal carcinogenesis.

DNA methyltransferases 1 and 3b expression in Huh-7 cells expressing HCV core protein of different genotypes.

BACKGROUND: Hepatitis C virus infects ~3% of the population and it is a risk factor for hepatocarcinogenesis. The epigenetic mechanisms of HCV-induced hepatocyte transformation towards malignancy in this context are unclear. AIMS: The purpose of this study was to evaluate the effect of HCV core proteins of different genotypes on DNA methyltransferases (DNMTs) induction. MATERIALS/METHODS: We investigated DNMT1, DNMT3b and E-Cadherin (CDH1) mRNA and protein expression levels in an in vitro model of Huh-7 cells expressing the HCV core protein of different genotypes: 1b, 2a, 3a, 4h and 5a. RESULTS: We found that both mRNA and protein expression levels of DNMT1 and 3b were upregulated in genotype 1b HCV core expressing cells as compared to control cells. DNMT3b mRNA levels did not change in genotypes 2a, 3a, 4h and 5a, but were upregulated at the protein level by genotype 1b, 2a, 3a. CDH1 mRNA expression was downregulated only in genotype 1b, whereas its protein expression resulted in downregulation by the HCV core of genotypes 1b, 2a and 3a. Conversely, no significant changes were observed for DNMTs and CDH1 investigated in Huh-7 cells expressing the genotypes 4h and 5a. Furthermore, we present evidence that HCV core 1b protein expression induces DNMTs overexpression through STAT3 protein as demonstrated by NSC74859 treatment. Moreover, SIRT1 inhibition affected DNMT1 and 3b expression only in HCV core protein genotype 1b expressing cells as demonstrated by treatment with its inhibitor sirtinol. CONCLUSIONS: Our findings suggest that HCV core protein could play a role in HCC development at least in part by altering DNMTs expression.

Genetic background and sex control the outcome of high-fat diet feeding in mice.

The sharp increase in obesity prevalence worldwide is mainly attributable to changes in physical activity and eating behavior but the metabolic and clinical impacts of these obesogenic conditions vary between sexes and genetic backgrounds. This warrants personalized treatments of obesity and its complications, which require a thorough understanding of the diversity of metabolic responses to high-fat diet intake. By analyzing nine genetically diverse mouse strains, we show that much like humans, mice exhibit a huge variety of physiological and biochemical responses to high-fat diet. The strains exhibit various degrees of alterations in their phenotypic makeup. At the transcriptome level, we observe dysregulations of immunity, translation machinery, and mitochondrial genes. At the biochemical level, the enzymatic activity of mitochondrial complexes is affected. The diversity across mouse strains, diets, and sexes parallels that found in humans and supports the use of diverse mouse populations in future mechanistic or preclinical studies on metabolic dysfunctions.

COX7A2L genetic variants determine cardiorespiratory fitness in mice and human.

Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3' untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.

The exercise-induced long noncoding RNA CYTOR promotes fast-twitch myogenesis in aging.

Skeletal muscle displays remarkable plasticity upon exercise and is also one of the organs most affected by aging. Despite robust evidence that aging is associated with loss of fast-twitch (type II) muscle fibers, the underlying mechanisms remain to be elucidated. Here, we identified an exercise-induced long noncoding RNA, CYTOR, whose exercise responsiveness was conserved in human and rodents. Cytor overexpression in mouse myogenic progenitor cells enhanced myogenic differentiation by promoting fast-twitch cell fate, whereas Cytor knockdown deteriorated expression of mature type II myotubes. Skeletal muscle Cytor expression was reduced upon mouse aging, and Cytor expression in young mice was required to maintain proper muscle morphology and function. In aged mice, rescuing endogenous Cytor expression using adeno-associated virus serotype 9 delivery of CRISPRa reversed the age-related decrease in type II fibers and improved muscle mass and function. In humans, CYTOR expression correlated with type II isoform expression and was decreased in aged myoblasts. Increased CYTOR expression, mediated by a causal cis­expression quantitative trait locus located within a CYTOR skeletal muscle enhancer element, was associated with improved 6-min walk performance in aged individuals from the Helsinki Birth Cohort Study. Direct CYTOR overexpression using CRISPRa in aged human donor myoblasts enhanced expression of type II myosin isoforms. Mechanistically, Cytor reduced chromatin accessibility and occupancy at binding motifs of the transcription factor Tead1 by binding, and hence sequestering, Tead1. In conclusion, the long noncoding RNA Cytor was found to be a regulator of fast-twitch myogenesis in aging.

