s-nitrosocysteine and Brain-Ischemia

Activation of sigma receptors at delayed time points has been shown to decrease injury following ischemic stroke. The mixed σ1/σ2 receptor agonist, 5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole (afobazole), provides superior long-term outcomes compared to other σ ligands in the rat middle cerebral artery occlusion (MCAO) stroke model. Experiments using the MCAO model were carried out to determine the molecular mechanism involved in the beneficial effects of afobazole. Administration of afobazole (3 mg/kg) at delayed time points post-stroke significantly increased the number of microglia and astrocytes detected in the ipsilateral hemisphere at 96 h post-surgery. Morphological analysis of the microglia indicated that a greater number of these cells were found in the ramified resting state in MCAO animals treated with afobazole relative to MCAO vehicle controls. Similarly, fewer reactive astrocytes were detected in the injured hemisphere of afobazole-treated animals. Both the enhanced survival and reduced activation of glial cells were abolished by co-application of either a σ1 (BD-1063) or a σ2 (SM-21) receptor antagonist with afobazole. To gain further insight into the mechanisms by which afobazole lessens stroke injury, we probed the brain sections for markers of neuroinflammation (tumor necrosis factor α) and nitrosative stress (S-nitrosocysteine). Data show that afobazole significantly reduces S-nitrosocysteine levels, but does not alter tumor necrosis factor α expression 96 h after an ischemic stroke. Taken together our data indicate that afobazole acting via both σ1 and σ2 receptors decreases stroke injury by enhancing glial cell survival, blocking ischemia-induced glial cell activation, and decreasing nitrosative stress.

Topics: Animals; Astrocytes; Benzimidazoles; Brain; Brain Ischemia; Butyrates; Cell Survival; Cysteine; Infarction, Middle Cerebral Artery; Macrophage Activation; Morpholines; Neuroglia; Neuroprotective Agents; Piperazines; Rats; Receptors, sigma; S-Nitrosothiols; Sigma-1 Receptor; Stroke; Tropanes

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line; Cells, Cultured; Cerebral Cortex; Cysteine; Enzyme Activation; Enzyme Precursors; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Molecular; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidation-Reduction; Phenylmercuric Acetate; Rats; Recombinant Proteins; Reperfusion; S-Nitrosothiols; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization