Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke1. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia2 and the tolerance conferred by ischemic preconditioning (IPC)3, would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Background The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) is an established 10-point quantitative topographic computed tomography scan score to assess early ischemic changes. We performed a non-inferiority trial between the e-ASPECTS software and neuroradiologists in scoring ASPECTS on non-contrast enhanced computed tomography images of acute ischemic stroke patients. Methods In this multicenter study, e-ASPECTS and three independent neuroradiologists retrospectively and blindly assessed baseline non-contrast enhanced computed tomography images of 132 patients with acute anterior circulation ischemic stroke. Follow-up scans served as ground truth to determine the definite area of infarction. Sensitivity, specificity, and accuracy for region- and score-based analysis, receiver-operating characteristic curves, Bland-Altman plots and Matthews correlation coefficients relative to the ground truth were calculated and comparisons were made between neuroradiologists and different pre-specified e-ASPECTS operating points. The non-inferiority margin was set to 10% for both sensitivity and specificity on region-based analysis. Results In total 2640 (132 patients × 20 regions per patient) ASPECTS regions were scored. Mean time from onset to baseline computed tomography was 146 ± 124 min and median NIH Stroke Scale (NIHSS) was 11 (6-17, interquartile range). Median ASPECTS for ground truth on follow-up imaging was 8 (6.5-9, interquartile range). In the region-based analysis, two e-ASPECTS operating points (sensitivity, specificity, and accuracy of 44%, 93%, 87% and 44%, 91%, 85%) were statistically non-inferior to all three neuroradiologists (all p-values <0.003). Both Matthews correlation coefficients for e-ASPECTS were higher (0.36 and 0.34) than those of all neuroradiologists (0.32, 0.31, and 0.3). Conclusions e-ASPECTS was non-inferior to three neuroradiologists in scoring ASPECTS on non-contrast enhanced computed tomography images of acute stroke patients.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1a levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.

N-Methyl-D-aspartate (NMDA)1 receptors are a subclass of excitatory, ionotropic glutamate neurotransmitter receptors.They are unique in that they require the binding of co-agonists, i.e. L-glutamate and glycine, together with the alleviation of a voltage-dependent blockade by magnesium ions for channel activation. NMDA receptors are critical mediators of excitatory neurotransmission in the brain, being pivotal for long term potentiation. They are also important as a therapeutic target post-ischemia. NMDA receptor channels are highly permeable to calcium ions, and thus overactivation leads to excitotoxic neuronal cell death (reviewed in Ref. 1). Seven genes encode NMDA receptor subunits NR1, NR2A-NR2D, and NR3A-NR3B. The NR1 subunit undergoes extensive splicing to yield eight variants NR1-1a,1b to NR1-4a,4b. Functional NMDA receptors are formed from the co-assembly of the obligatory NR1 glycine-binding subunit with NR2 and/or NR3 subunits, although it was recently shown (2) that NR1/NR3 subunits formed a novel, glycine-gated receptor. The quaternary structure of NMDA receptors is still not yet established. Experimental data support either a tetrameric structure comprising two NR1 and two NR2 subunits or a pentamer with reports of either three NR1 subunits co-assembled with two NR2s or two NR1s with three NR2s (1).All NMDA receptor subunits share the same transmembrane organization. They have an extracellular N-terminal domain of ϳ550 amino acids, 3 transmembrane domains, 1 re-entrant membrane domain M2 that is thought to form the inner lining of the cation channel, and an intracellular C-terminal tail. The N-terminal region can be subdivided into two discrete domains based on the amino acid sequence homology between the NMDA receptor subunits and amino acid-binding proteins of bacteria. Thus, the first ϳ400 amino acids have homology with the bacterial periplasmic leucine-isoleucine-valine-binding protein (LIVBP), whereas amino acids 420 -550 show similarity with the bacterial lysine-arginine-ornithine and glutaminebinding protein (reviewed in Refs. 1 and 3). The glycine-and glutamate-binding sites of the NR1 and NR2 subunits, respectively, have been localized to the N-terminal regions, i.e. amino acids 420 -550 (S1), that are proximal to the membrane and the S2 extracellular loop found between TM3 and TM4 by a combination of site-directed mutagenesis studies (4 -8) and by analogy with the crystal structure of the soluble extracellular domain of the non-NMDA, amino ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR2 glutamate receptor subunit (9). Most interesting, the N-terminal region distal to the membrane has been shown to be important for the assembly of non-NMDA receptors (10,11). This domain is requisite * This work was supported in part by the Biotechnology and Biological Sciences Research Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S...

