ABSTRACT
Hemoglobin is the major protein in red blood cells and transports oxygen from the lungs to oxygen-demanding tissues, like the brain. Mechanisms that facilitate the uptake of oxygen in the vertebrate brain are unknown. In invertebrates, neuronal hemoglobin serves as intracellular storage molecule for oxygen. Here, we show by immunohistochemistry that hemoglobin is specifically expressed in neurons of the cortex, hippocampus, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal hemoglobin distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels. Probing for low oxygen levels in the tissue, we provide evidence that hemoglobin alpha-positive cells in direct neighborhood with hemoglobin alpha-negative cells display a better oxygenation than their neighbors and can be sharply distinguished from those. Neuronal hemoglobin expression is upregulated by injection or transgenic overexpression of erythropoietin and is accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions. Thus we provide a novel mechanism for the neuroprotective actions of erythropoietin under ischemic-hypoxic conditions. We propose that neuronal hemoglobin expression is connected to facilitated oxygen uptake in neurons, and hemoglobin might serve as oxygen capacitator molecule.
Subject(s)
Cerebellum/metabolism , Cerebral Cortex/metabolism , Hemoglobins/biosynthesis , Hippocampus/metabolism , Neurons/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Cell Hypoxia , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Electrophoresis, Gel, Two-Dimensional , Erythropoietin/genetics , Erythropoietin/pharmacology , Female , Hippocampus/cytology , Hippocampus/drug effects , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/drug effects , Nitroimidazoles/pharmacology , Oxygen/metabolism , Rats , Rats, Wistar , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , Species SpecificityABSTRACT
Hypoxic-ischemic damage is a major challenge for neuronal tissue. In the present study, we investigated the effects of anoxia and glucose deprivation on adult neural stem cells (NSCs) in vitro. We assessed glucose deprivation, anoxia and the combination of the latter separately. After 24 h of anoxia, cell numbers increased up to 60% compared to normoxic controls. Whereas nearly all normoxic cells incorporated the mitotic marker BrdU (99%), only 85% of the anoxic cells were BrdU-positive. Counting of interphase chromosomes showed 8-fold higher cell division activity after anoxia. The number of necrotic cells doubled after anoxia (14% compared to 7% after normoxia). Apoptosis was measured by two distinct caspases assays. Whereas the total caspase activity was reduced after anoxia, caspase 3/7 showed no alterations. Glucose deprivation and oxygen glucose deprivation both reduced cell viability by 56 and 53%, respectively. Under these conditions, total caspases activity doubled, but caspase 3/7 activity remained unchanged. Erythropoietin, which was reported as neuroprotective, did not increase cell viability in normoxia, but moderately under oxygen glucose deprivation by up to 6%. Erythropoietin reduced total caspase activity by nearly 30% under all the conditions, whereas caspase 3/7 activity was not affected. Our results show that anoxia increases proliferation and viability of adult NSCs, although a fraction of NSCs does not divide during anoxia. In conclusion, anoxia increased cell viability, cell number and proliferation in NSCs from the rat brain. Anoxia turned out to be a highly stimulating environmental for NSCs and NSCs died only when deprived of glucose. We conclude that the availability of glucose but not of oxygen is a crucial factor for NSC survival, regulating apoptotic pathways via caspases activity other than the caspases 3/7 pathway. Therefore, we conclude that NSCs are dying from glucose deprivation, not from hypoxic-ischemic damage.
Subject(s)
Cell Hypoxia/physiology , Hypoxia-Ischemia, Brain/metabolism , Neuronal Plasticity/physiology , Neurons/metabolism , Recovery of Function/physiology , Stem Cells/metabolism , Animals , Apoptosis/physiology , Bromodeoxyuridine , Caspases/metabolism , Cell Count , Cell Differentiation/physiology , Cell Proliferation , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Erythropoietin/pharmacology , Glucose/deficiency , Male , Mitosis/physiology , Necrosis/etiology , Necrosis/metabolism , Neurons/cytology , Neuroprotective Agents/pharmacology , Rats , Rats, Wistar , Signal Transduction/physiology , Stem Cells/cytologyABSTRACT
On the basis of its inhibition by SB216763, we identified the multifunctional enzyme Glycogen Synthase Kinase 3beta (GSK3beta) as a central regulator for differentiation and cell survival of adult neural stem cells. Detected by proteomic approaches, members of the Wnt/beta-catenin signaling pathway appear to participate in enhanced neuronal differentiation and activated transcription of beta-catenin target genes during GSK3beta inhibition, associated with decreased apoptosis.
Subject(s)
Cell Differentiation , Cell Proliferation , Cerebral Ventricles/cytology , Glycogen Synthase Kinase 3/physiology , Neurons/cytology , Stem Cells/cytology , Animals , Apoptosis , Electrophoresis, Gel, Two-Dimensional , Glycogen Synthase Kinase 3 beta , Neurons/enzymology , Proteomics/methods , Rats , Stem Cells/enzymology , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.
