Fig. 6: (R)−4-HNE-adducted APP retrogrades to the Golgi apparatus aggravating Aβ40 generation.

a Representative high-magnification single-plane confocal images of HEK293T cells stained with C1/6.1 (red, APP) and RCAS1 (green, Golgi apparatus). HEK293T cells were pretreated with PBS or 2 μM (R)−4-HNE for 24 h. Scale bar, 5 μm. b and c Golgi apparatus was isolated from HEK293T cells pretreated with PBS or (R)−4-HNE by using a Golgi Apparatus Enrichment Kit (Invent, GO-037). b WB of APP and γ-secretase components, and c levels of Aβ40 and Aβ42 in PBS- or (R)−4-HNE-treated Golgi apparatus lysates. n = 3 biologically independent samples. d Scheme of SORL1 as a molecular link for retromer-dependent sorting of APP^34,35,36. Image was created using elements from ChemBioDraw Ultra software 14.0. e Immunoprecipitation of SORL1 and natural or 4-HNE modified APP, without or with 4-HNE treatment. Two independent biological replicates were performed. f The calculated protein-protein docking results of APP770 and SORL1 domains (YWTDs–CR cluster–FN repeats) by Rosetta software. The structures of APP770 and SORL1 are predicted by AlphaFold downloaded from UniProt (for APP770: AF-P05067-F1, for SORL1: AF-Q92673-F1). The CR cluster of SORL1 contains 11 complement-type repeat domains and is indicated as orange (CR 1−4), purple (CR 5-8), and blue (CR 9-11). g Relative mRNA and protein levels of VPS35 after ALDH2 knockdown by RNA silencing in HEK293T cells. n = 3 biologically independent samples. h–k VPS35 levels were suppressed by RNA silencing in HEK293T cells, and cells were then treated with PBS or 2 μM (R)−4-HNE for 24 h. h Verification of VPS35 knockdown by RT-qPCR and WB. n = 3 biologically independent samples. i Double staining with C1/6.1 (red, APP) and RCAS1 (green, Golgi apparatus) antibodies in the above cells. Scale bar, 10 μm. j WB of APP and PS1 in isolated Golgi apparatus lysates. k Levels of Aβ40 and Aβ42 in Golgi apparatus lysates. siNC, negative control small interfering RNA. n = 3 biologically independent samples. Data are presented as mean values ± SD. Statistical analysis was performed using a two-tailed Student’s t-test (c, g, h, k). Source data are provided as a Source Data file.