Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/greenbergLab © 2011 Buck Institute, All Rights Reserved Neuroglobin (Ngb) is a hypoxiainducible protein with cytoprotective effects in animal models of stroke, Alzheimer's disease, and related disorders, but the molecular mechanisms involved in its induction are unknown. We tested the hypothesis that hypoxiainducible factor1 (HIF1) regulates Ngb levels, using shRNAmediated knockdown and lentiviral vectormediated overexpression of the HIF1 subunit, in cultured neural (HN33) cells. HIF1 knockdown decreased and HIF1 overexpression increased Ngb levels, consistent with a connection between HIF1 and Ngb induction. These findings may have implications for understanding the hypoxiaresponse repertoire of neural cells and devising therapeutic strategies for neurologic disorders. http://www.ncbi.nlm.nih.gov/pubmed/22342914 Sat, 31 Dec 2011 00:00:00 -0800 Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGFtreated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngboverexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptordependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxiastimulated neuronal signaling pathways. http://www.ncbi.nlm.nih.gov/pubmed/22583764 Sat, 31 Dec 2011 00:00:00 -0800 Neuroglobin (Ngb) is an intracellular, oxygenbinding 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 shortchain fatty acids cinnamic acid and valproic acid (100 mol/l), but not by other shortchain 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. http://www.ncbi.nlm.nih.gov/pubmed/21228614 Fri, 31 Dec 2010 00:00:00 -0800 Little is known about the relationship between neuronal cell transplantation and endogenous neurogenesis after experimental stroke. We found previously that transplantation of neuronal precursors derived from BG01 human embryonic stem cells reduced infarct volume and improved behavioral outcome after distal middle cerebral artery occlusion (MCAO) in rats. In this study, transplantation was performed 14 days after distal MCAO and doublecortin (Dcx)expressing cells in the subventricular zone (SVZ) and subgranular zone of dentate gyrus (SGZ) were counted 60 days posttransplant. Transplantation increased neurogenesis (Dcx expression) in ipsilateral SVZ, but not in contralateral SVZ or either SGZ, in both young adult (3monthold) and aged (24monthold) rats. These findings suggest that cellbased therapy for stroke may be associated with changes in endogenous adaptive processes, including neurogenesis. http://www.ncbi.nlm.nih.gov/pubmed/21167824 Fri, 31 Dec 2010 00:00:00 -0800 We reported previously that Notch signaling is activated in human arteriovenous malformations (AVMs) and that intracerebral hemorrhage (ICH) in humans is accompanied by increased neurogenesis. The former phenomenon may be involved in AVM pathogenesis and the latter in the brain's response to ICHinduced injury. Here we describe increased expression of the hypoxiainducible neuroprotective protein, neuroglobin (Ngb), in neurons surrounding unruptured AVMs and in the perihematomal region adjacent to ICH. In these disorders, as in other clinical settings, such as ischemic stroke, AVM and ICHinduced overexpression of Ngb may be stimulated by ischemic hypoxia and may help to constrain brain injury. http://www.ncbi.nlm.nih.gov/pubmed/21725774 Fri, 31 Dec 2010 00:00:00 -0800 BACKGROUND AND PURPOSE: Interhemispheric inhibition via the corpus callosum has been proposed as an exacerbating factor in outcome from stroke. METHODS: We measured infarct volume and behavioral outcome after middle cerebral artery occlusion in callosotomized rats and acallosal mice. RESULTS: Neither callosotomy in rats nor callosal agenesis in mice improved infarct volume or behavioral outcome after middle cerebral artery occlusion. CONCLUSIONS: These findings argue against a role for transcallosal projections in exacerbating focal cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/21737800 Fri, 31 Dec 2010 00:00:00 -0800 BACKGROUND AND PURPOSE: The intraluminal suture technique for producing middle cerebral artery occlusion in rodents is the most commonly used method for modeling focal cerebral ischemia associated with clinical ischemic stroke. Synchrotron radiation angiography may provide a novel solution to directly monitor the success of middle cerebral artery occlusion. METHODS: Twenty adult SpragueDawley rats for middle cerebral artery occlusion models were prepared randomly with different suture head silicone coating. In vivo imaging was performed at beam line BL13W1, Shanghai Synchrotron Radiation Facility, Shanghai, China. RESULTS: Siliconecoated suture was superior to uncoated suture for producing consistent brain infarction. Additionally, silicone coating length was an important variable controlling the extent of the ischemic lesion: infarcts affected predominantly the caudateputamen with large variability (2 mm), both the cortex and caudateputamen (23.3 mm), and most of the hemisphere, including the hypothalamus (3.3 mm). CONCLUSIONS: Synchrotron radiation angiography provides a useful tool to observe hemodynamic changes after middle cerebral artery occlusion, and the physical properties of suture are critical to the success of the middle cerebral artery occlusion model. http://www.ncbi.nlm.nih.gov/pubmed/22180249 Fri, 31 Dec 2010 00:00:00 -0800 Neural precursor cell (NPC) transplantation may have a role in restoring brain function after stroke, but how aging might affect the brain's receptivity to such transplants is unknown. We reported previously that transplantation of human embryonic stem cell (hESC)derived NPCs together with biomaterial (Matrigel) scaffolding into the brains of young adult SpragueDawley rats 3 wks after distal middle cerebral artery occlusion (MCAO) reduced infarct volume, and improved neurobehavioral performance. In this study we compared the effect of NPC and Matrigel transplants in young adult (3moold) and aged (24moold) Fisher 344 rats from the National Institute on Aging's aged rodent colony. Distal MCAO was induced by electrocoagulation and hESCderived NPCs were transplanted into the infarct cavity 3 wks later. Aged rats developed larger infarcts, but infarct volume and performance on the cylinder and elevated body swing tests, measured 68 wks posttransplant, were improved by transplantation. We conclude that advanced age does not preclude a beneficial response to NPC and Matrigel transplantation following experimental stroke. http://www.ncbi.nlm.nih.gov/pubmed/20883527 Thu, 30 Sep 2010 00:00:00 -0700 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 patchclamp 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 4aminopyridine (4AP)/tetraethylammonium (TEA)sensitive and CdCl(2)sensitive inward currents type 2 cells, with Ca(2)sensitive K() and both 4AP/TEAsensitive and insensitive currents type 3 cells, with 4AP/TEAsensitive and insensitive K() and small Na() currents type 4 cells, with slowly activating, large linear outward current and sustained outward current without fastinactivating 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. http://www.ncbi.nlm.nih.gov/pubmed/20434436 Mon, 31 May 2010 00:00:00 -0700 Injury stimulates neurogenesis in the adult brain, but the role of injuryinduced neurogenesis in brain repair and recovery is uncertain. One strategy for investigating this issue is to ablate neuronal precursors and thereby prevent neurogenesis, but this is difficult to achieve in a specific fashion. We produced transgenic mice that express herpes simplex virus thymidine kinase (TK) under control of the promoter for doublecortin (Dcx), a microtubuleassociated protein expressed in newborn and migrating neurons. Treatment for 14 days with the antiviral drug ganciclovir (GCV) depleted Dcxexpressing and BrdUlabeled cells from the rostral subventricular zone and dentate gyrus, and abolished neurogenesis and associated neuromigration induced by focal cerebral ischemia. GCV treatment of DcxTK transgenic, but not WT, mice also increased infarct size and exacerbated postischemic sensorimotor behavioral deficits measured by rotarod, limb placing, and elevated body swing tests. These findings provide evidence that injuryinduced neurogenesis contributes to stroke outcome and might therefore be a target for stroke therapy. http://www.ncbi.nlm.nih.gov/pubmed/20385829 Wed, 31 Mar 2010 00:00:00 -0700 Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cellderived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed approximately 50 and approximately 60 reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase3, and by patchclamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event. http://www.ncbi.nlm.nih.gov/pubmed/19826433 Sun, 28 Feb 2010 00:00:00 -0800 BACKGROUND AND PURPOSE: We investigated whether neuroglobin, a neuronal protein that protects neurons from hypoxicischemic injury, is upregulated in ischemic stroke. METHODS: Neuroglobin immunoreactivity was measured in brain tissue from control subjects and patients with ischemic stroke. RESULTS: Neuroglobin was detected in several brain areas, and its expression was increased in the cortical periinfarct region after stroke. CONCLUSIONS: Ischemic stroke increases expression of the neuroprotective protein neuroglobin, suggesting neuroglobin may represent a novel target for stroke therapy. http://www.ncbi.nlm.nih.gov/pubmed/20075359 Sun, 28 Feb 2010 00:00:00 -0800 A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch1 ligands Jagged1 and Deltalike4 and the downstream Notch1 target Hes1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch1 activator. Our findings suggest that activation of Notch1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch1 in normal vasculature induces a proangiogenic state, which may contribute to the development of vascular malformations. http://www.ncbi.nlm.nih.gov/pubmed/19812212 Mon, 30 Nov 2009 00:00:00 -0800 The Notch1 signaling pathway is regarded as one of the main regulators of neural stem cell behavior during development, but its role in the adult brain is less well understood. We found that Notch1 was mainly expressed in doublecortin (DCX)positive cells corresponding to newborn neurons, whereas the Notch1 ligand, Jagged1, was predominantly expressed in glial fibrillary acidic protein (GFAP)positive astrocytic cells in the subventricular zone (SVZ) of the normal adult brain. These findings were confirmed by conditional depletion of DCXpositive cells in transgenic mice carrying herpes simplex virus thymidine kinase (HSVTK) under the control of the DCX promoter. In addition, the activated form of Notch1 (Notch intracellular domain, NICD) and its downstream transcriptional targets, Hes1 and sonic hedgehog (Shh), were also expressed in SVZ cells. Increased activation of Notch1 signaling increased SVZ cell proliferation, whereas inhibiting Notch1 signaling resulted in a reduction of proliferating cells in the SVZ. Levels of NICD, Hes1, and Shh were increased in the SVZ at 4 and 24 h after focal cerebral ischemia. Finally, ischemiainduced cell proliferation in the SVZ was blocked by inhibition of the Notch1 signaling pathway, suggesting that Notch1 signaling may have a key role in normal adult and ischemiainduced neurogenesis. http://www.ncbi.nlm.nih.gov/pubmed/19536070 Mon, 31 Aug 2009 00:00:00 -0700 VEGFB, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGFB is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGFB is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGFB targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGFB is achieved by regulating the expression of many vascular prosurvival genes via both NP1 and VEGFR1. Our work thus indicates that the function of VEGFB in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGFB inhibition may offer new therapeutic opportunities to treat neovascular diseases. http://www.ncbi.nlm.nih.gov/pubmed/19369214 Tue, 31 Mar 2009 00:00:00 -0700 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 (SVZ) with a lentivirusgreen fluorescent protein vector, produced stroke by occluding the middle cerebral artery, and detected newborn neurons 8 weeks later by fluorescence microscopy. Patchclamp studies on fluorescent neurons in the cortical region surrounding infarction showed tetrodotoxinsensitive Na() action potentials and spontaneous excitatory postsynaptic 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. http://www.ncbi.nlm.nih.gov/pubmed/18647640 Thu, 31 Jul 2008 00:00:00 -0700 Neurogenesis occurs in discrete regions of normal brains of adult mammals including humans, and is induced in response to brain injury and neurodegenerative disease. Whether intracerebral hemorrhage can also induce neurogenesis in human brain is unknown. Specimens were obtained from patients with primary intracerebral hemorrhage undergoing surgical evacuation of an intracerebral hematoma, and evaluated by twophoton laser scanning confocal microscopy. We found that neural stem/progenitor cellspecific protein markers were expressed in cells located in the perihematomal regions of the basal ganglia and parietal lobe of the adult human brain after primary intracerebral hemorrhage (n=5). Cells in this region also expressed cell proliferation markers, which colocalized to the same cells that expressed neural stem/progenitor cellspecific proteins. Our data suggest that intracerebral hemorrhage induces neurogenesis in the adult human brain. http://www.ncbi.nlm.nih.gov/pubmed/18446166 Thu, 31 Jul 2008 00:00:00 -0700 Neurogenesis occurs in a stem cell niche in which vascular elements, including endothelial cells (ECs), are thought to play an important role. Using coculture experiments, we investigated the effect of ECs on proliferation and functional neuronal differentiation of human embryonic stem (ES) cellderived neuronal precursor cells (NPCs). NPCs were cultured for 5 days in medium containing fibroblast growth factor2 (FGF2), with or without ECs. FGF2 and ECs were then removed, and NPCs were maintained in culture for additional periods. Compared to control NPC cultures, ECtreated NPC cultures showed increased cell proliferation at short intervals (5 days) after withdrawal of FGF2 and larger tetrodotoxinsensitive inward membrane currents at longer intervals (1014 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 cellimpermeable membrane. These findings indicate that ECs can regulate the proliferation and electrophysiological neuronal differentiation of human NPCs. http://www.ncbi.nlm.nih.gov/pubmed/18576913 Sat, 31 May 2008 00:00:00 -0700 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 (CTxB) 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. Neurons from Ngboverexpressing transgenic (NgbTg) 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 Rac1GDPdissociation inhibitor disassociation, and inhibition of actin assembly and deathsignaling module polarization. http://www.ncbi.nlm.nih.gov/pubmed/18198211 Sat, 31 May 2008 00:00:00 -0700 In response to injury, endogenous precursors in the adult brain can proliferate and generate new neurons, which may have the capacity to replace dysfunctional or dead cells. Although injuryinduced neurogenesis has been demonstrated in animal models of stroke, Alzheimer's disease (AD) and Huntington's disease (HD), studies of Parkinson's disease (PD) have produced conflicting results. In this study, we investigated the ability of adult mice to generate new neurons in response to the parkinsonian toxin 1methyl4phenyl1,2,3,6tetrahydropyridine (MPTP), which causes selective degeneration of nigrostriatal dopamine neurons. MPTP lesions increased the incorporation of 5bromo2'deoxyuridine5'monophosphate (BrdU), as well as the number of cells that coexpressed BrdU and the immature neuronal marker doublecortin (DCX), in two neuroproliferative regionsthe subgranular zone of the dentate gyrus (DG) and the rostral subventricular zone (SVZ). BrdUlabeled, DCXexpressing cells were not found in the substantia nigra (SN) of MPTPtreated mice, where neuronal cell bodies are destroyed, but were present in increased numbers in the striatum, where SN neurons lost in PD normally project. Fibroblast growth factor2 (FGF2), which enhances neurogenesis in a mouse model of HD, also increased the number of BrdU/DCXimmunopositive cells in the SN of MPTPtreated mice. Thus, MPTPinduced brain injury increases striatal neurogenesis and, in combination with FGF2 treatment, also stimulates neurogenesis in SN. http://www.ncbi.nlm.nih.gov/pubmed/18407421 Wed, 30 Apr 2008 00:00:00 -0700 Cerebral hypoxia and ischemia trigger endogenous protective mechanisms that can prevent or limit brain damage. Understanding these mechanisms may lead to new therapeutic strategies for stroke and related disorders. Neuroglobin (Ngb), a recently discovered protein that is distantly related to hemoglobin and myoglobin, is expressed predominantly in brain neurons, and appears to modulate hypoxicischemic brain injury. Evidence includes the observations that neuronal hypoxia and cerebral ischemia induce Ngb expression, that enhancing Ngb expression reducesand knocking down Ngb expression increaseshypoxic neuronal injury in vitro and ischemic cerebral injury in vivo, and that Ngboverexpressing transgenic mice are resistant to cerebral infarction. However, the mechanisms that underlie hypoxic induction of Ngb and neuroprotection by Ngb are still unclear. http://www.ncbi.nlm.nih.gov/pubmed/17942367 Thu, 31 Jan 2008 00:00:00 -0800 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 Ngbtransfected HN33 (mouse hippocampal neuronxN18TG2 neuroblastoma) cells with NO donors and compared their sensitivity to NOinduced cell death. Ngb overexpression shifted concentrationtoxicity 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. http://www.ncbi.nlm.nih.gov/pubmed/18035490 Mon, 31 Dec 2007 00:00:00 -0800 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 tetrodotoxinsensitive Na() channels and NmethylDaspartate receptor channels. These results support the feasibility of cellreplacement therapy for stroke using exogenous neuronal progenitors. http://www.ncbi.nlm.nih.gov/pubmed/19079668 Mon, 31 Dec 2007 00:00:00 -0800 Stroke stimulates neurogenesis in select regions of the adult brain, and the newborn neurons that result can migrate to areas of ischemic injury, where they may have the capacity to enhance brain recovery. These observations suggest that strokeinduced neurogenesis may contribute to endogenous brain repair after stroke, and that the mechanisms that underlie neurogenesis may represent potential therapeutic targets. Alternatively, transplantation of exogenously derived neural cells might also be an approach to the treatment of stroke. http://www.ncbi.nlm.nih.gov/pubmed/18045160 Wed, 31 Oct 2007 00:00:00 -0700 Neuroglobin (Ngb), a vertebrate globin expressed primarily in neurons, is induced by and protects against neuronal hypoxia and cerebral ischemia. To investigate the spectrum and mechanism of Ngb's neuroprotective action, we studied the effect of transgenic overexpression of Ngb on NMDA and betaamyloid (Abeta) toxicity in murine cortical neuron cultures in vitro and on the phenotype of Alzheimer's disease (AD) transgenic (APP(Sw,Ind)) mice. Compared with cortical neuron cultures from wildtype mice, cultures from Ngboverexpressing transgenic (NgbTg mice) were resistant to the toxic effects of NMDA and Abeta(2535), as measured by polarization of cell membrane lipid rafts, mitochondrial aggregation, lactate dehydrogenase release, and nuclear fragmentation. In addition, compared with APP(Sw,Ind) mice, doubletransgenic (NgbTg x APP(Sw,Ind)) mice showed reductions in thioflavinSstained extracellular Abeta deposits, decreased levels of Abeta(140) and Abeta(142), and improved behavioral performance in a Ymaze test of spontaneous alternations. These findings suggest that the spectrum of Ngb's neuroprotective action extends beyond hypoxicischemic insults. Ngb may protect neurons from NMDA and Abeta toxicity by inhibiting the formation of a deathsignaling membrane complex, and interventions that increase Ngb expression could have therapeutic application in AD and other neurodegenerative disorders. http://www.ncbi.nlm.nih.gov/pubmed/18025470 Wed, 31 Oct 2007 00:00:00 -0700 We investigated the effect of small cortical ischemic lesions, produced by intracerebral injection of the vasoconstrictor endothelin1, on neurogenesis in the adult mouse subventricular zone. Endothelin1 (0.51 microg) produced infarcts restricted to the cortex, and associated neurobehavioral deficits that largely resolved by 3 days. Bromodeoxyuridine labeling of proliferating cells in the subventricular zone was elevated by about 50 in endothelin1treated mice, and cells reactive for doublecortin, a marker for immature neurons, were similarly increased in number. These findings indicate that small ischemic lesions restricted to adult cerebral cortex can stimulate neuroproliferation at a distance. http://www.ncbi.nlm.nih.gov/pubmed/17669376 Tue, 31 Jul 2007 00:00:00 -0700 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 betaactin promoter. This mouse should prove helpful for studying Ngbmediated effects in vitro and in vivo. http://www.ncbi.nlm.nih.gov/pubmed/17537594 Sat, 30 Jun 2007 00:00:00 -0700 New neurons are generated continuously in the subventricular zone and dentate gyrus of the adult brain. Neuropathologic processes, including cerebral ischemia, can enhance neurogenesis, as can growth factors and other physiologic stimuli. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can promote neurogenesis, but it is unknown whether VEGF can enhance migration of newborn neurons toward sites of ischemic injury, where they might be able to replace neurons that undergo ischemic death. In the present study we produced permanent focal cerebral ischemia in transgenic (Tg) mice that overexpress VEGF. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (Brdu) labeling and immunostaining for cell typespecific markers. In VEGFTg mice, brains examined 728 days after cerebral ischemia showed markedly increased subventricular zone (SVZ) neurogenesis, chains of neuroblasts extending from the SVZ to the periinfarct cortex, and an increase in the number of newly generated cortical neurons at 1428 days after ischemia. In concert with these effects, VEGF overexpression reduced infarct volume and improved postischemic motor function. These findings provide evidence that VEGF increases SVZ neurogenesis and neuromigration, consistent with a possible role in repair. Our data suggest that in addition to its neuroprotective effects, which are associated with improved outcome in the acute phase after cerebral ischemia, VEGF enhances postischemic neurogenesis, which could provide a therapeutic target for more chronic brain repair. http://www.ncbi.nlm.nih.gov/pubmed/17243175 Wed, 31 Jan 2007 00:00:00 -0800 We sought genetic evidence for the involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF/). We report that SOD1(G93A)/VEGF/ doubletransgenic mice show delayed motor neuron loss, delayed motor impairment, and prolonged survival compared with SOD1(G93A) single transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS. http://www.ncbi.nlm.nih.gov/pubmed/17215390 Sun, 31 Dec 2006 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/17178470 Thu, 30 Nov 2006 00:00:00 -0800 Neuroglobin (Ngb), a protein related to myoglobin and hemoglobin but expressed predominantly in the brain, is induced by neuronal hypoxia and cerebral ischemia and protects against hypoxic or ischemic neuronal injury. We engineered transgenic mice that overexpress murine Ngb under the control of a chicken betaactin promoter, resulting in enhanced Ngb expression in multiple cell types and multiple tissues, including brain and heart. In Ngboverexpressing transgenic mice compared with wildtype littermates, the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by approximately 30, and the volume of myocardial infarcts produced by occlusion of the left anterior descending coronary artery was reduced by approximately 25. Ngb overexpression was associated with enhanced expression of endothelial nitric oxide synthase in vascular endothelial cells. These findings extend prior evidence for cytoprotection by Ngb and suggest both direct (parenchymatous) and indirect (vasomotor) protective mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/17098866 Tue, 31 Oct 2006 00:00:00 -0800 Current options for the treatment of stroke are extremely limited, partly because of the rapidity with which brain cells die when deprived of their blood supply. Several recent studies suggest that growth factors can produce improvement in animal models of stroke, even when administered at postischemic intervals of many hours to days, when conventional neuroprotective approaches are typically futile. Several growth factors can access the brain after systemic