Figure 4
APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates β-cleaved carboxy-terminal fragment of APP (βCTF)-induced enlargement and impaired transport of endosomes in neurons. (a) Overexpression of wild-type human APP (APPwt) or rab5 increases average cross-sectional areas of individual rhoB-positive endosomes in primary cultures of mouse cortical neurons (one-way analysis of variance (ANOVA), Tukey’s test, *P<0.05 and **P<0.01, respectively). (b) Rab5 or APPwt overexpression reduces average transport velocities of rhoB-positive endosomes in cultured neurons (one-way ANOVA, Tukey’s test, ***P<0.001). (c) Rab5 or APPwt overexpression has no effect on transport interruption of rhoB-positive endosomes in cultured neurons. (d–f) Raising βCTF levels by APPwt overexpression or exposure to the γ-secretase inhibitor L685 458 (L685) in cultured neurons increases average cross-sectional area of rab5-positive endosomes (d), decreases average speeds of rab5-positive endosomes (e) and increases numbers of stationary rab5 endosomes (f) (one-way ANOVA, Tukey’s test, **P<0.01 and ***P<0.001). Overexpressing the β-secretase cleavage-incompetent APPmv mutant does not alter these parameters (d–f). (g–i) Treatment with APPL1 small interfering RNA (siRNA) but not a control scrambled siRNA prevents from the increased cross-sectional area of rab5 endosomes (g), the reduced rab5 endosome transport velocity (h) and the transport interruption of rab5 endosomes (i.e. increased stationary endosome number) (i) that are induced in primary cultures of mouse cortical neurons by APPwt overexpression (unpaired two-tailed t-test, **P<0.01 and ***P<0.001). (j–l) The PTB domain of APPL1 is required for βCTF-induced endosome alterations in primary cortical neurons. Increased endosomal cross-sectional area (j), lowered transport rate of endosomes (k) and transport interruption of rab5 endosomes (elevated stationary endosomes) (l) are induced by overexpressing APPwt and APPL1wt (one-way ANOVA, Tukey’s test, **P<0.01 and ***P<0.001) but are not induced by overexpression of either APPL1wt with APPmv or APPwt with APPL1ΔPTB (PTB domain deleted APPL1 mutant). Results are presented as mean±s.e.m.
