Cyclic adenosine monophosphate response element- binding protein phosphorylation and neuroprotection by 4-phenyl-1-(4-phenylbutyl) piperidine (ppbp)

BACKGROUND: Previous studies show that the potent, prototypical σ1-receptor agonist 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) prevents cell death after oxygenglucose 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). METHODS: 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 σ1-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. RESULTS: 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. CONCLUSIONS: These data suggest that the mechanism of neuroprotection by PPBP may be linked to CREB phosphorylation.