Fig. 4

DnVPS4A causes increased accumulation and reduced secretion of Aβ42. Expression of control plasmid (ctr), wtVPS4A (wtVPS4) or the ATP hydrolysis deficient mutant dnVPS4A (dnVPS4) in Swe N2a cells (A-H) or wt primary mouse neurons (I) for 24 h. a Representative Western blot of APP, SDS-stable oligomeric Aβ species, β-CTFs and monomeric Aβ in cell lysate probed with antibody 6E10, and β-actin for protein normalization. b Western blot analysis of full length APP, β-CTFs and α-CTFs in cell lysate probed with the C-terminal specific APP antibody 369. c Western blot analysis of secreted αAPP (sAPPα) and Aβ in cell medium with antibody 6E10. d Densitometric quantification of A and C demonstrates that expression of dnVPS4, but not wtVPS4, increases intracellular Aβ compared to ctr. On the other hand, both wtVPS4A and dnVPS4A reduces secreted Aβ compared to ctr, although to a greater extent with dnVPS4A. DnVPS4 increases both cellular and secreted APP. Values are normalized against actin and expressed as percentage of control, n > 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. e Cellular full length APP is increased in cells transfected with dnVPS4A-FLAG (lower panel) compared to control plasmid Ctr-FLAG (upper panel). Scale bar 25 μm. f Antibody OC labelling of fibrillar oligomers and fibrils is increased in cells transfected with dnVPS4A (arrows) compared to control plasmid. Scale bar 25 μm. g Antibody OC labelling in dnVPS4-transfected cells is associated with enlarged vesicles positive for flotillin-1. Scale bar 15 μm. h Aβ42 accumulation in vesicles positive for ESCRT protein Tsg101 in dnVPS4A-transfected cells. Scale bar 25 μm. i Wt primary neurons at 12 DIV transfected with synapsin-dnVPS4A-FLAG or control synapsin-GFP plasmid for 24 h, immunolabelled for Aβ42 and the post-synaptic protein drebrin shows that Aβ42 labelling is increased with expression of dnVPS4A but not with control plasmid. Scale bar 50 μm