Funktionsanalyse der Reggie-Proteine während der neuronalen Differenzierung und Axon-Regeneration

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

Reggies/flotillins regulate retinal axon regeneration in the zebrafish optic nerve and differentiation of hippocampal and N2a neurons

The reggies/flotillins proteins upregulated during axon regeneration in retinal ganglion cells (RGCs) are scaffolding proteins of microdomains and involved in neuronal differentiation. Here, we show that reggies regulate axon regeneration in zebrafish (ZF) after optic nerve section (ONS) in vivo as well as axon/neurite extension in hippocampal and N2a neurons in vitro through signal transduction molecules modulating actin dynamics. ZF reggie-1a, -2a, and -2b downregulation by reggie-specific morpholino (Mo) antisense oligonucleotides directly after ONS significantly reduced ZF RGC axon regeneration: RGC axons from reggie Mo retinas were markedly reduced. Moreover, the number of axon-regenerating RGCs, identified by insertion of A488-coupled dextran, decreased by 69% in retinas 7 d after Mo application. At 10 and 14 d, RGCs decreased by 53 and 33%, respectively, in correlation with the gradual inactivation of the Mos. siRNA-mediated knockdown of reggie-1 and -2 inhibited the differentiation and axon/neurite extension in hippocampal and N2a neurons. N2a cells had significantly shorter filopodia, more cells had lamellipodia and fewer neurites, defects which were rescued by a reggie-1 construct without siRNA-binding sites. Furthermore, reggie knockdown strongly perturbed the balanced activation of the Rho family GTPases Rac1, RhoA, and cdc42, influenced the phosphorylation of cortactin and cofilin, the formation of the N-WASP, cortactin and Arp3 complex, and affected p38, Ras, ERK1/2 (extracellular signal-regulated kinases 1 and 2), and focal adhesion kinase activation. Thus, as suggested by their prominent re-expression after lesion, the reggies represent neuron-intrinsic factors for axon outgrowth and regeneration, being crucial for the coordinated assembly of signaling complexes regulating cytoskeletal remodeling

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1