administration, making them more attractive as therapeutic agents. Finally, growth factors are key mediators of neurogenesis in the adult brain, which could have a role in brain repair and functional recovery following stroke. http://www.ncbi.nlm.nih.gov/pubmed/17012059 Sat, 30 Sep 2006 00:00:00 -0700 Vascular endothelial growth factor (VEGF) is an angiogenesis factor with neurotrophic, neuroprotective and neuroproliferative effects. Depending on the dose, route and time of administration in relation to focal cerebral ischemia, VEGF can improve histological outcome and sensorimotor function in rodents. However, VEGF also increases vascular permeability, which can lead to brain edema and exacerbate ischemic brain injury. Thus, although VEGF is a candidate therapeutic for stroke and other ischemic disorders, its benefit relative to risk is uncertain. Considering that functional rather than histological measures of outcome are probably most relevant to therapeutic prospects for human stroke, we investigated the effects of VEGF after middle cerebral artery occlusion in rats using a series of behavioral tests. We report that VEGF improves functional outcome in ischemic rats, including both sensorimotor and cognitive deficiencies. http://www.ncbi.nlm.nih.gov/pubmed/16928361 Sat, 30 Sep 2006 00:00:00 -0700 Experimental stroke in rodents stimulates neurogenesis and migration of newborn neurons from their sites of origin into ischemic brain regions. We report that in patients with stroke, cells that express markers associated with newborn neurons are present in the ischemic penumbra surrounding cerebral cortical infarcts, where these cells are preferentially localized in the vicinity of blood vessels. These findings suggest that strokeinduced compensatory neurogenesis may occur in the human brain, where it could contribute to postischemic recovery and represent a target for stroke therapy. http://www.ncbi.nlm.nih.gov/pubmed/16924107 Thu, 31 Aug 2006 00:00:00 -0700 Cannabinoids, acting through the CB1 cannabinoid receptor (CB1R), protect the brain against ischemia and related forms of injury. This may involve inhibiting the neurotoxicity of endogenous excitatory amino acids and downstream effectors, such as nitric oxide (NO). Cannabinoids also stimulate neurogenesis in the adult brain through activation of CB1R. Because NO has been implicated in neurogenesis, we investigated whether cannabinoidinduced neurogenesis, like cannabinoid neuroprotection, might be mediated through alterations in NO production. Accordingly, we measured neurogenesis in dentate gyrus (DG) and subventricular zone (SVZ) of CB1Rknockout (KO) and wildtype mice, some of whom were treated with the cannabinoid agonist R()Win 552122 ()2,3dihydro5methyl3(morpholinyl)methylpyrrolo1,2,3de1,4benzoxazinyl(1naphthalenyl)methanone or the NO synthase (NOS) inhibitor 7nitroindazole (7NI). NOS activity was increased by approximately 25, whereas bromodeoxyuridine (BrdU) labeling of newborn cells in DG and SVZ was reduced by approximately 50 in CB1RKO compared with wildtype mice. 7NI increased BrdU labeling in both DG and SVZ and to a greater extent in CB1RKO than in wildtype mice. In addition, R()Win 552122 and 7NI enhanced BrdU incorporation into neuronenriched cerebral cortical cultures to a similar maximal extent and in nonadditive fashion, consistent with a shared mechanism of action. Doublelabel confocal microscopy showed coexpression of BrdU and the neuronal lineage marker doublecortin (Dcx) in DG and SVZ of untreated and 7NItreated CB1RKO mice, and 7NI increased the number of Dcx and BrdU/Dcximmunoreactive cells in SVZ and DG. Thus, cannabinoids appear to stimulate adult neurogenesis by opposing the antineurogenic effect of NO. http://www.ncbi.nlm.nih.gov/pubmed/16831955 Thu, 31 Aug 2006 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/16892029 Mon, 31 Jul 2006 00:00:00 -0700 VEGF is a canonical angiogenic factor. In addition, its role as a stimulator of neurogenesis was recently uncovered. Vascular and nervous networks share common molecular mechanisms underlying their morphogenesis. VEGF is likely to regulate both processes during development and in adult organisms. http://www.ncbi.nlm.nih.gov/pubmed/16787377 Wed, 31 May 2006 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/16705031 Sun, 30 Apr 2006 00:00:00 -0700 The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of betaamyloid peptides, a set of 3943 amino acid fragments derived proteolytically from betaamyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp664 to generate a second cytotoxic peptide, APPC31, but whether this Cterminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp664 caspase cleavage site. Surprisingly, whereas betaamyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp664. These results suggest that Asp664 plays a critical role in the generation of Alzheimerrelated pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via proteinprotein interactions. http://www.ncbi.nlm.nih.gov/pubmed/16641106 Sun, 30 Apr 2006 00:00:00 -0700 Alzheimer's disease (AD) is associated with increased production of new neurons (neurogenesis), which may be directed at brain repair. However, the effect of drugs used to treat AD on neurogenesis is unknown. We administered tacrine, galantamine or memantine to mouse cerebral cortical cultures in vitro, and to mice in vivo, and measured neurogenesis by labeling newborn cells with bromodeoxyuridine (BrdU) and confirming their neuronal lineage by celltypespecific protein expression. All three drugs increased BrdU incorporation into cortical cultures in vitro by up to 40, and increased BrdU labeling of cells in neuroproliferative regions of the adult mouse brain in vivo by 2645. BrdU labeling was associated with neuronal markers, such as Hu and betaIII tubulin. Thus, drugs used to treat AD increase cerebral neurogenesis both in vitro and in vivo, which may contribute to their therapeutic effects. http://www.ncbi.nlm.nih.gov/pubmed/16580645 Sun, 30 Apr 2006 00:00:00 -0700 Neurogenesis, or the production of new neurons from neuronal precursor cells, is a normal phenomenon in the adult brain, and is accentuated by brain injury. Forms of injury associated with increased neurogenesis include both acute (e.g., stroke) and chronic neurodegenerations. Studies on human postmortem material and transgenic mice overexpressing amyloid precursor protein mutations found in familial Alzheimer's disease (AD) suggest that AD is associated with enhanced neurogenesis. However, the mechanism responsible for this effect is unknown, as is what influence it may have on the clinical course of murine or human AD. If AD leads to the production of fully functional, mature neurons that can restore brain function, strategies aimed at further increasing endogenous neurogenesis may have therapeutic value. http://www.ncbi.nlm.nih.gov/pubmed/16472199 Tue, 31 Jan 2006 00:00:00 -0800 Reelin (Reln) is a protein involved in migration of newborn neurons during development. Reln mutations produce the reeler phenotype in mice, which is characterized by a defect in brain lamination, and autosomal recessive lissencephaly in humans. Reln expression persists in adult brain, but little is known about its function. We used reeler mice to investigate the effects of Reln deficiency on neurogenesis and the response to injury in the adult brain. Newborn neurons were decreased in number in the dentate gyrus and rostral migratory stream of reeler, compared to wildtype, mice. This was due, at least in part, to impaired cell migration. In addition, reeler mice showed increased susceptibility to ischemic brain injury. Cerebral infarcts from middle cerebral artery occlusion were larger in reeler than in wildtype mice, and associated neurobehavioral abnormalities were more severe. The brains of reeler mice also showed larger excitotoxic lesions after the intracerebral injection of NmethylDaspartate. Finally, despite the fact that reeler mice had larger cerebral infarcts, the ischemiainduced enhancement of neurogenesis observed in wildtype mice was attenuated. These findings suggest that, in addition to its neurodevelopmental effects, Reln deficiency continues to influence neurogenesis and ischemic neuronal injury in the adult brain. http://www.ncbi.nlm.nih.gov/pubmed/16438965 Tue, 31 Jan 2006 00:00:00 -0800 Cannabinoids protect neurons from excitotoxic injury. We investigated the mechanisms involved by studying NmethylDaspartate (NMDA) toxicity in cultured murine cerebrocortical neurons in vitro and mouse cerebral cortex in vivo. The cannabinoid agonist R()2,3dihydro5methyl3(morpholinyl)methylpyrrolo1,2,3de1,4benzoxazinyl(1naphthalenyl)methanone mesylate R()Win 55212 reduced neuronal death in murine cortical cultures treated with 20 microM NMDA, and its protective effect was attenuated by the CB1 cannabinoid receptor (CB1R) antagonist N(piperidin1yl)5(4chlorophenyl)1(2,4cichlorophenyl)4methyl1Hpyrazole3carboxamide hydrochloride (SR141716A). Cultures from CB1Rknockout mice were more sensitive to NMDA toxicity than were cultures from wildtype mice. The in vitro protective effect of R()Win 55212 was reduced by pertussis toxin, consistent with signaling through CB1Rcoupled Gproteins. The nitricoxide synthase (NOS) inhibitors 7nitroindazole (7NI) and NomeganitroLarginine methyl ester also reduced NMDA toxicity. In addition, CB1R and neuronal NOS were coexpressed in cultured cortical neurons, suggesting that cannabinoids might reduce NMDA toxicity by interfering with the generation of NO. NOS activity in cerebral cortex was higher in CB1Rknockouts than in wildtype mice, and 7NI reduced NMDA lesion size. R()Win 55212 inhibited NO production after NMDA treatment of wildtype cortical neuron cultures, measured with 4amino5methylamino2',7'difluorofluorescein diacetate, and this effect was reversed by SR141716A. In contrast, R()Win 55212 failed to inhibit NO production in cultures from CB1R knockouts. DibutyrylcAMP blocked the protective effect of R()Win 55212, and this was reversed by the protein kinase A (PKA) inhibitor N2((pbromocinnamyl)amino)ethyl5isoquinolinesulfonamide (H89). Cannabinoids seem to protect neurons against NMDA toxicity at least in part by activation of CB1R and downstream inhibition of PKA signaling and NO generation. http://www.ncbi.nlm.nih.gov/pubmed/16299067 Tue, 31 Jan 2006 00:00:00 -0800 Vascular endothelial growth factorB (VEGFB) is an angiogenic and neuroprotective protein that reduces hypoxic and ischemic neuronal injury. To determine if VEGFB also regulates neurogenesis in the adult brain, we studied the effects of VEGFB administration in vitro and in vivo, as well as the effect of VEGFB gene knockout (KO) in mice, on bromodeoxyuridine (BrdU) incorporation and expression of immature neuronal markers in the subgranular zone (SGZ) of the hippocampal dentate gyrus and the forebrain subventricular zone (SVZ). Intracerebroventricular VEGFB administration increased BrdU incorporation into cells of neuronal lineage both in vitro and in vivo, and VEGFBKO mice showed impaired neurogenesis, consistent with a neurogenesispromoting effect of VEGFB. In addition, intraventricular administration of VEGFB restored neurogenesis to wildtype levels in VEGFBKO mice. These results suggest a role for VEGFB in the regulation of adult neurogenesis, which could have therapeutic implications for diseases associated with central neuronal loss. http://www.ncbi.nlm.nih.gov/pubmed/16337622 Sat, 31 Dec 2005 00:00:00 -0800 Vascular endothelial growth factor (VEGF),which is prominently involved in angiogenesis, also exerts direct effects on neurons, leading to neurite extension, neuroprotection, and neurogenesis. However, the signal transduction pathways employed by VEGF in neurons are incompletely understood. We investigated the molecular mechanisms through which VEGF stimulates neurogenesis in primary cultures of rat cerebral cortical neurons. VEGF increased neurite outgrowth, measured using a colorimetric assay for cresyl violet staining of neuronal processes, with halfmaximal enhancement at 10 ng/mL and maximal, approximately 60 enhancement at 30100 ng/mL. The effect of VEGF was not reproduced by VEGFB or placental growth factor, but was blocked by SU1498, consistent with a VEGFR2 receptormediated process. VEGFinduced neurite outgrowth was also blocked by the ROK inhibitor Y27632 and the Rho inhibitors sulindac and Clostridium botulium exoenzyme C3, and was accompanied