HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH) 2 D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH) 2 D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH) 2 D not only increases calcium release from bone, but also inhibits calcium incorporation in bone. IntroductionIonized serum calcium levels are critical for the correct functioning of multiple vital cellular processes. Accordingly, the regulation of calcium homeostasis is directed at maintaining serum calcium levels within a narrow physiological range. Briefly, hypocalcemia leads to increased parathyroid hormone (PTH) secretion, which stimulates renal calcium reabsorption and bone resorption. PTH also enhances production of the active form of vitamin D, 1,25(OH) 2 vitamin D [1,25(OH) 2 D], which activates the vitamin D receptor (VDR) in the intestine to increase calcium absorption, and in bone to induce bone resorption (1).Because the diet is the only source of calcium to the body, dietary calcium content is critical for calcium homeostasis. At low dietary calcium intake, active calcium transport controlled by 1,25(OH) 2 D predominates, whereas at high calcium intake, calcium is absorbed via passive diffusion. This model is supported by the observation that intestinal calcium transport is reduced in systemic Vdr-null mice, leading to hypocalcemia, hypophosphatemia, and bone abnormalities that include rickets and hyperosteoidosis. The finding that a high calcium/lactose diet prevents this phenotype con-

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Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.

BackgroundCardiovascular magnetic resonance myocardial feature tracking (CMR-FT) is a promising technique for quantification of myocardial strain from steady-state free precession (SSFP) cine images. We sought to determine the variability of CMR-FT using a non-rigid elastic registration algorithm recently available in a commercial software package (Segment, Medviso) in a real-life clinical setting.MethodsFirstly, we studied the variability in a healthy volunteer who underwent 10 CMR studies over five consecutive days. Secondly, 10 patients were selected from our CMR database yielding normal findings (normal group). Finally, we prospectively studied 10 patients with known or suspected myocardial pathology referred for further investigation to CMR (patient group). In the patient group a second study was performed respecting an interval of 30 min between studies. All studies were manually segmented at the end-diastolic phase by three observers. In all subjects left ventricular (LV) circumferential and radial strain were calculated in the short-axis direction (EccSAX and ErrSAX, respectively) and longitudinal strain in the long-axis direction (EllLAX). The level of CMR experience of the observers was 2 weeks, 6 months and >20 years.ResultsMean contouring time was 7 ± 1 min, mean FT calculation time 13 ± 2 min. Intra- and inter-observer variability was good to excellent with an coefficient of reproducibility (CR) ranging 1.6% to 11.5%, and 1.7% to 16.0%, respectively and an intraclass correlation coefficient (ICC) ranging 0.89 to 1.00 and 0.74 to 0.99, respectively. Variability considerably increased in the test-retest setting with a CR ranging 4.2% to 29.1% and an ICC ranging 0.66 to 0.95 in the patient group. Variability was not influenced by level of expertise of the observers. Neither did the presence of myocardial pathology at CMR negatively impact variability. However, compared to global myocardial strain, segmental myocardial strain variability increased with a factor 2–3, in particular for the basal and apical short-axis slices.ConclusionsCMR-FT using non-rigid, elastic registration is a reproducible approach for strain analysis in patients routinely scheduled for CMR, and is not influenced by the level of training. However, further improvement is needed to reliably depict small variations in segmental myocardial strain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12968-017-0333-y) contains supplementary material, which is available to authorized users.

Summary The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network, nor to enhanced neurotrophin expression. Instead, PHD1−/− neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1−/− neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose from glycolysis. As a result, PHD1−/− neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a novel regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke.