GSK-3 inhibition through GLP-1R allosteric activation mediates the neurogenesis promoting effect of P7C3 after cerebral ischemic/reperfusional injury in mice

Abstract

An aminopropyl carbazole compound, P7C3, has been shown to be a potent neurogenesis promoting agent; however, its fundamental signaling action has yet to be elucidated. A cerebral ischemic/reperfusional (CI/R) injury model in mice was implemented to elucidate the neuronal protective mechanism(s) of P7C3. Treating CI/R mice using P7C3 (50–100 μg/kg, i.v.) significantly improved tracking distance and walking behavior, and reduced brain damage. Specifically, P7C3 promoted the expression of neurogenesis-associated proteins, including doublecortin, beta tubulin III (β-tub3), adam11 and adamts20, near the peri-infarct cortex, accompanied by glycogen synthase kinase 3 (GSK-3) inhibition and β-catenin upregulation. The application of a specific inhibitor against glucagon-like peptide 1 receptor (GLP-1R), exendin(9-39), revealed that the beneficial effects of P7C3 involved triggering the activation of GLP-1R-associated PKA/Akt signaling. P7C3 elicited the GLP-1R-dependent intracellular cAMP increment and the insulin secretion in cellular models. Surface plasmon resonance assay of P7C3 showed a Kd value of 0.53 μM for GLP-1R binding, and the docking of P7C3 to the putative active site on GLP-1R was successfully predicted by molecular modeling. Our findings indicate that P7C3 promotes the expression of neurogenesis proteins by activation of the cAMP/PKA-dependent and Akt/GSK3-associated β-catenin through positive allosteric stimulation of GLP-1R. Within the P7C3 class of neuroprotective molecules, this mechanism appears to be unique to the prototypical P7C3 molecule, as other active derivatives such as P7C2-A20 and P7C3-S243 they do not engage this same pathway and have been shown to work by nicotinamide phosphoribosyltransferase (NAMPT) stimulation.