by Y27632sensitive phosphorylation of cofilin, a downstream mediator of Rho/ROK signaling. We conclude that VEGF promotes neurite outgrowth from cerebral cortical neurons by interacting with VEGFR2 and activating Rho/ROK signaling pathways. http://www.ncbi.nlm.nih.gov/pubmed/16329123 Sat, 31 Dec 2005 00:00:00 -0800 Angiogenesisthe growth of new blood vesselsis a crucial force for shaping the nervous system and protecting it from disease. Recent advances have improved our understanding of how the brain and other tissues grow new blood vessels under normal and pathological conditions. Angiogenesis factors, especially vascular endothelial growth factor, are now known to have roles in the birth of new neurons (neurogenesis), the prevention or mitigation of neuronal injury (neuroprotection), and the pathogenesis of stroke, Alzheimer's disease and motor neuron disease. As our understanding of pathophysiology grows, these developments may point the way towards new molecular and cellbased therapies. http://www.ncbi.nlm.nih.gov/pubmed/16355213 Wed, 30 Nov 2005 00:00:00 -0800 There is no satisfactory treatment for Huntington's disease (HD), a hereditary neurodegenerative disorder that produces chorea, dementia, and death. One potential treatment strategy involves the replacement of dead neurons by stimulating the proliferation of endogenous neuronal precursors (neurogenesis) and their migration into damaged regions of the brain. Because growth factors are neuroprotective in some settings and can also stimulate neurogenesis, we treated HD transgenic R6/2 mice from 8 weeks of age until death by s.c. administration of FGF2. FGF2 increased the number of proliferating cells in the subventricular zone by approximately 30 in wildtype mice, and by approximately 150 in HD transgenic R6/2 mice. FGF2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6/2 mice. In the striatum, these neurons were DARPP32expressing medium spiny neurons, consistent with the phenotype of neurons lost in HD. FGF2 was neuroprotective as well, because it blocked cell death induced by mutant expanded Htt in primary striatal cultures. FGF2 also reduced polyglutamine aggregates, improved motor performance, and extended lifespan by approximately 20. We conclude that FGF2 improves neurological deficits and longevity in a transgenic mouse model of HD, and that its neuroprotective and neuroproliferative effects may contribute to this improvement. http://www.ncbi.nlm.nih.gov/pubmed/16326808 Wed, 30 Nov 2005 00:00:00 -0800 Heparinbinding epidermal growth factor (EGF)like growth factor (HBEGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with fulllength HBEGF, its HB or EGFlike domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10 by the HB domain and approximately 20 by the EGFlike domain, and the effects of the two domains were additive. Fulllength HBEGF was most effective (approximately 50 increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Nachlorate abolished cell proliferation induced by HBEGF, consistent with dependence on cellsurface heparan sulfate proteoglycans. The effect of HBEGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HBEGFinduced cell proliferation. The phosphatidylinositol 3'kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signalregulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HBEGFinduced BrdU incorporation into cultures, and HBEGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HBEGFstimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HBEGF operates. http://www.ncbi.nlm.nih.gov/pubmed/15952178 Sun, 31 Jul 2005 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15920461 Thu, 30 Jun 2005 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15941978 Tue, 31 May 2005 00:00:00 -0700 Neuroglobin (Ngb), a recently discovered intracellular respiratory globin in neurons, may play a crucial role in oxygen homeostasis in the brain. We report preliminary findings indicating the presence of functional neuroglobin in primary cultures of cerebral cortical astrocytes. Reverse transcription realtime polymerase chain reaction (RRTPCR) and immunostaining confirmed such presence in cultured astrocytes isolated from newborn mouse brain. Ngb antisense treatment increased apoptosis in ischemic astrocytes. The discovery of Ngb in astrocytes may provide some insight into how oxygen homeostasis is regulated in the brain. http://www.ncbi.nlm.nih.gov/pubmed/15657899 Thu, 31 Mar 2005 00:00:00 -0800 Nitric oxide (NO) influences infarct size after focal cerebral ischemia and also regulates neurogenesis in the adult brain. These observations suggest that therapeutic approaches to stroke that target NO signaling may provide neuroprotection and also enhance brain repair through cell replacement. However, ischemic injury and neurogenesis are both affected differently depending on which isoform of NO synthase is the source of NO. In addition, ischemia itself stimulates neurogenesis, and ischemiainduced neurogenesis may be regulated differently than neurogenesis in nonischemic brain. To determine how neuronal NO synthase affects ischemiainduced neurogenesis, transient focal cerebral ischemia was produced in wildtype mice and in knockout mice lacking neuronal NO synthase, and BrdU incorporation and doublecortin immunoreactivity were measured in the principal neuroproliferative regions of the adult brain. Knockout of neuronal NO synthase reduced infarct size and increased both basal and ischemiainduced neurogenesis, suggesting that NO from this source is an inhibitory regulator of neurogenesis in the ischemic brain. 7Nitroindazole, an NO synthase inhibitor that preferentially affects the neuronal isoform, also increased neurogenesis in rats when administered by the intracerebroventricular route. Selective inhibition of neuronal NO synthase may have the potential to both reduce infarct size and enhance neurogenesis in stroke. http://www.ncbi.nlm.nih.gov/pubmed/15689958 Mon, 28 Feb 2005 00:00:00 -0800 CB1 cannabinoid receptors (CB1Rs) are involved in protecting the brain from ischemia and related disorders. However, the underlying protective mechanisms are incompletely understood. We investigated the effect of CB1R activation on oxidative injury, which has been implicated in neuronal death after cerebral ischemia and neurodegenerative disorders, in mouse cortical neuron cultures. The CB1R agonist Win 552122 R()2,3dihydro5methyl3(morpholinyl)methylpyrrolo1,2,3de1,4benzoxazinyl(1naphthalenyl)methanone mesylate reduced neuronal death, measured by lactate dehydrogenase release, in cultures treated with 50 microM FeCl2, and its protective effect was attenuated by the CB1R antagonist SR141716A N(piperidin1yl)5(4chlorophenyl)1(2,4cichlorophenyl)4methyl1Hpyrazole3carboxamide hydrochloride. The endocannabinoid anandamide reproduced the effect of Win 552122, as did the antioxidant 6hydroxy2,5,7,8tetramethylchroman2carboxylic acid (Trolox). Neuronal injury was more severe after in vitro or in vivo administration of FeCl2 to CB1Rknockout compared with wildtype mice. Win 552122 reduced the formation of reactive oxidative species in cortical neuron cultures treated with FeCl2, consistent with an antioxidant action. Pertussis toxin reduced CB1Rmediated protection, which points to a protective mechanism that involves signaling through G(i/o) proteins. Since CB1Ractivated G protein signaling inhibits protein kinase A but activates phosphatidylinositol 3kinase (PI3K), we tested the involvement of these pathways in CB1Rmediated neuroprotection. Dibutyrylcyclic adenosine monophosphate (dbcAMP) blocked protection by Win 552122, whereas the PI3K inhibitor wortmannin did not, and the effect of dbcAMP was inhibited by the protein kinase A inhibitor H89 N2((pbromocinnamyl)amino)ethyl5isoquinolinesulfonamide ( or =10 nM). CB1Rinduced, SR141716A, pertussis toxin, and dbcAMPsensitive protection was also observed for two other oxidative insults, exposure to H2O2 or buthionine sulfoximine. Therefore, receptorstimulated inhibition of protein kinase A seems to be required for the neuroprotective effect of CB1R activation against oxidative neuronal injury. http://www.ncbi.nlm.nih.gov/pubmed/15626718 Mon, 28 Feb 2005 00:00:00 -0800 Cell replacement therapy may have the potential to promote brain repair and recovery after stroke. To compare how focal cerebral ischemia affects the entry, migration, and phenotypic features of neural precursor cells transplanted by different routes, we administered neuronal precursors from embryonic cerebral cortex of green fluorescent protein (GFP)expressing transgenic mice to rats that had undergone middle cerebral artery occlusion (MCAO) by the intrastriatal, intraventricular, and intravenous routes. MCAO increased the entry of GFPimmunoreactive cells, most of which expressed neuroepithelial (nestin) or neuronal (doublecortin) markers, from the ventricles and bloodstream into the brain, and enhanced their migration when delivered by any of these routes. Transplanted neural precursors migrated into the ischemic striatum and cerebral cortex. Thus, transplantation of neural precursors by a variety of routes can deliver cells with the potential to replace injured neurons to ischemic brain regions. http://www.ncbi.nlm.nih.gov/pubmed/15686965 Mon, 31 Jan 2005 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/15670206 Mon, 31 Jan 2005 00:00:00 -0800 Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescentonset IGEs. We report a singlenucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both casecontrol and familybased association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2centered nineSNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1 95 confidence interval 2.912.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genomecoded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter gammaaminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present. http://www.ncbi.nlm.nih.gov/pubmed/15532013 Fri, 31 Dec 2004 00:00:00 -0800 Neuroglobin (Ngb), a recently discovered O2binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxicischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in agerelated neurodegenerative disorders. Western blots from young adult (3 month), middleaged (12 month), and aged (24 month) rats showed an agerelated decline in Ngb expression in cerebral neocortex, hippocampus, caudateputamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to agerelated neurological disorders. http://www.ncbi.nlm.nih.gov/pubmed/15582755 Tue, 30 Nov 2004 00:00:00 -0800 The adult mammalian brain retains the capacity for neurogenesis, by which new neurons may be generated to replace those lost through physiological or pathological processes. However, neurogenesis diminishes with aging, and this casts doubt on its feasibility as a therapeutic target for cell replacement therapy in stroke and neurodegenerative disorders, which disproportionately affect the aged brain. In previous studies, neurogenesis was stimulated by cerebral ischemia in young rodents, and the neurogenesis response of the aged rodent brain to physiological stimuli, such as hormonal manipulation and growth factors, was preserved. To investigate the effect of aging on ischemiainduced neurogenesis, transient (60 min) middle cerebral artery occlusion was induced in young adult (3month) and aged (24month) rats, who were also given bromodeoxyuridine to label newborn cells. As found in prior studies, basal neurogenesis in control, nonischemic rats was reduced with aging. Ischemia failed to stimulate neurogenesis in the dentate gyrus (DG) subgranular zone (SGZ), in contrast to results obtained previously after more prolonged (90120 min) middle cerebral artery occlusion, but increased the number of BrdUlabeled cells in the forebrain subventricular zone (SVZ). This effect was less prominent in aged than in young adult rats, with foldstimulation of BrdU incorporation reduced by approximately 20 and the total number of cells generated diminished by approximately 50. BrdUlabeled cells in SVZ coexpressed neuronal lineage markers, consistent with newborn neurons. We conclude that ischemiainduced neurogenesis occurs in the aged brain, and that measures designed to augment this phenomenon might have therapeutic applications. http://www.ncbi.nlm.nih.gov/pubmed/15569354 Sun, 31 Oct 2004 00:00:00 -0700 Vascular endothelial growth factorB (VegfB) is an angiogenic protein related to VegfA, although it acts on a different set