Figure 4: Neu1 deficiency and consequent exacerbated lysosomal exocytosis promotes Aβ peptide release.

(a) Neurospheres isolated from WT^Arf and Neu1^−/−/Arf brains were maintained in culture in the presence of exogenous T-Aβ42 for 24 h and then imaged with confocal microscopy. T-Aβ42 fluorescence (red fluorescence of TAMRA-Aβ42) co-localized with lysotracker (green), marker of endosomes/lysosomes. (b) TIRF analysis shows that Neu1^−/−/Arf hippocampal neurospheres have enhanced T-Aβ levels in PM-docked lysosomes (lysostracker green). (c) Quantification of PM-docked lysosomes shows an increased number of clustered organelles at the PM of Neu1^−/−/Arf cells. (d) Quantification of T-Aβ levels present in PM-docked lysosomes shows higher amounts of T-Aβ in Neu1^−/−/Arf cells than in WT samples. (e) TAMRA fluorescence was assayed in neurospheres’ culture medium as an indicator of the levels of T-Aβ released by lysosomal exocytosis. (f,g) Extent of lysosomal exocytosis measured as β-hex enzyme activity and human Aβ-42 assayed in the culture medium of WT^Arf and Neu1^−/−/Arf neurospheres treated with the calcium ionophore calcimycin in the presence or absence of EGTA, which effectively blocks the lysosomal exocytosis. Asterisks on graphic bars indicate statistically significant results, as determined by the Student’s t-test. Data are represented as the mean±s.d. (error bars); n=3. (a,b) Scale bars, 20 μm.