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.

Figure 1.

Reggie downregulation reduces axon outgrowth. A, Mo antisense oligonucleotides labeled with lissamine were applied immediately after ONS. A maximum-intensity projection of a deconvoluted Z-stack from a retina, 3 d after ONS, illustrates that RGCs and axons are labeled by retrograde Mo transport. B, C, Reggie-2 Ab immunostaining is present in control Mo-treated retinas which did not alter reggie expression (B). Reggie Mos reduced reggie-2 Ab staining of RGCs 7 d after ONS (C, images were taken with the same microscope settings). Scale bars, 50 μm. D, Regeneration was assessed by quantifying axons from mini-explants isolated 4 d after ONS. Retina pairs (21) (control- and reggie–Mo-treated eyes from the same fish) were analyzed. The mean number of axons per explant was normalized to the control retina, and relative outgrowth efficiency is shown.

Figure 2.

Mo-mediated downregulation of reggie in RGCs leads to a reduction of regenerating axons. Mos were applied to the ZF optic nerves as described. Alexa-488–dextran was inserted into a second lesion site 2–3 mm distal from the first one at 7 d (A, B, C), 10 d (D, E, F), and 14 d (G, H, I), respectively. Counts of retrogradely labeled RGCs show that their number (A, D, G) was reduced in the reggie Mo-receiving RGCs (B, E, H) compared with the control Mo-containing RGCs (C, F, I): by 69% at 7 d, by 53% at 10 d, and by 33% at 14 d after ONS. Scale bars, 50 μm.

Figure 3.

Reggie-1 is required for hippocampal neuron differentiation. Representative wide-field images of transfected hippocampal neurons. A, Cells transfected with control (GL2) siRNA differentiated normally (insert) having long thin axons (to the right) and dendritic arborizations. B, C, Note the short and thick protrusions, large lamellipodia, and broad dendrites of reggie-1 (R1) siRNA-treated cells. D, Cells simultaneously transfected with the reggie-1 rescue construct (R1–EGFP rescue) and reggie-1 siRNAs differentiated normally. Scale bars, 20 μm. Histogram in E shows the quantification of the cells transfected with control GL2 siRNA, with reggie-1 siRNAs, and simultaneously transfected with R1–EGFP rescue construct and reggie-1 siRNAs.

Figure 4.

Reggie-1 knockdown impairs process formation in N2a cells. Cells transfected with either GL2 control siRNAs or a mix of reggie-1-specific siRNAs were stimulated with IGF-1, fixed 24 h after stimulation, and stained with phalloidin to visualize F-actin and cell morphology. Untreated and GL2 siRNA-transfected cells produced numerous filopodia (A, B), whereas reggie-1 downregulation (R1 siRNA) led to the formation of large lamellipodia (F). Significantly fewer cells formed neurites after reggie-1 siRNA transfection (E), which efficiently downregulated reggie-1 and reggie-2 expression compared with GL2 siRNA cells in Western blot experiments (G). D, N2a cells were simultaneously transfected with reggie-1 siRNA and the reggie-1 construct (R1–EGFP rescue) without siRNA-binding sites. A large proportion of the cells no longer exhibited the lamellipodia-rich phenotype but had instead many filopodia and neurites (D–F) much as control cells (A, B). Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Scale bars, 20 μm.

Figure 5.

Reggie-1 downregulation affects Rho GTPase activation in N2a cells. N2a cells starved overnight, stimulated with 50 ng/ml IGF-1 for 5 min, and assayed for GTP-loading of small GTPases. The histograms, A–D, show changes in the intensity (relative to controls) in the respective Western blots (n = 4), which are exemplified below each histogram. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Total Rac, cdc 42, RhoA, and Ras of crude lysates were used as loading control. A, B, In reggie-1 siRNA-treated cells, the activation patterns of Rac1 and cdc42 were significantly altered. IGF-1 application increased the activation of Rac1 in control-transfected cells, whereas siRNA-treated cells showed an elevated level of activated Rac1 without IGF-1, which increased further after IGF application. In contrast, cdc42 activity decreased during IGF application in control-transfected cells and remained at low levels in siRNA-transfected cells with or without IGF-1. C, RhoA activation increased during IGF application in control cells and further increased in reggie-1 siRNA-treated cells. D, Ras stimulation by IGF in control transfectants was absent in reggie–siRNA-treated cells.

Figure 6.

Coimmunoprecipitation assays for the analysis of the complex of Arp3, N-WASP, and cortactin and phosphorylation of cofilin and cortactin in reggie siRNA-treated N2a cells. A, In IGF-1 stimulated control cells, Abs against N-WASP and cortactin coprecipitate Arp3. In reggie-1 siRNA-treated cells, the Arp3, N-WASP, cortactin complex is present at lower levels and is almost disrupted during IGF stimulation. B, The phosphorylation of cortactin at Tyr466 is unchanged after IGF-1 treatment of control cells but is strongly reduced during IGF-1 treatment of reggie-1 siRNA cells. C, The phosphorylation of cofilin at Ser3 slightly decreases during IGF stimulation but is increased in reggie-1 siRNA-treated cells with or without IGF-1.

Figure 7.

Reggie-1 downregulation affects signaling in N2a cells. A, Activation of p38 was reduced in reggie siRNA-treated cells independently of IGF-1 stimulation in comparison with control transfectants. B, ERK1/2 activation was decreased in response to reggie siRNA. C, pFAK showed a significant activation during IGF-1 stimulation, and this stimulation was reduced in reggie-1 siRNA-treated cells. Total FAK was decreased in reggie-1 siRNA-treated cells. Total p38 (A), ERK1/2 (B), and GAPDH (C) were used as loading controls. Mean ± SEM; n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. D, JNK, PKC, and PKB phosphorylation was not affected by reggie-1 siRNA-treatment.