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1.  Pharmacological Induction of Neuroglobin Expression 
Pharmacology  2011;87(1-2):81-84.
Neuroglobin (Ngb) is an intracellular, oxygen-binding neuronal protein with protective effects against ischemia and related pathological processes. To identify small molecules capable of inducing Ngb protein expression, which might have therapeutic benefit, we examined Ngb expression by Western blot in cultured HN33 (mouse hippocampal neuron × N18TG2 neuroblastoma) cells. In addition to deferoxamine, which was shown previously to enhance Ngb levels, Ngb expression was increased by the short-chain fatty acids cinnamic acid and valproic acid (≥100 μmol/l), but not by other short-chain fatty acids, histone deacetylase inhibitors, or anticonvulsants. Drugs that stimulate the expression of neuroprotective proteins like Ngb may have therapeutic potential in the treatment of stroke and other neurological disorders.
doi:10.1159/000322998
PMCID: PMC3042117  PMID: 21228614
Neuroglobin; Cinnamic acid; Valproic acid; Ischemia
2.  Electrophysiological Properties of Subventricular Zone Cells in Adult Mouse Brain 
Brain research  2010;1340C:96-105.
The subventricular zone (SVZ) is a principal site of adult neurogenesis and appears to participate in the brain’s response to injury. Thus, measures that enhance SVZ neurogenesis may have a role in treatment of neurological disease. To better characterize SVZ cells and identify potential targets for therapeutic intervention, we studied electrophysiological properties of SVZ cells in adult mouse brain slices using patch-clamp techniques. Electrophysiology was correlated with immunohistochemical phenotype by injecting cells with lucifer yellow and by studying transgenic mice carrying green fluorescent protein under control of the doublecortin (DCX) or glial fibrillary acidic protein (GFAP) promoter. We identified five types of cells in the adult mouse SVZ: type 1 cells, with 4-aminopyridine (4-AP)/tetraethylammonium (TEA)-sensitive and CdCl2-sensitive inward currents; type 2 cells, with Ca2+-sensitive K+ and both 4-AP/TEA-sensitive and -insensitive currents; type 3 cells, with 4-AP/TEA-sensitive and -insensitive and small Na+ currents; type 4 cells, with slowly activating, large linear outward current and sustained outward current without fast-inactivating component; and type 5 cells, with a large outward rectifying current with a fast inactivating component. Type 2 and 3 cells expressed DCX, types 4 and 5 cells expressed GFAP, and type 1 cells expressed neither. We propose that SVZ neurogenesis involves a progression of electrophysiological cell phenotypes from types 4 and 5 cells (astrocytes) to type 1 cells (neuronal progenitors) to types 2 and 3 cells (nascent neurons), and that drugs acting on. ion channels expressed during neurogenesis might promote therapeutic neurogenesis in the injured brain.
doi:10.1016/j.brainres.2010.04.057
PMCID: PMC2884064  PMID: 20434436
doublecortin; potassium channels; sodium channels; neurogenesis; stroke
3.  VEGF overexpression delays neurodegeneration and prolongs survival in ALS mice 
We sought genetic evidence for involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase-1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF+/+). We report that SOD1G93A/VEGF+/+ double-transgenic mice show delayed motor neuron loss, delayed motor impairment and prolonged survival compared to SOD1G93A single-transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS.
doi:10.1523/JNEUROSCI.4433-06.2007
PMCID: PMC2830908  PMID: 17215390
vascular endothelial growth factor; amyotrophic lateral sclerosis; motor neuron; superoxide dismutase-1; transgenic; neurodegeneration
4.  Endothelium-Induced Proliferation and Electrophysiological Differentiation of Human Embryonic Stem Cell-Derived Neuronal Precursors 
Stem cells and development  2008;17(3):565-572.
