Functional Analysis of Reggie Proteins during Neuronal Differentiation and Axon Regeneration

Abstract

The reggies are scaffolding proteins of membrane microdomains and involved in various cellular processes including neuronal differentiation. Reggie-1 and -2 were originally discovered as proteins upregulated during axon regeneration in retinal ganglion cells (RGCs) after optic nerve injury, suggesting a function of these proteins in axon regeneration. Loss-of-function studies via small interfering RNAs (siRNAs) or morpholino antisense oligonucleotides (Mos) against reggie-1 and -2 were performed to clarify, whether the reggies are causally linked to neuronal differentiation in cell cultures and/or axonal regeneration after optic nerve section (ONS) in zebrafish. Silencing of reggie-1 with siRNAs caused a simultaneous loss of reggie-2 protein by proteasomal degradation in N2a cells. Depletion of both reggies in differentiating N2a cells led to significantly shorter filopodia, more cells with lamellipodia and fewer with neurites, a defect which was rescued by a reggie-1 construct without siRNA binding sites. Furthermore, reggie knockdown strongly perturbed the balanced activation of the Rho family GTPases RhoA, Rac1 and cdc42 and affected activation of MAP kinases p38 and ERK1/2, Ras and focal adhesion kinase (FAK). Downregulation of zebrafish reggie-1a, -2a and -2b expression in vivo by application of reggie-specific Mos directly after ONS significantly reduced the capability of zebrafish RGCs to regenerate axons. In an outgrowth assay, the number of re-growing RGC axons in vitro from reggie Mo-treated retinae was markedly reduced compared to controls. Moreover, the number of axon regenerating RGCs in vivo, identified by insertion of A488-coupled dextran 7d after Mo-application, decreased by 69% in reggie Mo-retinae as opposed to controls. At 10 and 14 d, labeled RGCs decreased by 53 and 33%, respectively, in correlation with the gradual loss of the Mos. Thus, as suggested by their prominent re-expression upon lesion, the reggies represent neuron-intrinsic factors for axon outgrowth and regeneration in vitro and in vivo by coordinating signal transduction pathways to control cytoskeletal remodeling.