Fig. 7: Endolysosomal imbalance in hippocampal neurons deficient for PSEN2 or expressing the PSEN2 N141I mutant.

a Representative images of DIV14 hippocampal neurons immunostained for full length APP, APPCTF/Aβ (82e1) and LAMP1. Arrowheads in zoomed insets highlight co-localization. b Western blot of DIV14 neuronal lysates showing increased APP-CTF levels. c Quantification of a for triple overlap between LAMP1 and APP/82e1 positive puncta. d Quantification of ratio APP: APP-CTF ratio of blots from (b). e Representative images of immunolabeling of early (EEA1) and recycling endosomes (VPS35), and LE/Lys (LAMP1). White arrowheads in zoomed insets show overlap between three compartments. f–h Quantification of total area of (f) LAMP1, (g) EEA1 and (h) VPS35 as percentage of total neuronal soma area. i Representative images of lysosomal Ca²⁺ measured through feeding cells with dextran coupled Alexa488 and Ca²⁺ sensitive Rhodamine-coupled dextran, and quantified in (j) as a Rhodamine/Alexa488 ratio. k LAMP1 surface labelling combined with Phalloidin staining, quantified in (l). m Schematic of the dual labelled mCherry-GFP-LC3: in autophagosomes both mCherry and GFP give yellow fluorescence; whereas in lysosomes, GFP is quenched resulting in red fluorescence. Created in BioRender. Vrancx, C. (2024) https://BioRender.com/j26q595. n Representative images of DIV14 hippocampal neurons transfected with mCherry-GFP-LC3, quantified in (o) as total number of mCherry+ LC3 puncta normalized to APPKI neurons and in (p) as total number of mCherry + /GFP + LC3 puncta. Graphs (c–f–g–h–j–l–o–p) were statistically analyzed using one-way ANOVA with Tukey’s correction for multiple testing compared to APPKI. All graphs are represented as mean ± SEM, with triangles and circles representing the average per neuronal culture and individual cells, respectively. Scale bars are indicated in the figure.