Figure 5
APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif)- and βCTF (β-cleaved carboxy-terminal fragment of APP)-dependent endosomal abnormalities in Down syndrome (DS) fibroblasts and Alzheimer's disease (AD) brain. (a) Greater APPL1 colocalization with rab5 in DS fibroblasts compared with control (ctrl) fibroblasts is seen by immunocytochemistry as shown in representative images of cells double immunolabeled with antibodies to APPL1 (green) and rab5 (red). The graph shows a higher APPL1 and rab5 colocalization coefficient (R) in DS fibroblasts, as calculated by Pearson's correlation coefficient in 30 DS and 30 control cells (mean±s.e.m., unpaired two-tailed t-test *P<0.05). (b) APPL1 mediates endosomal enlargement in DS cells. Treatment with APPL1 small interfering RNA (siRNA), but not scrambled control siRNA, blocks the increase in cross-sectional area of endosomes in DS fibroblasts but has no effect on normal endosomes in age-matched 2N ctrl fibroblasts (n=30 cells, one-way analysis of variance (ANOVA), Tukey’s test, mean±s.e.m., *P<0.05 and ***P<0.001). A representative blot with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control and graphic quantitation from three separate experiments are shown. (c) APPL1 mediates the abnormally elevated endocytosis in DS fibroblasts.7 Fluorescent horseradish peroxidase (HRP)-positive puncta are higher in DS fibroblasts at 30 min after the addition of HRP to the medium compared with control cells and are reduced in DS cells treated with APPL1 siRNA compared with cells treated with scrambled (ctrl) siRNA, whereas APPL1 siRNA has no effect on HRP uptake in control fibroblasts. Quantitative analysis of fluorescence at 0, 5, 15 and 30 min after the addition of HRP to the medium shows significantly reduced HRP uptake after APPL1 siRNA compared with cells treated with scrambled (ctrl) siRNA (ctrl–ctrl-siRNA; 15, 15, 20 and 19 cells; ctrl-siRNA: 16, 28, 17 and 25 cells; DS-ctrl-siRNA: n=26, 10, 18 and 36 cells; DS-APPL1 siRNA: n=12, 16, 39 and 42 cells in 0, 5, 15 and 30 min from two experiments, mean±s.e.m., one-way ANOVA, Tukey’s test, mean±s.e.m., ***P<0.001). (d) APPL1 mediates abnormally increased nuclear factor-κB (NF-κB) pathway activation in DS fibroblasts. Functional evidence for APPL1-mediated rab5 activation on endosomes is reflected in greater APPL1/rab5-endosomal-mediated nuclear localization of the NF-κB p65 subunit (p65/RelA).16 Levels of nuclear p65/RelA normalized to nuclear histone H3 as a loading control (third lane) are elevated in DS fibroblasts compared with those in control (ctrl) cells (first lane). This elevation is reversed by APPL1 siRNA (fourth lane) but not scrambled (ctrl) siRNA as shown on an immunoblot representative of three experiments quantified in the graph (mean±s.e.m., two-way ANOVA with interaction, *P<0.05). (e) βCTF levels, but not full-length APP (flAPP) or αCTF levels, are significantly elevated in AD brain compared with age-matched control (ctrl) brain, as shown on an immunoblot representative of three experiments quantified in the graph. C1/6.1 antibody was used to measure βCTF and αCTF, which are normalized to the level of APP (n=13 control and 13 AD brains, mean±s.e.m., unpaired two-tailed t-test, *P<0.05). Identities of βCTF (C1–C4) and αCTF (C5) were confirmed using 6E10 (data not shown). Actin is used as a loading control. (f) Membrane-associated APPL1 levels are increased in AD brains compared with control (ctrl) brains as shown on an immunoblot representative of three experiments quantified in the graph as a ratio of membrane-associated APPL1 in a 50 μg pellet (P) of brain homogenate to the APPL1 level in the 50 μg supernatant (S) (n=13 control and 13 AD brains, mean±s.e.m., unpaired two-tailed t-test, *P<0.05). (g) APPL1 association with rab5-positive endosomes is increased in AD brain. Double immunofluorescence labeling confirms rab5-positive endosome enlargement in neocortical pyramidal neurons of AD brain8 and demonstrates greater APPL1 colocalization (arrowheads) in these enlarged endosomes as compared with neuropathologically normal control brains. The graph shows a higher APPL1 and rab5 colocalization coefficient (R) in AD brains, as calculated by Pearson's correlation coefficient (n=90 cells, mean±s.e.m., unpaired two-tailed t-test, *P<0.05). (h) Recruitment of APPL1 to rab5 endosomes is higher in neuronal endosomes of AD brain compared with that in control (ctrl) brain, as reflected by an increased percent of rab5 endosome surface area occupied by APPL1-immunoreactive signal quantified from images similar to those in panel g. Comparison of rab5 endosomes of different size ranges demonstrates increased APPL1 colocalization on endosomes of greater size in AD brains (n=90 cells; 3824, 408 and 224 endosomes for control, 3194, 269 and 156 endosomes for AD in each size bin, mean±s.e.m., unpaired two-tailed t-test, ***P<0.001).