Neurogenesis occurs in a stem cell niche in which vascular elements, including endothelial cells (ECs), are thought to play an important role. Using co-culture experiments, we investigated the effect of ECs on proliferation and functional neuronal differentiation of human embryonic stem (ES) cell-derived neuronal precursor cells (NPCs). NPCs were cultured for 5 days in medium containing fibroblast growth factor-2 (FGF-2), with or without ECs. FGF-2 and ECs were then removed, and NPCs were maintained in culture for additional periods. Compared to control NPC cultures, EC-treated NPC cultures showed increased cell proliferation at short intervals (5 days) after withdrawal of FGF-2 and larger tetrodotoxin-sensitive inward membrane currents at longer intervals (10–14 days), but a similar pattern of development of neuronal differentiation markers. The effects of ECs appeared to result from the release of soluble factors rather than from cell contact, because they were observed despite the physical separation of NPCs from ECs by a cell-impermeable membrane. These findings indicate that ECs can regulate the proliferation and electrophysiological neuronal differentiation of human NPCs.
doi:10.1089/scd.2007.0124
PMCID: PMC2810207  PMID: 18576913
5.  Regulation of Hypoxic Neuronal Death Signaling by Neuroglobin 
The signal transduction pathways involved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx-B) subunit Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal membranes and aggregation of microdomains with the subjacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consistent with an early role in death signal transduction. Ngb-expressing neurons and neurons from Ngb-overexpressing transgenic (Ngb-Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Rac1-GDI disassociation, and inhibition of actin assembly and death-signaling module polarization.
doi:10.1096/fj.07-100784
PMCID: PMC2746758  PMID: 18198211
soma; polarity; disc-death inducing signaling complex
6.  Electrophysiological neurodifferentiation of subventricular zone-derived precursor cells following stroke 
Neuroscience letters  2008;442(3):305-308.
Stroke in rodents is associated with increased neurogenesis and the migration of newborn neurons to sites of brain ischemia, where they may participate in repair and recovery. To determine if neurogenesis following stroke yields functional new neurons, we labeled neuronal precursors in the mouse subventricular zone with a lentivirus-green fluorescent protein vector, produced stroke by occluding the middle cerebral artery, and detected newborn neurons 8 weeks later by fluorescence microscopy. Patch-clamp studies on fluorescent neurons in the cortical region surrounding infarction showed tetrodotoxin-sensitive Na+ action potentials and spontaneous excitatory post-synaptic currents, suggesting that ischemia led to functional neurogenesis with synaptic integration. These findings support the hypothesis that enhancing endogenous neurogenesis after stroke might have therapeutic benefit.
doi:10.1016/j.neulet.2008.07.032
PMCID: PMC2547133  PMID: 18647640
Neurogenesis; subventricular zone; ischemia; stroke; action potential; differentiation
7.  Endothelium-Induced Proliferation and Electrophysiological Differentiation of Human Embryonic Stem Cell-Derived Neuronal Precursors 
Stem Cells and Development  2008;17(3):565-572.
Abstract
Neurogenesis occurs in a stem cell niche in which vascular elements, including endothelial cells (ECs), are thought to play an important role. Using co-culture experiments, we investigated the effect of ECs on proliferation and functional neuronal differentiation of human embryonic stem (ES) cell-derived neuronal precursor cells (NPCs). NPCs were cultured for 5 days in medium containing fibroblast growth factor-2 (FGF-2), with or without ECs. FGF-2 and ECs were then removed, and NPCs were maintained in culture for additional periods. Compared to control NPC cultures, EC-treated NPC cultures showed increased cell proliferation at short intervals (5 days) after withdrawal of FGF-2 and larger tetrodotoxin-sensitive inward membrane currents at longer intervals (10–14 days), but a similar pattern of development of neuronal differentiation markers. The effects of ECs appeared to result from the release of soluble factors rather than from cell contact, because they were observed despite the physical separation of NPCs from ECs by a cell-impermeable membrane. These findings indicate that ECs can regulate the proliferation and electrophysiological neuronal differentiation of human NPCs.
doi:10.1089/scd.2007.0124
PMCID: PMC2810207  PMID: 18576913
8.  Neuroglobin Protects Against Nitric Oxide Toxicity 
Neuroscience letters  2007;430(2):135-137.
