Fig 2: Cross-correlations between HCS and GWAS data. a Mean variations in mCherry fluorescence intensity after the silencing of genes associated with the AD risk in the IGAP’s meta-analysis. Eight genes (in red) were included in the best 5% variations, based on the HCS data. b Impact of the silencing of the 8 hits on the Aß1-X secretion level in the medium of the HEK293-mCherry-APP-YFP cell line. Histograms indicate mean ± SD. *p < 0.05, non-parametric test. c Validation of FERMT2 silencing after transfection with the siRNA-FERMT2 SMARTpool used in HCS. d Representative fluorescence microscopy images and quantification showing the impact of FERMT2 silencing on mCherry and YFP intensity based on HCS data. Scale bar 10 µm

Fig 3: FERMT2 silencing inhibits APP degradation and promotes APP recycling at the plasma membrane. a HEK293-APP695WT cells transiently transfected with siFERMT2 or non-targeting siRNA were treated with bafilomycin A1 (BafA1, 50 nM) for the indicated times. Cell extracts were then analyzed by WB. Densitometric analyses and mature APP levels for three independent experiments are shown. Graphs indicate the mean ± SD. *p < 0.05, non-parametric test. b The time course of APP endocytosis and degradation was indirectly visualized by internalization of 6E10 antibody. Cells were incubated with 6E10 antibody at 4 °C for 1 h. The temperature was then shifted to 37 °C, and cells were processed for immunofluorescence at the indicated times. Scale bar 10 µm. c Relative fluorescence intensity from 6E10 staining, showing the time course of APP degradation. d A zoom-in (the square in b) for the indicated times (0 and 20 min at 37 °C). Co-staining with anti-Rab4 antibody was used to visualize the APP within Rab4-positive endosomes involving in recycling. e Co-localization of 6E10 staining with Rab4 staining, as a guide to the APP level within recycling endosomes at the indicated times. f Cells transiently transfected with anti-FERMT2 in the presence or absence of siRab4. Extracts were analyzed by WB using anti-APP C-terminal, anti-FERMT2, anti-Rab4 or anti-actin antibodies

Fig 4: Characterization of the impact of FERMT2 on APP metabolism. a Impact of FERMT2 silencing on APP metabolism in the HEK293-APP695WT cell line. Cells transiently transfected with siFERMT2 or non-targeting siRNA were analyzed by WB using anti-APP C-terminal, anti-FERMT2 or anti-actin antibodies. sAPPa, sAPPß and Aß1-X secreted into conditioned medium were assayed using an AlphaLISA. ma. APP, mature APP; im. APP, immature APP. Densitometric analyses and WB quantifications from three independent experiments are shown. Histograms indicate the mean ± SD. a.u., arbitrary units. *p < 0.05, non-parametric test. b Impact of FERMT2 silencing on the mature APP levels in HEK293 cells endogenously expressing APP. c Quantification of mature and immature APP levels after lentiviral transduction with shRNA against FERMT2 in a primary neuronal culture endogenously expressing APP