Fig. 5

Increased cellular levels of Aβ with dnVPS4A expression is consistent with decreased lysosomal degradation. a Bafilomycin A1 (BafA1) treatment had similar effects on Aβ as dnVPS4a, markedly increasing intracellular levels of Aβ and reducing Aβ secretion. Representative Western blot analysis of cell lysates and cell medium from Swe N2a cells treated with 5 nM BafA1 for 24 h and blotted for 6E10 for Aβ and β-actin. b Densitometric quantification of A. Values are normalized against actin and expressed as percentage of untreated control, n = 3, *** p < 0.001. c When the ability of lysosomes to degrade Aβ is abolished by BafA1, blocking Aβ on route to the lysosomes via dnVPS4 does not lead to further Aβ accumulation. Representative Western blots are shown of lysates (left) and medium (right) from Swe N2a cells treated with 5 nM BafA1 6 h after transfection with mock (no plasmid), control plasmid ctrGFP, wtVPS4A or dnVPS4A. Cells were collected 24 h post-transfection, n = 3. d Western blot analysis of isolated exosomes secreted from Swe N2a cells transfected with mock (no plasmid), ctrGFP, wtVPS4 or dnVPS4 for 48 h. Representative blots demonstrate that exosome secretion is not reduced with dnVPS4. Instead, increased amounts of CD63, an exosomal marker postulated to be involved in ESCRT independent ILV formation, is seen in the exosomal fraction from dnVPS4 transfected cells. Flotillin-1, an exosomal marker associated with cholesterol-enriched lipid rafts is also increased in the exosomal fraction from dnVPS4 transfected cells, while the ESCRT-I protein and exosomal marker Tsg101 is not changed, n = 3