Figure 6.

C9ORF72 regulates autophagy in neuronal cell lines. (A) Neuro2a cells were cotransfected with DsRed-LC3 for 48 h. Fluorescence microscopy revealed that C9ORF72 appeared as punctate structures that colocalized with DsRed-LC3, indicating autophagosomes, inset demonstrates higher magnification (×100) of the areas highlighted to illustrate autophagosomes, Scale bar: 10 μm. (B) Cells were treated with 100 nm bafilomycin for 4 h to inhibit fusion of autophagosomes to the lysosome. White arrow represents C9ORF72 colocalization with autophagosomes. Scale bar: 10 μm. (C) Quantification of the percentage of cells in which C9ORF72 colocalized with DsRed-LC3 revealed elevation in the numbers of LC3-positive structures, indicating autophagosomes, colocalizing with C9ORF72 in bafilomycin treated cells. Data are represented as mean ± SEM; *P < 0.05 versus untreated cells by unpaired t-test, 50 cells were scored, n = 2. (D) Human SH-SY5Y cells were treated with 100 nm bafilomycin for 4 h to test examine autophagic flux by LC3 immunoblotting. (E) Human SH-SY5Y cells were transfected with human C9ORF72-targeting or control siRNA for 72 h. Immunoblotting revealed depletion of C9ORF72 by 75% compared with control siRNA-treated cells. Immunoblotting for LC3 was performed on lysates harvested from cells treated with control or C9ORF72-targetd siRNA, indicating the formation of autophagosomes. (F) Quantification of the ratio of phosphatidylethanolamine-modified product of LC3II, relative to LC3I by densitometry of immunoblots, demonstrated a reduction in this ratio by 45% in C9ORF72-siRNA-treated cells, whereas there was no change in control siRNA-treated cells compared with untreated cells. Data are represented as mean ± SEM; **P < 0.001 versus untreated cells by unpaired t-test, n = 3. (G) C9ORF72 is associated with lysosomes in Neuro 2A cells. Cells were transfected with C9ORF72-GFP and treated with Lysotracker for 20 min, Scale bar: 10 μm.