Anales RANF

P.59 P2X7 AND BDNF RECEPTORS CONVERGE TO SHAPE ERK SIGNALLING IN NEURONS THROUGH THE MODULATION OF ERK-DIRECTED PROTEIN PHOSPHATASES M.J. Queipo, J.C. Gil-Redondo, R. Gómez-Villafuertes, E.G. Delicado, M.T. Miras- Portugal and R. Pérez-Sen School of Veterinary Sciences, Complutense University of Madrid (UCM), Madrid, Spain, Neurochemistry Research Institute, IUIN, UCM, Madrid, Spain, Sanitary Research Institute of the San Carlos Clinical Hospital, IdiSSC, UCM, Madrid, Spain. Nucleotide receptors share signalling mechanisms with neurotrophins modulating several biological responses. In cerebellar neurons, P2X7 and BDNF receptors activate ERK signalling and promote neuronal differentiation and survival. The distinct pattern of ERK activation triggered by P2X7 and BDNF receptors reveals disparities in the extent they regulate the inactivation mechanisms, through the ERK-selective phosphatases. The family of dual specificity phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases are emerging as key regulators of the intensity and duration of MAPK signalling. The ERK- targeted phosphatases DUSP1 and DUSP6 were studied in primary cerebellar neurons, to analyse the changes in expression and activity of these proteins upon stimulation with P2X7 agonist and BDNF. Quantitative RT-PCR studies revealed that DUSP1 and DUSP6 transcripts were induced during neuronal differentiation and increased in response to BzATP and BDNF. Interestingly, P2X7 receptors correlated with DUSP6 protein along the culture, and dynamically regulated DUSP6 expression levels and activity. At short-term stimulations, DUSP6 protein decreased and was targeted to proteasome degradation, prolonging cytosolic ERK activity. This was followed by the recovery of protein levels through dusp6 gene induction that terminated ERK signalling. Regarding the nuclear phosphatase DUSP1, whereas BzATP only elicited a transient response, BDNF produced a sustained increase in DUSP1 protein levels that involved several mechanisms, enhanced transcription of dusp1 gene and protein stabilization, which was mediated by ERK-dependent Ser 359 phosphorylation of DUSP1. The divergent behaviour in DUSP1 regulation by P2X7 and BDNF receptors could explain the different modifications on the dendritic and axonal branching observed with BzATP and BDNF. These effects were lost in cultured neurons in which Dusp1 was down- regulated by shRNA. In conclusion, the spatio-temporal regulation of ERK signalling elicited by P2X7 and BDNF receptors through DUSP1 and DUSP6 phosphatases provides a mechanism for fine-tune modulation of the neuronal differentiation. References: Miras-Portugal MT, Queipo MJ et al. Brain Res Bull. 2018, Dec 26. doi: 10.1016/j.brainresbull.2018.12.012. “P2 receptor interaction and signalling cascades in neuroprotection”. Queipo MJ, Gil-Redondo JC, et al., Front Mol Neurosci. 2018, Jan 10;10:448. “P2X7 Nucleotide and EGF Receptors Exert Dual Modulation of the Dual-Specificity Phosphatase 6 (MKP-3) in Granule Neurons and Astrocytes, Contributing to Negative Feedback on ERK Signaling”.