The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK‐3beta: connecting neurotransmission with neuroprotection

In this study, we provide evidence that the muscarinic M1 receptor targets NF‐E2‐related factor‐2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase‐1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase‐1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2‐aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Gαq, phospholipase C‐β and the classical PKC‐γ isoenzyme, as responsible for the regulation of Nrf2. A PKC‐insensitive Nrf2S40A single‐point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase‐3β (GSK‐3β) augmented M1‐dependent activation of AREs while a PKC‐insensitive mutant of GSK‐3β (GSK‐3β‐Δ9) blocked this effect and prevented M1‐induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Gαq/phospholipase C‐β/PKC/GSK‐3β axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer’s disease, require a reinforcement of the cell antioxidant capacity.