Cyclic Adenosine Monophosphate Response Element-Binding Protein Phosphorylation and Neuroprotection by 4-Phenyl-1-(4-Phenylbutyl) Piperidine (PPBP)

Abstract

Previous studies show that the potent, prototypical sigma(1)-receptor agonist 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) prevents cell death after oxygen-glucose deprivation (OGD) in primary cortical neuronal cultures. We tested the hypothesis that PPBP protects neurons by a mechanism involving activation of the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB). Primary cultured cortical neurons were exposed to 2 h of OGD and allowed to recover for 24 h, and PPBP treatment was initiated 15 min before the insult in the presence and absence of the sigma(1)-receptor antagonist rimcazole and inhibitors against protein kinases known to activate signal transduction cascades that result in CREB phosphorylation, such as H89 (protein kinase A inhibitor), LY294002 (PI3K inhibitor), U0126 (MEK1/2 inhibitor), or KN62 calmodulin kinase II inhibitor). Neuronal cell death was assayed by lactate dehydrogenase measurement 24 h after OGD. CREB phosphorylation was measured by immunoblot analysis at 30 min, 1 h, and 3 h of reoxygenation. Blots were quantitatively analyzed using Quantity One image analysis software. PPBP increased CREB phosphorylation at 1 h after recovery from OGD, which was abolished by rimcazole (1.7 +/- 0.2 in PPBP and 0.8 +/- 0.1 in PPBP plus rimcazole with OGD compared with 0.9 +/- 0.1 in OGD alone, p-CREB/CREB). The PPBP-induced increase in CREB phosphorylation was blocked by H89 (0.5 +/- 0.07) but not U0126, KN62, or LY294002. PPBP treatment prevented OGD-induced cell death and pretreatment with H89 blocked this protection (0.18 +/- 0.02 in PPBP and 0.27 +/- 0.03 in PPBP plus H89 with OGD compared with 0.33 +/- 0.02 in OGD alone, lactate dehydrogenase assay). Pretreatment with LY294002, UO126, or KN62 had no effect on neuronal protection by PPBP. These data suggest that the mechanism of neuroprotection by PPBP may be linked to CREB phosphorylation.