Fig 2: Knockdown of Protrudin and its interactors attenuates RTN3-induced DRGN neurite outgrowth. (A) Timeline for experimental analysis of Protrudin and its interactors in RTN3-overexpressed DRGN. Representative Western blots to confirm knockdown of (B) Protrudin, (C) FYCO1, (D) RAB7 and (E) SYT7. ß-actin is used as a protein loading control. Representative images to show neurite outgrowth in (F) RTN3 overexpressed DRGN controls and after knockdown of (G) Protrudin, (H) FYCO1, (I) RAB7 and (J) SYT7 in RTN3-overexpressed cultures. (K) Quantification to show attenuation of RTN3-overexpressed DRGN neurite outgrowth by shProtrudin, shFYCO1, shRAB7 and shSYT7. (L) DRGN survival remains unaffected by knockdown of Protrudin, FYCO1, RAB7 and SYT7 in RTN3-overexpressed DRGN. n = 9 wells/condition Scale bars in (F–J) = 100 µm. Data are means ± SEM. *** p = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Fig 3: RTN3 overexpression promotes RGC axon regeneration and functional recovery after ONC, which is also protrudin-dependent. (A) Western blot and (B) quantification to show that RTN3 plasmids significantly upregulate RTN3 protein in the retina after intravitreal injection in vivo. (C) Immunohistochemistry to localise RTN3 protein to the RGCs (green) in the ganglion cell layer (GCL) and the inner plexiform and inner nuclear layer (INL). ONL = outer nuclear layer. (D) High power images to show RTN3 protein localised to ßIII-tubulin+ RGCs (arrows) in the GCL. (E) GAP43 immunohistochemistry and (F) quantification to show that in control optic nerve, few if any GAP43+ axons pass beyond the lesion site (*) whilst in RTN3 overexpressed eyes, significant GAP43+ axons are present beyond the lesion site, an effect which is obliterated when Protrudin is knocked out at the same time as RTN3 overexpression. (G) Representative images to show FluorGold (FG) backfilled RGC in retinal wholemounts after RTN3 overexpression. (H) Quantification of the number of FG+ RGC in retinal wholemounts shows that overexpression of RTN3 is significantly neuroprotective, an effect that is independent of protrudin. (I) Representative ERG traces and (J) quantification of the pSTR amplitude shows significant improvements in RTN3 overexpressed eyes which are ablated after protrudin knockdown. Arrows show the peak of the pSTR. Data are means ± SEM. n = 12 eyes/optic nerves/treatment. Scale bars in (C) = 100 µm, scale bars in (D) = 25 µm, scale bars in (E) = 200 µm, scale bars in (G) = 50 µm. *** p = 0.0001, one-way ANOVA with Dunnett’s post hoc test.