of tyrosine kinase receptors. Like VegfA, VegfB is expressed in the brain and is induced at sites of brain injury. VegfA has neuroprotective and angiogenic effects, but VegfAknockout mice die in utero, so the effect of endogenous VegfA signaling in neuropathologic states, such as cerebral ischemia, cannot be tested directly. In contrast, VegfBknockout mice survive to adulthood with little abnormality in the absence of pathologic stresses. To determine if VegfB regulates the severity of cerebral ischemia, the middle cerebral artery was occluded in VegfBknockout, heterozygous, and wildtype mice, and the volume of the resulting cerebral infarcts and associated impairment of neurologic function were measured. Infarct volume was increased by approximately 40 and neurologic impairment was more severe in VegfBknockout mice, implying that endogenous VegfB acts to protect the brain from ischemic injury. VegfB also protected cultured cerebral cortical neurons from hypoxic injury, suggesting that its protective action is mediated at least in part through a direct effect on neurons. http://www.ncbi.nlm.nih.gov/pubmed/15529014 Sun, 31 Oct 2004 00:00:00 -0700 Neurogenesis continues in the adult brain and is increased in certain pathological states. We reported recently that neurogenesis is enhanced in hippocampus of patients with Alzheimer's disease (AD). We now report that the effect of AD on neurogenesis can be reproduced in a transgenic mouse model. PDGFAPP(Sw,Ind) mice, which express the Swedish and Indiana amyloid precursor protein mutations, show increased incorporation of BrdUrd and expression of immature neuronal markers in two neuroproliferative regions: the dentate gyrus and subventricular zone. These changes, consisting of approximately 2fold increases in the number of BrdUrdlabeled cells, were observed at age 3 months, when neuronal loss and amyloid deposition are not detected. Because enhanced neurogenesis occurs in both AD and an animal model of AD, it seems to be caused by the disease itself and not by confounding clinical factors. As neurogenesis is increased in PDGFAPP(Sw,Ind) mice in the absence of neuronal loss, it must be triggered by more subtle disease manifestations, such as impaired neurotransmission. Enhanced neurogenesis in AD and animal models of AD suggests that neurogenesis may be a compensatory response and that measures to enhance neurogenesis further could have therapeutic potential. http://www.ncbi.nlm.nih.gov/pubmed/15340159 Tue, 31 Aug 2004 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15284846 Sat, 31 Jul 2004 00:00:00 -0700 Pharmacological studies suggest a role for CB1 cannabinoid receptors (CB1R) in regulating neurogenesis in the adult brain. To investigate this possibility, we measured neurogenesis by intraperitoneal injection of bromodeoxyuridine (BrdU), which labels newborn neurons, in wildtype and CB1Rknockout (CB1RKO) mice. CB1RKO mice showed reductions in the number of BrdUlabeled cells to approximately 50 of wildtype (WT) levels in dentate gyrus and subventricular zone (SVZ), suggesting that CB1R activation promotes neurogenesis. To test this further, WT mice were given the CB1R antagonist N(piperidin1yl)5(4chlorophenyl)1(2,4dichlorophenyl)4methyl1Hpyrazole3carboximide hydrochloride (SR141716A) before measuring neurogenesis with BrdU. SR141716A paradoxically increased the number of BrdUlabeled cells by approximately 50 in SVZ another CB1R antagonist, 1(2,4dichlorophenyl)5(4iodophenyl)4methylN1piperidinyl1Hpyrazole3carboxamide (AM251), had a similar effect. To investigate this discrepancy, SR141716A was given to CB1RKO mice, in which it still stimulated neurogenesis, indicating involvement of a nonCB1 receptor. Action at one such nonCB1, SR141716Asensitive site, the VR1 vanilloid receptor, was tested by administering SR141716A to VR1KO mice, in which the ability of SR141716A to enhance neurogenesis was abolished. Thus, CB1 and VR1 receptors both seem to have roles in regulating adult neurogenesis. http://www.ncbi.nlm.nih.gov/pubmed/15266010 Wed, 30 Jun 2004 00:00:00 -0700 We used highthroughput Western blotting to identify proteins that are up or downregulated by neuronal hypoxia in vitro. Exposure to hypoxia for 24 h produced or = 1.5fold increases in the expression of 10/700 proteins (1.4) and decreases in the expression of 16/700 proteins (2.3). Upregulated proteins included Arc, doublecortin/calmodulin kinaselike 1, integrin alpha(v), and fibronectin downregulated proteins included nuclear autoantigenic sperm protein, protein kinase Crelated kinase 2, and E2F transcription factor 1. The prominence of cytoskeletonrelated proteins among those showing altered expression highlights the role of the cytoskeleton in neuronal responses to hypoxia. http://www.ncbi.nlm.nih.gov/pubmed/15176469 Mon, 31 May 2004 00:00:00 -0700 Heparinbinding epidermal growth factorlike growth factor (HBEGF) is a hypoxiainducible, neuroprotective protein that also stimulates proliferation of neuronal precursor cells. Accordingly, HBEGF may contribute to recovery from cerebral injury through direct neuroprotective effects, by enhancing neurogenesis, or both. When administered by the intracerebroventricular route 13 days after focal cerebral ischemia in adult rats, HBEGF decreased the volume of the resulting infarcts and reduced postischemic neurological deficits. HBEGF also increased the incorporation of bromodeoxyuridine into cells expressing the immature neuronal marker protein TUC4 in the dentate subgranular and rostral subventricular zones, consistent with increased proliferation of neuronal precursors. However, HBEGF decreased the number of newborn neurons that migrated into the ischemic striatum, perhaps partly because reduction of infarct size by HBEGF also reduced the stimulus to migration. To determine if HBEGF might also directly inhibit migration of neuronal precursors, we cocultured subventricular zone (SVZ) explants treated with HBEGF or vehicle together with hypoxic cerebral cortical explants, and measured cell migration from the former toward the latter. HBEGF reduced directed migration of SVZ cells toward the cortical explants, possibly due to a local chemoattractant effect on neuronal precursor cells, which may be mediated through the HBEGFspecific receptor, Narginine dibasic convertase. The delayed neuroprotective effect of HBEGF may have implications for efforts to prolong the therapeutic window for intervention in stroke. http://www.ncbi.nlm.nih.gov/pubmed/15087709 Wed, 31 Mar 2004 00:00:00 -0800 Stathmin is a developmentally regulated cytosolic protein expressed at high levels in the brain. Twodimensional differential ingel electrophoresis and mass spectroscopy of proteins expressed in immature and mature cultures from embryonic rat cerebral cortex identified stathmin among several differentially expressed proteins, consistent with a possible role in neurogenesis. Stathmin immunohistochemistry in adult rodent brain revealed prominent expression in neuroproliferative zones and neuronal migration pathways, a pattern that resembles the expression of doublecortin, which is implicated in neuronal migration. Stathmin immunoreactivity was also associated with neurons undergoing ectopic chain migration into the ischemic striatum and cerebral cortex following focal cerebral ischemia. Reducing the expression of stathmin or doublecortin with an antisense oligonucleotide inhibited the migration of new neurons from the subventricular zone to the olfactory bulb via the rostral migratory stream. These results suggest a role for stathmin in the migration of newborn neurons in the adult brain. http://www.ncbi.nlm.nih.gov/pubmed/14769823 Sat, 31 Jan 2004 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/14720577 Wed, 31 Dec 2003 00:00:00 -0800 Neurogenesis, which persists in the adult mammalian brain, may provide a basis for neuronal replacement therapy in neurodegenerative diseases like Alzheimer's disease (AD). Neurogenesis is increased in certain acute neurological disorders, such as ischemia and epilepsy, but the effect of more chronic neurodegenerations is uncertain, and some animal models of AD show impaired neurogenesis. To determine how neurogenesis is affected in the brains of patients with AD, we investigated the expression of immature neuronal marker proteins that signal the birth of new neurons in the hippocampus of AD patients. Compared to controls, Alzheimer's brains showed increased expression of doublecortin, polysialylated nerve cell adhesion molecule, neurogenic differentiation factor and TUC4. Expression of doublecortin and TUC4 was associated with neurons in the neuroproliferative (subgranular) zone of the dentate gyrus, the physiological destination of these neurons (granule cell layer), and the CA1 region of Ammon's horn, which is the principal site of hippocampal pathology in AD. These findings suggest that neurogenesis is increased in AD hippocampus, where it may give rise to cells that replace neurons lost in the disease, and that stimulating hippocampal neurogenesis might provide a new treatment strategy. http://www.ncbi.nlm.nih.gov/pubmed/14660786 Wed, 31 Dec 2003 00:00:00 -0800 Bone marrow cells (BMC) can be induced to express neuronal phenotypic features in vitro, but the extent to which they can transdifferentiate to mature, functional neurons is uncertain. We examined the effects of different growth factors and combinations thereof on the expression of neuronal marker proteins in cultures of BMC enriched in marrow stromal cells. Patterns of neuronal marker expression varied depending on the growth factor or factors to which BMC cultures were exposed. Cultures treated for up to 5 weeks with epidermal growth factor, fibroblast growth factor2, retinoic acid, and nerve growth factor displayed neuronlike cellular processes and expressed neuronal markers, including the neuronal nuclear antigen NeuN, microtubuleassociated protein 2, tau, synaptophysin, alpha(1A) and alpha(1B) calcium channel subunits, NR2A glutamate receptor subunits, and gammaaminobutyric acid. However, the intracellular distribution of these markers was distinct from their usual distribution in mature neurons. We conclude that a variety of growth factors can drive BMC toward a neuronal phenotype or phenotypes, but that morphological neuronal features and the ectopic expression of neuronal proteins and neurotransmitters may not equate with the ability to execute normal neuronal functions. http://www.ncbi.nlm.nih.gov/pubmed/14637082 Fri, 31 Oct 2003 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/14552865 Tue, 30 Sep 2003 00:00:00 -0700 Pathological processes, including cerebral ischemia, can enhance neurogenesis in the adult brain, but the fate of the newborn neurons that are produced and their role in brain repair are obscure. To determine if ischemiainduced neuronal proliferation is associated with migration of nascent neurons toward ischemic lesions, we mapped the migration of cells labeled by cell proliferation markers and antibodies against neuronal marker proteins, for up to 2 weeks after a 90min episode of focal cerebral ischemia caused by occlusion of the middle cerebral artery. Doublecortinimmunoreactive cells in the rostral subventricular zone, but not the dentate gyrus, migrated into the ischemic penumbra of the adjacent striatum and, via the rostral migratory stream and lateral cortical stream, into the penumbra of ischemic cortex. These results indicate that after cerebral ischemia, new neurons are directed toward sites of brain injury, where they might be in a position to participate in brain repair and functional recovery. http://www.ncbi.nlm.nih.gov/pubmed/14550778 Tue, 30 Sep 2003 00:00:00 -0700 BACKGROUND AND PURPOSE: Endoglin (CD105) is a membrane glycoprotein that is mutated in hereditary hemorrhagic telangiectasia (OslerRenduWeber disease) and shows increased expression in proliferating endothelial cells during angiogenesis. METHODS: We investigated the effect of hypoxia on endoglin expression in murine cerebral microvascular endothelial (bEND.3) cells in vitro and the possible involvement of mitogenactivated protein kinase (MAPK) pathways. RESULTS: Hypoxia increased endoglin mRNA and protein expression in bEND.3 cells, which was associated with phosphoactivation of extracellular signalrelated kinase (ERK), p38 MAPK, and Jun aminoterminal kinase (JNK). Inhibitors of p38 decreased hypoxic induction of endoglin expression, as