6-cyano-7-nitroquinoxaline-2-3-dione and Infarction--Middle-Cerebral-Artery

Recent studies have indicated that dynamic alterations in the structure of postsynaptic density (PSD) are involved in the pathogenesis of many central nervous system disorders, including ischemic stroke. Homer is the newly identified scaffolding protein located at PSD and regulates synaptic function. Homer1a, an immediate early gene, has been shown to be induced by several stimulations, such as glutamate, brain-derived neurotrophic factor, and trauma. However, whether acidosis mediated by acid-sensing ion channels (ASICs) and hypoxia during cerebral ischemia can change Homer1a expression remains to be determined.. We investigated that acidosis and hypoxia selectively and rapidly upregulated Homer1a expression, but not Homer1b/c in cultured cortical neurons. We also found that Homer1a exhibited induction expression in brain cortex of the middle cerebral artery occlusion (MCAO) rats. Additionally, acid-evoked Homer1a mRNA induction depended on extracellular signal-regulated kinase1/2 (ERK1/2) and Akt activity, and ASIC1a-mediated calcium influx whereas hypoxia depended only on ERK1/2 activity. Also, we demonstrated that continuous acidosis and hypoxia resulted in pronounced cell injury and Homer1a knockdown with small interfering RNA aggravated this damage induced by 3 h acid and hypoxia incubation in neuro-2a cells.. Homer1a might act as an activity-dependent regulator responding to extracellular stimuli during cerebral ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Amiloride; Animals; Carrier Proteins; Cell Survival; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Homer Scaffolding Proteins; Hypoxia; Infarction, Middle Cerebral Artery; Neurons; Peptides; Rats; Rats, Sprague-Dawley; Signal Transduction; Spider Venoms; Time Factors; Up-Regulation

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²⁺-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Infarction; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Cells, Cultured; Cerebral Cortex; CREB-Binding Protein; Disease Models, Animal; Disks Large Homolog 4 Protein; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glucose; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Neurons; Nimodipine; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors

Excessive presynaptic glutamate release after cerebral ischaemia leads to neuronal death mainly through excessive calcium entry of N-methyl-D-aspartate receptors (NMDARs). Our recent study reported that cerebroside can open large-conductance Ca²⁺-activated K⁺ (BKCa) channels. The present study evaluated the effects of cerebroside-A (CS-A), a single molecule isolated from an edible mushroom, on brain injury after focal or global ischaemia in adult male mice and rats. We herein report that treatment with CS-A after 60-min middle cerebral artery occlusion dose-dependently reduced the cerebral infarction with at least a 6-h efficacious time-window, which was partially blocked by the BKCa channel blocker charybdotoxin (CTX). Treatment with CS-A after 20 min global cerebral ischaemia (four-vessel occlusion) significantly attenuated the death of pyramidal cells in hippocampal CA1 area, which was also sensitive to CTX. CS-A, by opening the BKCa channel, could prevent excessive glutamate release after oxygen-glucose deprivation (OGD). In addition, CS-A could inhibit NMDAR Ca²⁺ influx, which did not require the activation of the BKCa channel. Furthermore, CS-A blocked the OGD-induced NMDAR-dependent long-term potentiation in hippocampal CA1 region. These findings indicate that treatment with CS-A after stroke exerts potent neuroprotection through prevention of excessive glutamate release and reduction of Ca²⁺ influx through NMDARs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Brain Ischemia; Calcium; Cerebral Infarction; Cerebrosides; Charybdotoxin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glucose; Glutamic Acid; Hippocampus; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neuroprotective Agents; Neurotoxins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Valine