Fig. 5
C9orf72 DPRs and RAN expression induce Golgi fragmentation in Neuro2a cells. (A) Fluorescent microscopy images of the Golgi apparatus following immunocytochemistry for GM130 in UT cells or populations expressing EV or codon-optimised C9orf72 GAx40, GRx40, GPx40 or PRx40 DPRs. Scale bar: 5 µm. (B) Quantification of the proportion of cells expressing pcDNA3.1 empty vector (EV) or C9orf72 DPRs with Golgi fragmentation in (A). Mean ± SEM, n = 3, one-way ANOVA followed by post-hoc Tukey test, ***p < 0.001. Significantly more cells with fragmented Golgi were present in populations expressing GAx40, GRx40, GPx40 or PRx40 DPRs compared to UT and EV cells. (C) Design of constructs for RAN translation of the C9orf72 HRE. Constructs contain either 0, 3 (control) or 40 repeats (G4C2RANx0, G4C2RANx3, G4C2RANx40) with a 3′-3xFLAG, HA and MYC tag (each in a different reading frame) and a mCherry fluorophore, but they lack an ATG start codon. Hence, the C9orf72 HRE can undergo RAN translation in any forward frame. The black hexagon represents a stop codon at the end of the Flag, HA and mCherry tags (if frame 3 is used, translation of the MYC tag is prevented by the mCherry stop codon). P2A sequence induces ribosomal skipping during translation. mCherry sequence has a start codon and thus is also translated in the absence of RAN translation. (D) Immunofluorescent microscopy images of cells expressing RAN C9orf72 constructs G4C2RANx0, G4C2RANx3, or G4C2RANx40 in Neuro2A cells following immunostaining for polyGP and displaying mCherry. G4C2RANx40 produces polyGP by RAN translation in Neuro2A cells. Scale bar: 10 µm. (E) Western blotting of C9orf72 DPRs using anti-polyGP antibody (GP) in insoluble lysate fractions prepared from untransfected (UT) Neuro2A cells or cells expressing mCherry-tagged C9orf72 G4C2RANx0, G4C2RANx3, or G4C2RANx40 constructs. PolyGP was expressed in cells transfected with G4C2RANx40, but not G4C2RANx0 or G4C2RANx3. (F) Western blotting of C9orf72 DPRs using an anti-polyGP antibody (GP) in soluble lysate fractions prepared from untransfected (UT) Neuro2A cells or cells expressing mCherry-tagged C9orf72 G4C2RANx0, G4C2RANx3, or G4C2RANx40 constructs. GAPDH was used as a loading control. (G) Fluorescent confocal microscopy images of mCherry-tagged C9orf72 DPRs and the Golgi apparatus following immunocytochemistry for polyGP and GM130 in untransfected Neuro2A cells (UT), or cells transfected with either G4C2RANx0, G4C2RANx3, or G4C2RANx40 constructs. Arrows: fragmented Golgi were identified by condensed punctate structures. Scale bar: 10 µm. (H) Quantification of the proportion of cells in (F) with Golgi fragmentation. Mean ± SEM, n = 3; *p < 0.05, one-way ANOVA followed by post-hoc Tukey test. Significantly more cells with fragmented Golgi were present in populations transfected with G4C2RANx40, expressing polyGP, compared to those transfected with controls G4C2RANx0, G4C2RANx3 or UT cells