One approach for protecting neurons from excitotoxic damage in stroke is to attenuate receptor activity with specific antagonists. S-Methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), the active metabolite of disulfiram, has been shown to be a partial antagonist of glutamate receptors and effective in reducing seizure. First, we investigated neuroprotective effect of DETC-MeSO on primary cortical neuronal culture under hypoxia/reoxygenation condition in vitro. Then, DETC-MeSO was administered subcutaneously for 4 and 8 days with the first injection occurring 1 h before or 24 h after reperfusion in the rat middle cerebral artery occlusion stroke model. Rats were subjected to the neuroscore test, and the brain was analyzed for infarct size. Monitoring neurotransmitter release was carried out by microdialysis. Heat shock proteins, key proteins involved in apoptosis and endoplasmic reticulum (ER) stress, were analyzed by immunoblotting. DETC-MeSO greatly reduced both cell death following hypoxia/reoxygenation and brain infarct size. It improved performance on the neuroscore test and attenuated proteolysis of αII-spectrin. The level of pro-apoptotic proteins declined, and anti-apoptotic and HSP27 protein expressions were markedly increased. Levels of the ER stress protein markers p-PERK, p-eIF2α, ATF4, JNK, XBP-1, GADD34, and CHOP significantly declined after DETC-MeSO administration. Microdialysis data showed that DETC-MeSO increased high potassium-induced striatal dopamine release indicating that more neurons were protected and survived under ischemic insult in the presence of DETC-MeSO. We also showed that DETC-MeSO can prevent gliosis. DETC-MeSO elicits neuroprotection through the preservation of ER resulting in reduction of apoptosis by increase of anti-apoptotic proteins and decrease of pro-apoptotic proteins.

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Taurine, as a free amino acid, is found at high levels in many tissues including brain, heart and skeletal muscle and is known to demonstrate neuroprotective effects in a range of disease conditions including stroke and neurodegenerative disease. Using in vitro culture systems we have demonstrated that taurine can elicit protection against endoplasmic reticulum stress (ER stress) from glutamate excitotoxicity or from excessive reactive oxygen species in PC12 cells or rat neuronal cultures. In our current investigation we hypothesized that taurine treatment after stroke in the rat middle cerebral artery occlusion (MCAO) model would render protection against ER stress processes as reflected in decreased levels of expression of ER stress pathway components. We demonstrated that taurine elicited high level protection and inhibited both ATF-6 and IRE-1 ER stress pathway components. As ischemic stroke has a complex pathology it is likely that certain combination treatment approaches targeting multiple disease mechanisms may have excellent potential for efficacy. We have previously employed the partial NMDA antagonist DETC-MeSO to render protection against in vivo ischemic stroke using a rat cerebral ischemia model. Here we tested administration of subcutaneous administration of 0.56 mg/kg DETC-MeSO or 40 mg/kg of taurine separately or as combined treatment after a 120 min cerebral ischemia in the rat MCAO model. Neither drug alone demonstrated protection at the low doses employed. Remarkably however the combination of low dose DETC-MeSO plus low dose taurine conferred a diminished infarct size and an enhanced Neuroscore (reflecting decreased neurological deficit). Analysis of ER stress markers pPERK, peIF-2-alpha and cleaved ATF-6 all showed decreased expression demonstrating that all 3 ER stress pathways were inhibited concurrent with a synergistic protective effect by the post-stroke administration of this DETC-MeSO-taurine combination treatment.

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