title: Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation.
abstract: Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H(2)O(2)). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H(2)O(2) treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H(2)O(2) treatment vs. the other cell lines. Pre-treatment with 250 microM or 125 microM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H(2)O(2) treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H(2)O(2) exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H(2)O(2) exposure did not increase ERK phosphorylation over control, demonstrating a different response to H(2)O(2) vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H(2)O(2) exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H(2)O(2) induced increase in ERK phosphorylation.
authors.affiliation: Department of Schizophrenia Research, Merck Research Laboratories, Merck & Co, Inc, West Point, PA 19486-0004, USA. brian_magliaro@merck.com
authors.forename: Brian C
authors.initials: BC
authors.lastname: Magliaro
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Saldanha
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003024:Clozapine / Q000031:analogs & derivatives / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors* / Q000633:toxicity; D007202:Indicators and Reagents; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072105:Superoxide Dismutase-1
chemical_list: D014150:Antipsychotic Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D018696:Neuroprotective Agents; C000606290:SOD1 protein, human; C058272:norclozapine; D006861:Hydrogen Peroxide; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D048049:Extracellular Signal-Regulated MAP Kinases; D003024:Clozapine