Reggies/Flotillins Regulate Retinal Axon Regeneration in the Zebrafish Optic Nerve and Differentiation of Hippocampal and N2a Neurons

Abstract

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.

Function of Flotillins in Receptor Tyrosine Kinase Signaling and Endocytosis: Role of Tyrosine Phosphorylation and Oligomerization

“…Recent studies have confirmed the relevance of flotillins for axon regeneration in zebrafish as well as in mammalian cell cultures. Apparently, downregulation of flotillins by flotillin specific morpholinos in the zebrafish resulted in a massive reduction in the number of regenerating axons (60). Since the role of flotillins in neuronal regeneration has recently been directly reviewed, we here omit a long discussion of the topic.…”

“…Their original discovery as "reggies" suggested a function in neuronal regeneration in the goldfish (2,45), which has been later verified by further studies from the same group (57,60,61). However, the regenerative capacity of the optic nerve is poorly conserved in mammals, making a generalization of the regenerative function of flotillins in the nervous system of mammals difficult.…”

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

“…As an additional control for the specificity of the effect, shR1 cells were transfected with a reggie-1 rescue construct (R1-rescue) which is resistant to the shRNA (Munderloh et al, 2009). The non-rescued cells ( Fig.…”

“…Reggies were also needed for growth cone formation and elongation (Koch et al, 2013;Munderloh et al, 2009) and the targeted delivery of N-cadherin as one of many growth and guidance receptors (Bodrikov et al, 2011) in elongating neurites. The Rab11/ARF-6 dependent recycling of integrins was demonstrated by Eva and colleagues as an essential molecular mechanism for the regeneration of axons in mammals (Eva et al, 2010(Eva et al, , 2012.…”

“…Indeed, a proteomic analysis previously recognized that reggie-1 and reggie-2 are part of the FA complex (Kuo et al, 2011). In addition, the downregulation of reggie and overexpression of a dominant negative (DN) reggie-1 construct disturbed the localization of overexpressed prion protein in structures resembling FAs (Schrock et al, 2009), affected Src and FAK and impaired axon growth (Langhorst et al, 2008a;Munderloh et al, 2009). As reggies were, furthermore, implicated in the delivery of the T cell receptor to the T cell cap (Stuermer et al, 2004) and in Rab11a-dependent E-cadherin recycling , we concluded that reggies might regulate as well integrin recycling and FA dynamics through Rab11a (Stuermer, 2010).…”