NMDA receptors are a subclass of excitatory, ionotropic glutamate receptors. They are composed of obligatory NR1 subunits co-assembled with NR2 subunits of which there are four types yielding four major NMDA receptor subclasses NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D (reviewed in e.g. Cull-Candy et al. 2001). NMDA receptors are clustered at synapses via their interaction with the scaffolding protein, post-synaptic density (PSD)-95 (Kornau et al. 1995). The association between NMDA receptors and PSD-95 is mediated via the motif, ES(D/E)V that is common to all NR2 subunit C-termini. A PSD-95 binding motif, threonine serine valine valine, is also present in the C2' exon of NR1-3a,b and NR1-4a,b splice variants. Co-transfection of PSD-95 with NMDA NR1/NR2A or NR1/NR2B NMDA receptor clones has been shown to enhance in an ES(D/E)Vdependent manner, the expression of NR2A and NR2B subunits resulting in an increased cell surface expression of assembled NR1/NR2A and NR1/NR2B subtypes (Rutter and Stephenson 2000;Rutter et al. 2002;Lin et al. 2004). Received August 8, 2007; accepted September 18, 2007. Address correspondence and reprint requests to F. Anne Stephenson, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK. E-mail: anne.stephenson@pharmacy.ac.uk 1 The present address of Michalis Papadakis is the Acute Stroke Programme, Nuffield Department of Clinical Medicine, Level 7, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.Abbreviations used: ESDV, glutamate, serine, aspartate, valine; MAGUK, membrane associated guanylate kinase; PBS, phosphate buffered saline; PDZ, post-synaptic density protein (PSD95), Drosophila disc large tumour suppressor (DlgA), and zo-1 protein; PSD, postsynaptic density; SAP, synapse associated protein. AbstractNMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C a...

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Key points• Cerebral ischaemia results in the activation of multiple pathways that can independently lead to neuronal death. Agents targeting a number of processes at one time are likely to be translated into stroke therapy.• Hypoxia-inducible factor (HIF) is a transcription complex which responds to changes in oxygen. HIF levels are tightly regulated by a group of prolyl hydroxylases (PHDs).• In this study, we investigated the function of each of the HIF-PHDs in the first 24 hours following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed.• We found that the PHD1 −/− , PHD2 +/− , PHD3 −/− mice had different outcomes after inducing ischaemia. In particular, the PHD2 +/− mice had an improved rCBF response post-reperfusion with better behavioural scores. The PHD3 −/− mice have worse rCBF but no behavioural change.• The information gained enhances understanding of the biological processes involved and informs strategies for therapeutic targeting of the PHD enzymes.Abstract This study investigated the function of each of the hypoxia inducible factor (HIF) prolyl-4-hydroxylase enzymes (PHD1-3) in the first 24 h following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed. Male, 8-to 12-week old PHD1 −/− , PHD2 +/− and PHD3 −/− mice and their wild-type (WT) littermate were subjected to 45 min of middle cerebral artery occlusion (MCAO). During the experiments, regional cerebral blood flow (rCBF) was recorded by laser Doppler flowmetry. Behaviour was assessed at both 2 h and 24 h after reperfusion with a common neuroscore. Infarct volumes, blood-brain barrier (BBB) disruption, cerebral vascular density, apoptosis, reactive oxygen species (ROS), HIF1α, and glycogen levels were then determined using histological and immunohistochemical techniques. When compared to their WT littermates, PHD2 +/− mice had significantly increased cerebral microvascular density and more effective restoration of CBF upon reperfusion. PHD2 +/− mice showed significantly better functional outcomes and higher activity rates at both 2 h and 24 h after MCAO, associated with significant fewer apoptotic cells in the penumbra and less BBB disruption; PHD3 −/− mice had impaired rCBF upon early reperfusion but comparable functional outcomes; PHD1 −/− mice did not show any significant changes following the MCAO. Production of ROS, HIF1α staining and glycogen content in the brain were not different in any comparison. Life-long genetic inhibition of PHD enzymes produces different effects on outcome in the first 24 h after R-L. Chen and S. Nagel contributed equally to this publication. C. W. Pugh and A. M. Buchan are joint senior authors. transient cerebral ischaemia. These need to be considered in optimizing therapeutic effects of PHD inhibitors, particularly when isoform specific inhibitors become available.

This study investigated the effects of 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8-to 12-week-old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood-brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up-regulated HIF-1a, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up-regulated HIF-1a and delocalized ZO-1. Pre-treating IOX3 on RBE4 cells 24 h before oxygenglucose deprivation had a protective effect on endothelial barrier preservation with ZO-1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia. Keywords: BBB, EPO, HIF, IOX3, preconditioning, stroke. J. Neurochem. (2014) 131, 177-189.The hypoxia-inducible factors (HIFs) are transcription factors that regulate gene expression in response to cellular hypoxia (Semenza 1999;Pugh and Ratcliffe 2003;Kaelin and Ratcliffe 2008;Greer et al. 2012). A mechanism by which the human HIFs 'sense' oxygen levels is enabled by four oxygen-sensitive hydroxylases: three prolyl hydroxylases (PHDs) and one asparaginyl hydroxylase, factor inhibiting HIF (FIH) (Schofield and Ratcliffe 2004). In normoxia, hydroxylation of either of two prolyl residues in the oxygen-dependent degradation domains of HIF-1a promotes its interaction with the von Hippel-Lindau ubiquitin E3 ligase complex. Thereafter, HIF-1a is targeted