The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins

Abstract

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5–15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor κB (NF-κB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75NTR, in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-κB-mediated control of neurite growth in developing neurons.

This work was funded by Ministerio de Sanidad y Consumo (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) Grant CB06/05/1104), Ministerio de Educación y Ciencia Grant SAF2007-60287, Generalitat de Catalunya (Suport als Grups de Recerca Consolidats) (J.X.C.), Ministerio de Ciencia e Innovación Grants BFU2008-01328 (V.J.Y.) and BFU2008-3980 (E.S.), and Fundació Caixa Catalunya Obra Social (E.S.). C.S. was supported by postgraduate fellowships from Ministerio de Educación y Ciencia and Fondo de Investigación Sanitaria. V.I.-G. holds a postgraduate fellowship from the Generalitat de Catalunya. R.S.M. and V.J.Y. are under the Juan de la Cierva and Ramon y Cajal programs, respectively, from the Ministerio de Educación y Ciencia (Spain), cofinanced by the European Social Fund. We thank D. Trono (Geneva, Switzerland) for providing the lentiviral plasmids and Dr. Joaquin Lopez-Soriano and Patricia Ortega for excellent technical support.