Methazolamide and Melatonin Inhibit Mitochondrial Cytochrome C Release and Are Neuroprotective in Experimental Models of Ischemic Injury

Abstract

Background and Purpose-The identification of a neuroprotective drug for stroke remains elusive. Given that mitochondria play a key role both in maintaining cellular energetic homeostasis and in triggering the activation of cell death pathways, we evaluated the efficacy of newly identified inhibitors of cytochrome c release in hypoxia/ischemia induced cell death. We demonstrate that methazolamide and melatonin are protective in cellular and in vivo models of neuronal hypoxia. Methods-The effects of methazolamide and melatonin were tested in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons. Mitochondrial membrane potential, release of apoptogenic mitochondrial factors, pro-IL-1␤ processing, and activation of caspase -1 and -3 were evaluated. Methazolamide and melatonin were also studied in a middle cerebral artery occlusion mouse model. Infarct volume, neurological function, and biochemical events were examined in the absence or presence of the 2 drugs. Results-Methazolamide and melatonin inhibit oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1␤ processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, they decrease infarct size and improve neurological scores after middle cerebral artery occlusion in mice. Conclusions-We demonstrate that methazolamide and melatonin are neuroprotective against cerebral ischemia and provide evidence of the effectiveness of a mitochondrial-based drug screen in identifying neuroprotective drugs. Given the proven human safety of melatonin and methazolamide, and their ability to cross the blood-brain-barrier, these drugs are attractive as potential novel therapies for ischemic injury.

N-Acetyl-Serotonin Offers Neuroprotection through Inhibiting Mitochondrial Death Pathways and Autophagic Activation in Experimental Models of Ischemic Injury

“…OHSCs were treated by H 2 O 2 for 18 h while OGD was induced (Pringle et al, 1997;Pellegrini-Giampietro et al, 1999) in an airtight chamber in a serum-and glucose-free balanced salt solution containing the following (in mmol/l): NaCl 124, KCl 5, MgCl 1.3, NaH 2 PO 4 1.25, NaHCO 3 26, CaCl 2 2, pH 7.4. Dishes were sealed in an airtight chamber with anaerobic system envelopes with palladium catalyst (BD BBL GasPak Plus, Becton Dickinson) Wang et al, 2009). Following induction of hypoxia and hypoglycemia, OHSCs were transferred to normal serum-free medium (75% MEM, 25% Hanks balanced salt solution, 5 mg/ml glucose) with 0.25 mg/ml propidium iodide (PI) and replaced in the incubator under normoxic conditions.…”

“…PI images were analyzed, and the intensity of PI fluorescence in the selected region of interest (CA1, CA3, and dentate gyrus) was used as an index of cell death. Cell death of PCNs, PHNs, and OHSCs was also quantitatively evaluated by the lactate dehydrogenase (LDH) assay as previously described according to the manufacturer's instructions (Roche; Wang et al, 2009Wang et al, , 2011.…”

“…Wild-type Atg5 (autophagy-competent) mouse embryonic fibroblasts (Atg5 ϩ/ϩ MEFs) and Atg5-deficient (autophagy-deficient) mouse embryonic fibroblasts (MEFs; Atg5 Ϫ/Ϫ ) were cultured as previously described (Kuma et al, 2004) and subjected to H 2 O 2 for 18 h. Cell death was quantified by the MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Roche; Wang et al, 2011).…”

“…Apoptosis, necrosis, inflammation, and oxidative stress contribute to the development of ischemic cascade Pandya et al, 2011). Autophagy leads to degradation of proteins and removes harmful product after ischemia, especially dysfunctional mitochondria (Wen et al, 2008;Pan, 2013). Interestingly, autophagy not only has a protective effect on neuronal survival, but also promotes cell death, known as "autophagic cell death," in a wide range of important situations including cerebral ischemia (Koike et al, 2008;Puyal et al, 2009;Nopparat et al, 2010;Gao et al, 2012;Sheng et al, 2012;Shi et al, 2012).…”

“…N-acetylserotonin (NAS) is a naturally occurring chemical intermediate that is produced from serotonin and is converted to melatonin, an agent that has been reported by us and other researchers to be neuroprotective in experimental models of ischemic stroke and other neurological diseases Wang et al, 2009Wang et al, , 2011Pandya et al, 2013;Zhang et al, 2013). Both NAS and melatonin belong to a library of 1040 FDA-approved compounds assembled by the National Institute of Neurological Disorders and Stroke (the NINDS Custom Collection).…”