did dominant negative MAPK kinase 3 (MKK3), which activates p38. In contrast, constitutively active MKK3 or JNK1 potentiated the hypoxic induction of endoglin. CONCLUSIONS: These results indicate that hypoxia induces the expression of endoglin at both the mRNA and protein levels and that induction is regulated by the p38 and perhaps also JNK pathways. http://www.ncbi.nlm.nih.gov/pubmed/12947156 Tue, 30 Sep 2003 00:00:00 -0700 Neurogenesis, which may contribute to the ability of the adult brain to function normally and adapt to disease, nevertheless declines with advancing age. Adult neurogenesis can be enhanced by administration of growth factors, but whether the aged brain remains responsive to these factors is unknown. We compared the effects of intracerebroventricular fibroblast growth factor (FGF)2 and heparinbinding epidermal growth factorlike growth factor (HBEGF) on neurogenesis in the hippocampal dentate subgranular zone (SGZ) and the subventricular zone (SVZ) of young adult (3month) and aged (20month) mice. Neurogenesis, measured by labelling with bromodeoxyuridine (BrdU) and by expression of doublecortin, was reduced by approximately 90 in SGZ and by approximately 50 in SVZ of aged mice. HBEGF increased BrdU labelling in SGZ at 3 months by approximately 60 and at 20 months by approximately 450, which increased the number of BrdUlabelled cells in SGZ of aged mice to approximately 25 of that in young adults. FGF2 also stimulated BrdU labelling in SGZ, by approximately 25 at 3 months and by approximately 250 at 20 months, increasing the number of newborn neurones in older mice to approximately 20 of that in younger mice. In SVZ, HBEGF and FGF2 increased BrdU incorporation by approximately 140 at 3 months and approximately 170 at 20 months, so the number of BrdUlabelled cells was comparable in untreated 3monthold and growth factortreated 20monthold mice. These results demonstrate that the aged brain retains the capacity to respond to exogenous growth factors with increased neurogenesis, which may have implications for the therapeutic potential of neurogenesis enhancement in ageassociated neurological disorders. http://www.ncbi.nlm.nih.gov/pubmed/12882410 Mon, 30 Jun 2003 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/12835424 Mon, 30 Jun 2003 00:00:00 -0700 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 vWFimmunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 13 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. http://www.ncbi.nlm.nih.gov/pubmed/12813020 Sat, 31 May 2003 00:00:00 -0700 Many changes in neuronal gene expression occur in response to ischemia, and these may play a role in determining the fate of ischemic neurons. To identify genes induced in the rat brain following cerebral ischemia, a strategy was used that combines subtractive hybridization and differential screening. Among the genes identified was one referred to as global ischemiainducible gene 11(Giig11). Sequence analysis indicated that Giig11 exhibited 97 and 91 identity to the known Ero1L (S. cereviseae ero1like oxidoreductase) of mouse and human origin, which is involved in oxidative endoplasmic reticulum protein folding. Rat Ero1L/Giig11 also contains a l07bp sequence that is nearly identical ( 95) to the known dispersed repetitive identifier (ID), but which is lacking in mouse and human Ero1L. Northern blotting showed that expression of the ID element and Ero1L/Giig11 mRNA increased after global cerebral ischemia. In situ hybridization demonstrated increased expression of Ero1L/Giig11 in the brain following ischemic injury, with the highest levels in the vulnerable hippocampal CA1 pyramidal neurons. Transfection of cultured primary hippocampal neurons with a plasmid containing green fluorescent protein (gfp) and Ero1L/Giig11 cDNA (with and without the ID element) produced a gfpEro1L/Giig11 fusion protein, and more fusion protein was localized into dendrites in the presence of the ID element, suggesting that the ID element promotes Ero1L/Giig11 protein localization to dendrites. Therefore, Ero1L/Giig11 may have a role in ischemiainduced neuronal repair or survival mechanisms directed at counteracting abnormalities in protein folding, maturation and distribution. http://www.ncbi.nlm.nih.gov/pubmed/12694393 Wed, 30 Apr 2003 00:00:00 -0700 Neuroglobin (Ngb) is an O(2)binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O(2), detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngbexpressing adenoassociated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke. http://www.ncbi.nlm.nih.gov/pubmed/12621155 Fri, 28 Feb 2003 00:00:00 -0800 Neurogenesis persists in the adult brain, where it may contribute to repair and recovery after injury, but the lack of methods for noninvasive stimulation of cerebral neurogenesis limits its potential for clinical application. We report that intranasal administration of either fibroblast growth factor2 or heparinbinding epidermal growth factorlike growth factor increases neurogenesis, measured by the incorporation of bromodeoxyuridine into cells that express the early neuronal marker protein doublecortin in the subventricular zone of mouse brain. These findings indicate that intranasal growth factors may have potential as neurogenesispromoting therapeutic agents. http://www.ncbi.nlm.nih.gov/pubmed/12601711 Fri, 31 Jan 2003 00:00:00 -0800 A recombinant adenoassociated virus (rAAV) vector was used to overexpress the antiapoptotic Bcl2family protein, BCLw, in rat brain. Three weeks after injecting the vector into cerebral cortex and striatum on one side, temporary focal ischemia was induced by occlusion of the ipsilateral middle cerebral artery for 90 min, followed by reperfusion for 24 h. BCLw expression was increased in cerebral cortex and striatumand in neurons, astroglia and endothelial cellsin the brains of rats that received the rAAVBCLw vector, compared to rats given phosphatebuffered saline or a control vector containing the gene for green fluorescent protein. Recipients of the rAAVBCLw vector also showed a 30 reduction in infarct size and a 3340 improvement in neurological function, compared to the control groups. These results provide evidence for a role of BCLw in regulating histological and functional outcome after focal cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/12571640 Fri, 31 Jan 2003 00:00:00 -0800 Neurogenesis, or the production of new neurons, is regulated by physiological and pathological processes including aging, stress, and brain injury. Many mitogenic and trophic factors that regulate proliferation of nonneuronal cells are also involved in neurogenesis. These include vascular endothelial cell growth factor (VEGF), which stimulates the incorporation of bromodeoxyuridine (BrdU) into neuronal precursor cells in vitro and in the adult rat brain in vivo. Using BrdU labeling as an index of cell proliferation, we found that the in vitro neuroproliferative effect of VEGF was associated with upregulation of E2F family transcription factors, cyclin D1, cyclin E, and cdc25. VEGF also increased nuclear expression of E2F1, E2F2, and E2F3, consistent with regulation of the G1/S phase transition of the cell cycle. The proliferative effect of VEGF was inhibited by the extracellular signalregulated kinase kinase (MEK) inhibitor PD98059, the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF102390X, and the phosphatidylinositol 3kinase (PI3K) inhibitor wortmannin, indicating involvement of multiple signaling pathways. These findings help to provide a molecular basis for some of the recently identified neuronal effects of VEGF. http://www.ncbi.nlm.nih.gov/pubmed/12554697 Tue, 31 Dec 2002 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/12461187 Sat, 30 Nov 2002 00:00:00 -0800 Endogenous cannabinoid signaling pathways have been implicated in protection of the brain from hypoxia, ischemia, and trauma, but the mechanism for these protective effects is uncertain. We found that in CB1 cannabinoid receptor knockout mice, mortality from permanent focal cerebral ischemia was increased, infarct size and neurological deficits after transient focal cerebral ischemia were more severe, cerebral blood flow in the ischemic penumbra during reperfusion was reduced, and NMDA neurotoxicity was increased compared with wildtype littermates. These findings indicate that endogenous cannabinoid signaling pathways protect mice from ischemic stroke by a mechanism that involves CB1 receptors, and suggest that both blood vessels and neurons may be targets of this protective effect. http://www.ncbi.nlm.nih.gov/pubmed/12427832 Thu, 31 Oct 2002 00:00:00 -0800 We used cDNA microarray gene expression profiling to characterize the transcriptional response to exposure of cultured mouse cerebral cortical neurons to hypoxia for 24 hr. Of 11,200 genes examined, 1,405 (12.5) were induced or repressed at least 1.5fold, whereas 26 known genes were induced and 20 known genes were repressed at least 2.5fold. The most strongly induced genes included genes coding for endoplasmic reticulum proteins (Ero1L/Giig11, Sac1p, Ddit3/Gadd153), proteins involved in ubiquitination (Arih2, P4hb), proteins induced by hypoxia in nonneuronal systems (Gpi1, Aldo1, Anxa2, Hig1), and proteins that might promote cell death (Gas5, Egr1, Ndr1, Vdac2). These findings reinforce the importance of endoplasmic reticulumbased mechanisms and of proteinubiquitination pathways in the neuronal response to hypoxia. http://www.ncbi.nlm.nih.gov/pubmed/12462408 Mon, 30 Sep 2002 00:00:00 -0700 Neuroglobin is a newly identified vertebrate globin that binds O(2) and is expressed in cerebral neurons. We found recently that neuronal expression of neuroglobin is stimulated by hypoxia and ischemia and protects neurons from hypoxic injury. Here we report that, like hemoglobin and myoglobin, neuroglobin expression can also be induced by hemin. Induction was concentration dependent and time dependent, with maximal (about 4fold) increases in neuroglobin mRNA and protein levels occurring with 50 microM hemin and at 8 to 24 hours. The inductive effect of hemin was attenuated by the protein kinase G inhibitor KT5823 and the soluble guanylate cyclase inhibitor LY83583, was mimicked by treatment with 8bromocyclic guanosine 3',5'monophosphate, and was accompanied by a greater than 10fold increase in cGMP levels, suggesting that it is mediated through protein kinase G and soluble guanylate cyclase. In contrast, hypoxic induction of neuroglobin was blocked by the mitogenactivated protein kinase/extracellular signalregulated kinase kinase inhibitor PD98059, indicating that hemin and hypoxia regulate neuroglobin expression by different mechanisms. These results provide evidence for regulation of neuroglobin expression by at least 2 signal transduction pathways. http://www.ncbi.nlm.nih.gov/pubmed/12239161 Sat, 31 Aug 2002 00:00:00 -0700 To identify novel genes involved in cannabinoid receptormediated signaling, we used cDNA microarrays to detect changes in mRNA expression in the forebrains of mice 12 h after they were given a single intraperitoneal dose of the naturallyoccurring Cannabis sativa alkaloid Delta(9)tetrahydrocannabinol (Delta(9)THC) or the synthetic cannabinoid receptor agonist (R)()2,3dihydro5methyl3(morpholinyl)methyl pyrrolo1,2,3de1,4benzoxazinyl1naphtalenylmethanone mesylate R()WIN 55,2122. Of approximately 11,000 genes from a mouse brain cDNA library that were probed, 65 showed altered (increased or decreased at least 2fold) expression after exposure to Delta(9)THC, 41 after exposure to R()WIN 55,2122, and 20 genes after exposure to both drugs. Genes affected similarly by Delta(9)THC and R()WIN 55,2122 were considered likely to reflect cannabinoid receptor activation, and expression of the protein products of two such genes not previously implicated in cannabinoid signalingmelanocytespecific generelated gene 1 (MRG1) and hexokinase 4 (glucokinase, GK)was measured by Western blotting and immunohistochemistry. Western blots showed approximately 2fold increases in the levels of both proteins in mouse forebrain. Immunohistochemistry revealed preferential localization of MRG1 to cerebral blood vessels and of GK to hypothalamic neurons. These findings suggest that MRG1 and GK are cannabinoidregulated genes and that they may be involved in the vascular and hypothalamic effects of cannabinoids, respectively. http://www.ncbi.nlm.nih.gov/pubmed/12237329 Sat, 31 Aug 2002 00:00:00 -0700 Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain in vivo. VEGF (10 ng/ml) stimulated 5bromo2'deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 2030. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk1, but not VEGFR1/Flt1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment in vivo include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well. http://www.ncbi.nlm.nih.gov/pubmed/12181492 Sat, 31 Aug 2002 00:00:00 -0700 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 caspase3 activation. Hypoxiaconditioned medium and stem cell factor (SCF), which was present in hypoxiaconditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, ckit, 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. http://www.ncbi.nlm.nih.gov/pubmed/12163450 Wed, 31 Jul 2002 00:00:00 -0700 Heparinbinding epidermal growth factor (EGF)like growth factor (HBEGF) is found in cerebral neurons, and its expression is increased after hypoxic or ischemic injury, which also stimulates neurogenesis. To investigate the possible role of HBEGF in hypoxicischemic induction of neurogenesis, we measured its expression, effects, and target receptors in embryonic murine cerebral cortical cultures and in adult rat brain. Hypoxia increased HBEGF expression by approximately 50 in cortical cultures, where expression was associated with mature and immature neurons. HBEGF (5100 ng/ml) stimulated by approximately 80 the incorporation of bromodeoxyuridine (BrdU) into cultured cells that expressed the HBEGF receptors epidermal growth factor receptor (EGFR)/avian erythroblastic leukemia viral oncogene homolog 1 (ErbB1) and Narginine dibasic convertase (NRDc). Intracerebroventricular administration of HBEGF in adult rats increased BrdU labeling in the subventricular zone and in the subgranular zone of dentate gyrus, where EGFR/ErbB1 and NRDc were also expressed and where ischemiainduced neurogenesis is observed. We conclude that HBEGF stimulates neurogenesis in proliferative zones of the adult brain that are also affected in ischemia and that it does so by interacting with EGFR/ErbB1 and possibly NRDc. Therefore, HBEGF may help to trigger proliferation of neuronal precursors in brain after hypoxic or ischemic injury. http://www.ncbi.nlm.nih.gov/pubmed/12097488 Sun, 30 Jun 2002 00:00:00 -0700 Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splicevariants of caspase2 mRNA were identified, caspase2S and caspase2L, which were highly homologous with the sequences of human and mouse caspase2S and caspase2L genes, respectively. RTPCR demonstrated an increase in expression of both caspase2S and caspase2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the proapoptotic caspase2L splice variant revealed an increase in procaspase2 (51 kDa) protein from 4 to 72 h following ischemia compared with shamoperated controls. Furthermore, an approximately 30kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase2like inhibitor (VDVADFMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenowlabeling assay) and increased the number of healthyappearing CA1 neurons (cresyl violet) compared with vehicletreated controls. Taken together, the data suggest that caspase2 induction and activation are important mediators of delayed neuronal death following transient global ischemia. http://www.ncbi.nlm.nih.gov/pubmed/12067235 Fri, 31 May 2002 00:00:00 -0700 The multifunctional tumor suppressor protein, p53, inhibits cell growth and promotes differentiation and programmed cell death. p53 activity is controlled by transcriptional, translational, and posttranslational regulation. A major pathway for posttranslational regulation of p53 comprises its nucleocytoplasmic transport and subsequent proteasomal degradation, which involves binding to the oncoprotein, murine double minute2 (Mdm2). Hypoxia and other stress signals cause cellular injury partly through the action of p53. In this study, we show that hypoxia induces downregulation of Mdm2 as well as serine 15 phosphorylation and nuclear accumulation of p53 in cultured cortical neurons from E16 mice. These effects are diminished by the p38 mitogenactivated protein kinase inhibitors SB203580 and SB202190, but not by the inactive analog SB202474, and by a dominantinterfering mutant of the p38activating kinase mitogenactivated protein kinase kinase 3 (MKK3). Hypoxic neuronal death was also reduced by p38 inhibitors, by dominantinterfering MKK3, and by a p53antisense oligodeoxynucleotide and was increased by a constitutively active form of p38 and by an Mdm2antisense oligodeoxynucleotide. These results demonstrate that p38 and Mdm2 have roles in coupling hypoxicischemic neuronal insults to activation of p53 and hypoxic cell death. http://www.ncbi.nlm.nih.gov/pubmed/11948180 Fri, 31 May 2002 00:00:00 -0700 Hypoxia and ischemia regulate the expression of several important genes at the level of transcription and of mRNA stability. Two isoforms of a 40kDa poly(C)binding protein, previously identified as RNAbinding proteins, bind to a hypoxiainducible proteinbinding site in the 3'untranslated region of erythropoietin and tyrosine hydroxylase mRNAs and regulate mRNA stability. To determine if poly(C)binding proteins show changes in expression which might regulate mRNA stability in hypoxic or ischemic neuronal cells, we examined poly(C)binding protein 1 and poly(C)binding protein 2 expression in hypoxic cortical neuron cultures and in rat cerebral cortex after focal ischemia. Reverse transcriptionpolymerase chain reaction and western blotting showed hypoxic upregulation of poly(C)binding protein 1, and downregulation of poly(C)binding protein 2, mRNA and protein expression. Hypoxiainducible expression of poly(C)binding protein 1 was mediated by p38 mitogenactivated protein kinase, while hypoxiareducible expression of poly(C)binding protein 2 was mediated by protein kinase C. Immunostaining showed that poly(C)binding protein 1, but not poly(C)binding protein 2, expression was increased in the ischemic boundary zone (penumbra) of the frontal cortex after 90 min of ischemia, and persisted for at least 72 h after reperfusion. These results demonstrate that poly(C)binding protein 1 and poly(C)binding protein 2 in cortical neurons are differentially affected by hypoxic/ischemic insults, suggesting that there are functional differences between poly(C)binding protein isoforms. Since we observed no poly(C)binding protein expression in astroglia, alternative mRNA stability mechanisms may exist in these cells. http://www.ncbi.nlm.nih.gov/pubmed/11958862 Sun, 31 Mar 2002 00:00:00 -0800 We investigated the role of mitogenactivated protein kinase (MAPK) pathways in hypoxic neuronal injury using primary cultures from murine cerebral cortex. Hypoxia caused the death of approximately 50 of neurons at 16 h and approximately 65 of neurons at 24 h. This was associated with phosphoactivation of the MAPK/extracellular signalregulated kinase (ERK) kinase MEK1/2 and its downstream target ERK1/2, but not p38 MAPK or cJun Nterminal kinase (JNK), as detected by western blotting. The MEK1/2 inhibitor, PD98059, increased neuronal death in hypoxic cultures, suggesting that MEK1/2 promotes neuronal survival, whereas the p38 inhibitors, SB202190 and SB203580, had no effect. To identify downstream effects of ERK1/2 that might regulate hypoxic neuronal death, we measured hypoxiainduced phosphorylation of three ERK1/2 targets: the 90kDa ribosomal protein S6 kinase (RSK), the transcription factor ELK1, and the proapoptotic Bcl2 family protein Bad. We observed increased abundance of inactivated (phospho)Bad, but no change in phosphoRSK or phosphoELK1. Moreover, the MEK inhibitor PD98059 reduced phosphoinactivation of Bad in hypoxic cultures. These findings suggest that a cellsurvival program involving phosphoactivation of MEK1/2 and ERK1/2 and inactivation of Bad is mobilized in hypoxic neurons, and may help to regulate neuronal fate following hypoxicischemic injury. http://www.ncbi.nlm.nih.gov/pubmed/11796750 Mon, 31 Dec 2001 00:00:00 -0800 Globins are oxygenbinding heme proteins present in bacteria, protists, fungi, plants, and animals. Their functions have diverged widely in evolution, and include binding, transport, scavenging, detoxification, and sensing of gases like oxygen, nitric oxide, and carbon monoxide. Neuroglobin (Ngb) is a recently discovered monomeric globin with high affinity for oxygen and preferential localization to vertebrate brain. No function for Ngb is known, but its affinity for oxygen and its expression in cerebral neurons suggest a role in neuronal responses to hypoxia or ischemia. Here we report that Ngb expression is increased by neuronal hypoxia in vitro and focal cerebral ischemia in vivo, and that neuronal survival after hypoxia is reduced by inhibiting Ngb expression with an antisense oligodeoxynucleotide and enhanced by Ngb overexpression. Both induction of Ngb and its protective effect show specificity for hypoxia over other stressors. We conclude that hypoxiainducible Ngb expression helps promote neuronal survival from hypoxicischemic insults. http://www.ncbi.nlm.nih.gov/pubmed/11742077 Fri, 30 Nov 2001 00:00:00 -0800 The recent demonstration that biochemical pathways from diverse organisms are arranged in scalefree, rather than random, systems Jeong et al., Nature 407 (2000) 651654, emphasizes the importance of developing methods for the identification of biochemical nexusesthe nodes within biochemical pathways that serve as the major input/output hubs, and therefore represent potentially important targets for modulation. Here we describe a bioinformatics approach that identifies candidate nexuses for biochemical pathways without requiring functional gene annotation we also provide proofofprinciple experiments to support this technique. This approach, called Nexxus, may lead to the identification of new signal transduction pathways and targets for drug design. http://www.ncbi.nlm.nih.gov/pubmed/11741594 Fri, 30 Nov 2001 00:00:00 -0800 Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemiainduced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia. http://www.ncbi.nlm.nih.gov/pubmed/11740203 Fri, 30 Nov 2001 00:00:00 -0800 Cell deathregulatory genes like caspases and bcl2 family genes are involved in delayed cell death in the CA1 sector of hippocampus after global cerebral ischemia, but little is known about the mechanisms that trigger their expression. The authors found that expression of Fas and Fasligand messenger ribonucleic acid and protein was induced in vulnerable CA1 neurons at 24 and 72 hours after global ischemia. Fasassociating protein with a novel death domain (FADD) also was upregulated and immunoprecipitated and colocalized with Fas. Caspase10 was activated and interacted with FADD protein to an increasing extent as the duration of ischemia increased. Moreover, caspase10 colocalized with both FADD and caspase3. These findings suggest that Fasmediated death signaling may play an important role in signaling hippocampal neuronal death in CA1 after global cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/11740202 Fri, 30 Nov 2001 00:00:00 -0800 Vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective as well as angiogenic properties, but the pathways involved in VEGFmediated neuronal survival have not been identified. We found previously that VEGF protects cultured neural cells from death induced by serum withdrawal or hypoxia via the activation of VEGF2/fetal liver kinase1 receptors, phosphatidylinositol 3'kinase, Akt and nuclear factorkappa B. We now report that in mouse cortical neuron cultures subjected to hypoxia, the neuroprotective effect of VEGF involves suppression of celldeath pathways mediated by caspase3. Exposure to hypoxia for 24 h caused the death of 71/4 of cultured neurons this was reduced to 40/1 by VEGF (n=3, P0.005) and to 44/1 by the caspase3 inhibitor benzyloxycarbonylDEVDfluoromethyl ketone (n=3, P0.005). VEGF inhibited the activation of caspase3 as measured by the 1720kDa caspase3 cleavage product, and immunolocalization of VEGF and activated caspase3 showed segregated expression in separate neuronal populations. An antisense, but not sense, oligodeoxyribonucleotide directed against VEGF increased the proportion of neurons expressing activated caspase3, and correspondingly reduced the viability of hypoxic neurons by 37/2 (n=3, P0.005). These findings suggest that VEGF protects neurons from hypoxic injury by inhibiting the activation