Neuroglobin (Ngb) is a novel vertebrate globin expressed principally in neurons. Ngb expression is induced by hypoxia and ischemia, and Ngb protects neurons against these insults. The mechanism of Ngb's protective action is unknown, but its ability to bind NO suggests that NO scavenging might be involved. To test this hypothesis, we treated wild type and Ngb-transfected HN33 (mouse hippocampal neuron × N18TG2 neuroblastoma) cells with NO donors and compared their sensitivity to NO-induced cell death. Ngb overexpression shifted concentration-toxicity curves to the right, indicating reduced susceptibility to NO or is metabolites. The results suggest that the ability of Ngb to neutralize the neurotoxic effects of reactive nitrogen species may be an important contributor to its neuroprotective properties.
doi:10.1016/j.neulet.2007.10.031
PMCID: PMC2265792  PMID: 18035490
Neuroglobin; nitric oxide; hypoxia
9.  A neuroglobin-overexpressing transgenic mouse 
Gene  2007;398(1-2):172-176.
Neuroglobin (Ngb) is a recently discovered vertebrate globin expressed primarily in neurons. Ngb expression is induced by hypoxia and ischemia, and Ngb protects neurons from these insults. However, its normal physiological role and the mechanism underlying its neuroprotective action are uncertain. We report production of a transgenic mouse in which Ngb is overexpressed under the control of the chicken β-actin promoter. This mouse should prove helpful for studying Ngb-mediated effects in vitro and in vivo.
doi:10.1016/j.gene.2007.02.044
PMCID: PMC2098872  PMID: 17537594
oxygen; globin; hypoxia; ischemia; brain; neuron
10.  Electrophysiological Properties of Mouse Cortical Neuron Progenitors Differentiated In Vitro and In Vivo 
Central neurons are highly vulnerable to injury and have limited ability to regenerate. Therefore, transplantation of exogenous neuronal progenitor cells has been considered a potential therapy for the restoration of lost neurons and associated brain function. In a previous study, we found that when injected into rat brain following focal ischemia, cortical neuronal progenitor cells cultured from mouse brain can migrate into ischemic areas and differentiate into cells with morphological and biochemical features of neurons. However, no direct electrophysiological evidence was provided to indicate that these cells become functional neurons in vivo.In this study, we measured the electrophysiological properties of neuronal progenitor cells from embryonic mouse cerebral cortex, both in cell culture and in rat brain slices following intracerebral injection. We demonstrate that some of these cells differentiate to express electrophysiological properties expected of mature neurons, including tetrodotoxin-sensitive Na+ channels and N-methyl-D-aspartate receptor channels. These results support the feasibility of cell-replacement therapy for stroke using exogenous neuronal progenitors.
PMCID: PMC2596322  PMID: 19079668
Progenitor cell; differentiation; neuron; patch-clamp; ion channel; ischemia
11.  VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia 
Journal of Clinical Investigation  2003;111(12):1843-1851.
Vascular endothelial growth factor (VEGF) is an angiogenic protein with therapeutic potential in ischemic disorders, including stroke. VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the manner in which these effects may interact in the ischemic brain is poorly understood. We produced focal cerebral ischemia by middle cerebral artery occlusion for 90 minutes in the adult rat brain and measured infarct size, neurological function, BrdU labeling of neuroproliferative zones, and vWF-immunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 1–3 of reperfusion. VEGF reduced infarct size, improved neurological performance, enhanced the delayed survival of newborn neurons in the dentate gyrus and subventricular zone, and stimulated angiogenesis in the striatal ischemic penumbra, but not the dentate gyrus. We conclude that in the ischemic brain VEGF exerts an acute neuroprotective effect, as well as longer latency effects on survival of new neurons and on angiogenesis, and that these effects appear to operate independently. VEGF may, therefore, improve histological and functional outcome from stroke through multiple mechanisms.
doi:10.1172/JCI200317977
PMCID: PMC161428  PMID: 12813020
12.  Stem cell factor stimulates neurogenesis in vitro and in vivo 
Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.
doi:10.1172/JCI15251
PMCID: PMC151087  PMID: 12163450

Results 1-12 (12)