Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.This work was supported by grants SAF2014-52737-P to T.I., SAF2013-45258-P to M.R. C., BFU2016-77885-P to F.H., SAF2014-54070-JIN to A.M. from Ministerio de Economía, Industria y Competitividad (Spain). It was also funded by P2010/BMD-2332 (Neurodegmodels) from Comunidad de Madrid to T.I., F.H. and J.A.) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, Spain) to T.I., F.H., J.A., and I.F.). J.P.-U. is a recipient of a predoctoral contract from SAF2014-52737-P; L.G.-G. was funded by a contract from CIBERNED; A.G.-M. and A.S.S. were funded by contracts from CIBERNED cooperative projects 2013/07 and CIBERNED 2015-2/06, respectively. A.M., A.D.P. is a recipient of a Juan de la Cierva formación fellowship (Ministerio de Economía, Industria y Competitividad, Spain) associated to CIBERNED. J.J.-A. was funded by a predoctoral contract from CSIC (JAEPredoc program) and by CIBERNED. The cost of this publication has been paid in part by FEDER (European Funds for Regional Development) funds. We are grateful to Professor Eric N. Olson (Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, USA) for kindly providing Prkd1-floxed mice; Professor Jianping Ye (Pennington Biomedical Research Center, Baton Rouge, LA, USA) for his generous gift of RelA−/− mouse embryo fibroblasts; Dr. Marta Nieto (Centro Nacional de Biotecnología, Madrid, Spain) for providing reagents; Dr. C. López- Menéndez for scientific advice an critical reading of this manuscript as well as members of our laboratories for constructive suggestions. We also acknowledge Dr. L. Sánchez- Ruiloba for her valuable support with confocal microscopy, Drs. M. Díaz-Guerra and S. Gascón for providing lentivirus containing synapsin promoter and helpful discussion, Drs. Ana Guadaño-Ferraz and S. Bárez-López for valuable advice in human and mouse brain immunohistochemical approaches. We also thank M. Rodríguez-Martínez and T. Navarro for performing MRI analysis; J. Pérez for image design; and I. Santana, S. Ramirez, S. Valle, G. Cano-García, J. Deyell, and J. Henández-Bermúdez for their help and technical support