of caspase3, and could therefore act as an endogenous neuroprotective factor in cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/11734367 Fri, 30 Nov 2001 00:00:00 -0800 The brain's response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotidebased microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7fold or greater increases in the expression of 57 genes and 1.7fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injuryresponse genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1 the IGF1 receptor and the IGF1binding protein IGFBP2 Rb, the Rbbinding protein E2F1, and the E2Frelated transcription factor, TFDP1 the CACNB3 and CACNB4 betasubunits of the voltagegated calcium channel and caspase3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemiaGRB2, an adapter protein involved in growthfactor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain. http://www.ncbi.nlm.nih.gov/pubmed/11456315 Sat, 30 Jun 2001 00:00:00 -0700 Bax is a proapoptotic Bcl2 family protein that regulates programmed cell death through homodimerization and through heterodimerization with Bcl2. Bax alpha is encoded by six exons and undergoes alternative splicing. Bax kappa, a splice variant of Bax with conserved BH1, BH2 and BH3 binding domains and a Cterminal transmembrane domain (TM), but with an extra 446bp insert between exons 1 and 2 leading to loss of an Nterminal ART domain, was identified from an ischemic rat brain cDNA library. Expression of Bax kappa mRNA and protein was upregulated in hippocampus after cerebral ischemic injury. The increased Bax kappa mRNA was distributed mainly in selectively vulnerable hippocampal CA1 neurons that are destined to die after global ischemia. Overexpression of Bax kappa protein in HN33 mouse hippocampal neuronal cells induced cell death, which was partially abrogated by cooverexpression of Bcl2. Moreover, cooverexpression of Bax kappa and Bax alpha increased HN33 cell death. The results suggest that the Bax kappa may have a role in ischemic neuronal death. http://www.ncbi.nlm.nih.gov/pubmed/11413234 Thu, 31 May 2001 00:00:00 -0700 Cyclic AMP (cAMP) response element binding protein (CREB) is a transcription factor that has been implicated in neuronal responses to ischemia. We examined the effect of global cerebral ischemia in the rat on the expression of CREB, its transcriptionally active phosphorylated form (pCREB), and the nuclear adaptor protein, CREB binding protein (CBP). Global ischemia induced the expression of pCREB and CBP in vulnerable neurons of the hippocampal CA1 sector. In primary cultures of murine cortical neurons subjected to hypoxia, CBP was selectively expressed in cells with morphologically intact cell nuclei, and not in cells with condensed or fragmented nuclei indicative of irreversibly damaged neurons. These results support a role for transcriptional activation by CREB and CBP in neuronal cellsurvival programs following cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/11345520 Mon, 30 Apr 2001 00:00:00 -0700 Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5bromo2'deoxyuridine5'monophosphate (BrdUrd) over 2day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regionsthe subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemiainduced neurogenesis in what may be adaptive processes that contribute to recovery after stroke. http://www.ncbi.nlm.nih.gov/pubmed/11296300 Sat, 31 Mar 2001 00:00:00 -0800 Vascular endothelial growth factor is an angiogenic peptide that binds to tyrosine kinase receptors on target cells to activate signal transduction pathways involving phosphatidylinositol 3'kinase and the serinethreonine protein kinase, Akt. To determine whether this signaling pathway is activated in cerebral ischemia, we examined the expression of vascular endothelial growth factor receptors 1 and 2, and phosphatidylinositol 3'kinaseactivated phosphoAkt, in the cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Western blot analysis and immunocytochemistry demonstrated induction of vascular endothelial growth factor receptor 1 and 2 expression, and of antiphosphatidylinositol 3'kinaseimmunoprecipitated phosphoAkt, in vulnerable regions of the cortex and hippocampus after 15 min of global ischemia and 472 h of reperfusion. These findings demonstrate that vascular endothelial growth factor receptors and receptorcoupled signal transduction pathways are induced in ischemic brain in vivo, and could therefore participate in endogenous neuroprotective responses to ischemia. http://www.ncbi.nlm.nih.gov/pubmed/11036205 Sun, 31 Dec 2000 00:00:00 -0800 Vascular endothelial growth factor (VEGF) is an angiogenic factor with neurotrophic effects in the peripheral nervous system. To determine if VEGF can also promote the survival of central neurons, we examined its effect on HN33 (mouse hippocampal neuron x neuroblastoma) cells deprived of serum. Serumdeprived HN33 cells expressed VEGFR2 receptors, which, in the presence of VEGF, interacted with the downstream signaling molecules phosphatidylinositol 3'kinase and phosphoAkt, as demonstrated by immunoprecipitation and Western blotting. Treatment of serumdeprived HN33 cells with VEGF also stimulated the phosphorylation of IkappaBalpha and nuclear translocation of the transcription factor NFkappaB. Withdrawal of serum for 24 h reduced HN33 cell viability by approximately 50 in the absence of VEGF, but by only approximately 20 in the presence of 100 ng/mL of VEGF. These findings support a neurotrophic role for VEGF in the central nervous system, which may be mediated through VEGFR2 receptors, the protein kinases phosphatidylinositol 3'kinase and Akt, and the transcription factor NKkappaB. Thus, VEGF, like other neurotrophic factors, could exert protective effects in acute or chronic neurodegenerative disorders. http://www.ncbi.nlm.nih.gov/pubmed/10984196 Thu, 30 Nov 2000 00:00:00 -0800 Vascular endothelial growth factor is an angiogenic and neurotrophic peptide whose expression is transcriptionally induced in hypoxic tissues through the action of hypoxiainducible factor1alpha. To determine if this signaling pathway is activated in the ischemic brain, and might therefore participate in adaptive processes such as angiogenesis and neuroprotection, we examined the expression of vascular endothelial growth factor and hypoxiainducible factor1alpha in cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Northern analysis showed ischemiainducible expression of multiple vascular endothelial growth factor messenger ribonucleic acid splice variants between 4 and 24h. Western analysis and immunocytochemistry demonstrated the concerted induction of vascular endothelial growth factor and hypoxiainducible factor1alpha in the same, apparently neuronal, cells in vulnerable regions of cortex and hippocampus after 15min of ischemia, which persisted for as long as 4 to 72h of reperfusion. These findings demonstrate that hypoxiasensitive vascular endothelial growth factor signaling can be induced in neurons in global cerebral ischemia in vivo, and are consistent with the hypothesis that ischemic insults trigger hypoxiasensing and adaptive downstream molecular responses in central neurons. http://www.ncbi.nlm.nih.gov/pubmed/11029549 Sat, 30 Sep 2000 00:00:00 -0700 In addition to its betterknown hemopoietic action, erythropoietin (Epo) has neurotrophic properties and neuroprotective effects in some models of hypoxicischemic injury. To define further the cellular mechanisms underlying neuroprotection by Epo, we studied the effects of Epo on hypoxia with glucose deprivation in cultured rat cortical neurons and astroglia and on exposure to excitotoxins in cultured rat cortical neurons. Epo (30 pM) reduced neuronal, but not astroglial, cell death from hypoxia with glucose deprivation, and also attenuated the neurotoxic effect of (/)alphaamino3hydroxy5methylisoxazole4propionic acid (AMPA), but not other excitotoxins. Epo appears to protect against cerebral ischemia through a direct effect on neurons that may be mediated in part by AMPA receptors. http://www.ncbi.nlm.nih.gov/pubmed/10963901 Sat, 30 Sep 2000 00:00:00 -0700 Vascular endothelial growth factor (VEGF) is a hypoxiainducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR2 receptors and neuropilin1, but not VEGFR1 receptors. The neuropilin1 ligand placenta growth factor2 failed to reproduce the protective effect of VEGF, pointing to VEGFR2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3'kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3'kinase/Akt signal transduction system in VEGFmediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxicischemic injury. http://www.ncbi.nlm.nih.gov/pubmed/10963684 Sat, 30 Sep 2000 00:00:00 -0700 Cannabinoids protect cortical neurons from ischemic injury by interacting with CB1 receptors. Because a variety of neuroprotective genes are induced in cerebral ischemia, we examined the effect of experimental stroke, produced by 20 minutes of middle cerebral artery occlusion in rats, on CB1 receptor expression. Western blotting and immunohistochemistry showed that CB1 expression on neurons was increased in the arterial boundary zone of the cortical mantle, beginning by 2 hours and persisting for 72 hours or more after ischemia These findings are consistent with a neuroprotective role for endogenous cannabinoid signaling pathways and with a potential therapeutic role in stroke for drugs that activate CB1 receptors. http://www.ncbi.nlm.nih.gov/pubmed/10939579 Mon, 31 Jul 2000 00:00:00 -0700 Tcell restricted intracellular antigenrelated protein (TIAR) is an RNA recognition motiftype RNAbinding protein that has been implicated in the apoptotic death of Tlymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Doublelabeling studies showed that TIAR protein expression was colocalized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury. http://www.ncbi.nlm.nih.gov/pubmed/10700014 Fri, 31 Mar 2000 00:00:00 -0800 The endogenous cannabinoids (endocannabinoids) anandamide and 2arachidonylglycerol increased cell viability in cerebral cortical neuron cultures subjected to 8 h of hypoxia and glucose deprivation. This effect was observed at nanomolar concentrations, was reproduced by a nonhydrolyzable analog of anandamide, and was unaltered by CB1 or CB2 cannabinoid receptor antagonists. Like synthetic cannabinoids, endocannabinoids can protect neurons from hypoxic injury, and may represent endogenous neuroprotective molecules in cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/10653017 Mon, 31 Jan 2000 00:00:00 -0800 Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calciumbinding proteins have been implicated in calciumregulated signal transduction. Apoptosislinked gene 2 is a calciumbinding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosislinked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosislinked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosislinked gene 2 protein was also upregulated in the ischemic borderzone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably overexpressed in the caudateputamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosislinked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay. Our results suggest that apoptosislinked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia. http://www.ncbi.nlm.nih.gov/pubmed/10683420 Fri, 31 Dec 1999 00:00:00 -0800 Calcium channels that are regulated by voltage have an important role in linking cellular stimulation to physiological responses in the nervous system. In addition, a number of autoimmune or genetic disorders that affect calcium channels (channelopathies) have been identified, including several that affect neuromuscular function. These include the LambertEaton myasthenic syndrome, which is associated with autoantibodies directed against neuronal calcium channels, as well as at least two inherited neuromuscular diseaseshypokalemic periodic paralysis and some varieties of malignant hyperthermiathat affect calcium channels in skeletal muscle. Preliminary progress has been achieved in understanding the relationship between these immunological or genetic abnormalities and the alterations in channel function that they produce. A major challenge that remains is to determine how calcium channelopathies lead to the curious assortment of paroxysmal and progressive disorders that are observed clinically. http://www.ncbi.nlm.nih.gov/pubmed/10487899 Thu, 30 Sep 1999 00:00:00 -0700