Tom Dresselaers

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

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 Pparα 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-2α 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.

Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization

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.

Cell labeling and tracking for experimental models using Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI), as one of the most powerful methods in clinical diagnosis, has emerged as an additional method in the field of molecular and cellular imaging. Compared to established molecular imaging methods, MRI provides in vivo images with high resolution. In particularly in the field of cell-based therapy, non-invasively acquired information on temporal changes of cell location linked to high-resolution anatomical information is of great interest. Relatively new approaches like responsive contrast agents or MR imaging reporter gene expression are MRI applications beyond temporal and spatial information on labeled cells towards investigations on functional changes of cells in vivo. MRI-based cell monitoring and tracking studies require prior labeling of the cells under investigation for excellent contrast against the background of host tissue. Here, an overview is provided on contrast generation strategies for MRI of cells. This includes MR contrast agents, various approaches of cell labeling and MRI as well as MR spectroscopic methods used for cell tracking in vivo. Advantages and disadvantages of the particular labeling approaches and methods are discussed. In addition to description of the methods, the emphasis is on the potential but also challenges and shortcomings of this imaging technique for applications that aim to visualize cellular processes in vivo.

Matrix-binding vascular endothelial growth factor (VEGF) isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1

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.

Effects of MRI Contrast Agents on the Stem Cell Phenotype

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.

FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry

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.

Quantitative evaluation of MRI-based tracking of ferritin-labeled endogenous neural stem cell progeny in rodent brain

Endogenous neural stem cells have the potential to facilitate therapy for various neurodegenerative brain disorders. To increase our understanding of neural stem and progenitor cell biology in healthy and diseased brain, methods to label and visualize stem cells and their progeny in vivo are indispensable. Iron oxide particle based cell-labeling approaches enable cell tracking by MRI with high resolution and good soft tissue contrast in the brain. However, in addition to important concerns about unspecific labeling and low labeling efficiency, the dilution effect upon cell division is a major drawback for longitudinal follow-up of highly proliferating neural progenitor cells with MRI. Stable viral vector-mediated marking of endogenous stem cells and their progeny with a reporter gene for MRI could overcome these limitations. We stably and efficiently labeled endogenous neural stem/progenitor cells in the subventricular zone in situ by injecting a lentiviral vector expressing ferritin, a reporter for MRI. We developed an image analysis pipeline to quantify MRI signal changes at the level of the olfactory bulb as a result of migration of ferritin-labeled neuroblasts along the rostral migratory stream. We were able to detect ferritin-labeled endogenous neural stem cell progeny into the olfactory bulb of individual animals with ex vivo MRI at 30 weeks post injection, but could not demonstrate reliable in vivo detection and longitudinal tracking of neuroblast migration to the OB in individual animals. Therefore, although LV-mediated labeling of endogenous neural stem and progenitor cells resulted in efficient and stable ferritin-labeling of stem cell progeny in the OB, even with quantitative image analysis, sensitivity remains a limitation for in vivo applications.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T2/T2* (spin–spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T2*-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Metabolic and Type 1 cannabinoid receptor imaging of a transgenic rat model in the early phase of Huntington disease

Several lines of evidence imply early alterations in metabolic and endocannabinoid neurotransmission in Huntington disease (HD). Using [(18)F]MK-9470 and small animal PET, we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding in vivo in pre-symptomatic and early symptomatic rats of HD (tgHD), in relation to glucose metabolism, morphology and behavioral testing for motor and cognitive function. Twenty-three Sprague-Dawley rats (14 tgHD and 9 wild-types) were investigated between the age of 2 and 11 months. Relative glucose metabolism and parametric CB1 receptor images were anatomically standardized to Paxinos space and analyzed voxel-wise. Volumetric microMRI imaging was performed to assess HD neuropathology. Within the first 10 months, bilateral volumes of caudate-putamen and lateral ventricles did not significantly differ between genotypes. Longitudinal- and genotype evolution showed that relative [(18)F]MK-9470 binding progressively decreased in the caudate-putamen and lateral globus pallidus of tgHD rats (-8.3%, p≤1.1×10(-5) at 5 months vs. -10.9%, p<1.5×10(-5) at 10 months). In addition, relative glucose metabolism increased in the bilateral sensorimotor cortex of 2-month-old tgHD rats (+8.1%, p≤1.5×10(-5)), where it was positively correlated to motor function at that time point. TgHD rats developed cognitive deficits at 6 and 11 months of age. Our findings point to early regional dysfunctions in endocannabinoid signalling, involving the lateral globus pallidus and caudate-putamen. In vivo CB1 receptor measurements using [(18)F]MK-9470 may thus be a useful early biomarker for HD. Our results also provide evidence of subtle motor and cognitive deficits at earlier stages than previously described.

Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism

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. 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 away 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 regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke.