FIGURE 5.

In vitro FAD mutation promotes APP/Aβ mitochondrial accumulation and mitochondrial dysfunction. A, mitochondrial containing and cytosolic fractions were prepared and blotted for mitochondrial markers Cox IV and VDAC, while β-tubulin was used to verify cytosolic fractions. Western blot analysis for human APP (6E10) revealed a significant increase in APP/Aβ derivatives in SWE (S) mitochondrial fractions compared with WT APP (W). B, ATP levels were assessed with an ATP luciferase assay in both W and S cell lines. ATP production was significantly decreased in the SWE cell line compared with WT. In addition, SWE cells were more sensitive to mitochondrial uncoupler (FCCP), complex III inhibitor (antimycin), and the ATP synthase inhibitor oligomycin compared with WT. C, 5 μg of protein from either WT or SWE whole cell lysates and MCF fractions were subjected to the DNP derivative assay followed by Western blotting. SWE cells exhibited increased DNP activity compared with WT cells (n = 8). All data points, *, p < 0.05 SWE cells compared with WT APP cells. D, co-IP experimentation by pulling down APP/Aβ (4G8) and blotting for HSP60 showed increases in APP/Aβ binding to HSP60 in APP_SWE mitochondrial-containing fractions (MCF) compared with APP_WT (n = 6). HSP60 IP followed by 4G8 immunoblot shows increased APP binding to HSP60 in APP_SWE cells compared with APP_WT. 4G8 and HSP60 immunoblots show equal amounts of HSP60 and 4G8 were immunoprecipitated for each experiment.