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. 2012 Feb 1;32(5):1714–1729. doi: 10.1523/JNEUROSCI.5433-11.2012

Figure 10.

Figure 10.

Accumulation of APP-CTF through γ-secretase inhibition induce neurite outgrowth. N2a cells and cortical neurons coexpressing YFP and EV or APP-FL were treated with Compound E (10 nm; 24 h) and/or adenylate cyclase inhibitor MDL-12,330A (10 nm; 30 min) and neurite extensions were quantified as described in Materials and Methods. Representative images of N2a cells (a) or inverted images of 8 DIV cortical neurons (c) coexpressing YFP and APP-FL are shown. b, d, Morphological changes were quantified and plotted relative to untreated EV/YFP cells. e–g, N2a cells stably expressing PS1-wt or PS1-D385A were transiently transfected with EV, APP-FL, APP-C99–6myc, or mAICD. e, The levels of PS1 and APP expression were analyzed by Western blotting using PS1NT and CTM1 antibodies, respectively. f, Representative images of N2a cells coexpressing YFP and APP-FL are shown. g, Quantitative analysis of total neurite area relative to EV control cells demonstrated that the loss of PS1 function affects neurite outgrowth in N2a cells. h–j, APP-CTF accumulation through differential secretase processing. N2a cells (h, j) and COS cells (i) were transiently transfected with EV, APP-FL, APP-M596V (APP β-site cleavage mutant), or APP-F615P (APP α-site cleavage mutant) and treated with or without Compound E (24 h; 10 nm). To detect α- and β-CTFs of APP, Western blotting analysis of Tris-Tricine gels was performed using rabbit polyclonal CTM1 and mouse monoclonal 82E1 antibodies. j, N2a cells were transiently transfected with EV or the indicated APP-FL variants. The total neurite area was quantified and plotted relative to untreated EV cells. Statistical analysis was performed using ANOVA Kruskal–Wallis test followed by Dunn's post hoc multiple-comparison analysis. **p < 0.001, compared with stable PS1-wt N2a cells expressing APP-FL. ##p < 0.001, compared with untreated cells or PS1-wt control cells within the same transfected condition. The total number of cells, quantified from at least three independent experiments, is shown in parentheses. Error bars indicate SEM.