Asperuloside Improves Obesity and Type 2 Diabetes through Modulation of Gut Microbiota and Metabolic Signaling.

Asperuloside (ASP) is an iridoid glycoside that is extracted from Eucommia leaves. Eucommia is used in traditional Chinese medicine and has a long history of benefits on health and longevity. Here, we investigated the impact of ASP on obesity-related metabolic disorders and show that ASP reduces body weight gain, glucose intolerance, and insulin resistance effectively in mice fed with a high-fat diet (HFD). Intestinal dysbiosis is closely linked with metabolic disorders. Our data indicate that ASP achieves these benefits on metabolic homeostasis by reversing HFD-induced gut dysbiosis and by changing gut-derived secondary metabolites and metabolic signaling. Our results indicate that ASP may be used to regulate gut microbiota for the treatment of obesity and type 2 diabetes.

Sustained Melanopsin Photoresponse Is Supported by Specific Roles of ß-Arrestin 1 and 2 in Deactivation and Regeneration of Photopigment.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are indispensable for non-image-forming visual responses that sustain under prolonged illumination. For sustained signaling of ipRGCs, the melanopsin photopigment must continuously regenerate. The underlying mechanism is unknown. We discovered that a cluster of Ser/Thr sites within the C-terminal region of mammalian melanopsin is phosphorylated after a light pulse. This forms a binding site for ß-arrestin 1 (ßARR1) and ß-arrestin 2. ß-arrestin 2 primarily regulates the deactivation of melanopsin; accordingly, ßαrr2-/- mice exhibit prolonged ipRGC responses after cessation of a light pulse. ß-arrestin 1 primes melanopsin for regeneration. Therefore, ßαrr1-/- ipRGCs become desensitized after repeated or prolonged photostimulation. The lack of either ß-arrestin attenuates ipRGC response under prolonged illumination, suggesting that ß-arrestin 2-mediated deactivation and ß-arrestin 1-dependent regeneration of melanopsin function in sequence. In conclusion, we discovered a molecular mechanism by which ß-arrestins regulate different aspects of melanopsin photoresponses and allow ipRGC-sustained responses under prolonged illumination.

The RNA-Binding Protein NONO Coordinates Hepatic Adaptation to Feeding.

The mechanisms by which feeding and fasting drive rhythmic gene expression for physiological adaptation to daily rhythm in nutrient availability are not well understood. Here we show that, upon feeding, the RNA-binding protein NONO accumulates within speckle-like structures in liver cell nuclei. Combining RNA-immunoprecipitation and sequencing (RIP-seq), we find that an increased number of RNAs are bound by NONO after feeding. We further show that NONO binds and regulates the rhythmicity of genes involved in nutrient metabolism post-transcriptionally. Finally, we show that disrupted rhythmicity of NONO target genes has profound metabolic impact. Indeed, NONO-deficient mice exhibit impaired glucose tolerance and lower hepatic glycogen and lipids. Accordingly, these mice shift from glucose storage to fat oxidation, and therefore remain lean throughout adulthood. In conclusion, our study demonstrates that NONO post-transcriptionally coordinates circadian mRNA expression of metabolic genes with the feeding/fasting cycle, thereby playing a critical role in energy homeostasis.

Diurnal transcriptome atlas of a primate across major neural and peripheral tissues.

Diurnal gene expression patterns underlie time-of-the-day-specific functional specialization of tissues. However, available circadian gene expression atlases of a few organs are largely from nocturnal vertebrates. We report the diurnal transcriptome of 64 tissues, including 22 brain regions, sampled every 2 hours over 24 hours, from the primate Papio anubis (baboon). Genomic transcription was highly rhythmic, with up to 81.7% of protein-coding genes showing daily rhythms in expression. In addition to tissue-specific gene expression, the rhythmic transcriptome imparts another layer of functional specialization. Most ubiquitously expressed genes that participate in essential cellular functions exhibit rhythmic expression in a tissue-specific manner. The peak phases of rhythmic gene expression clustered around dawn and dusk, with a "quiescent period" during early night. Our findings also unveil a different temporal organization of central and peripheral tissues between diurnal and nocturnal animals.