Figure 1.

A genome-wide RNAi screen in cultured Drosophila cells for modifiers of Aβ secretion. A, Tris–bicine urea SDS-PAGE immunoblot of conditioned media from Drosophila S2 cells expressing pAc5:SP-C99. Standard peptides confirmed that Aβ species corresponding to Aβ₃₈, Aβ₄₀, and Aβ₄₂ were generated. Aβ species were generated in a similar ratio to human cells, with Aβ₄₀ the predominant species and Aβ₃₈ and Aβ₄₂ minor species. B, S2 cells after treatment with dsRNAs against GFP, Drosophila Psn, and Kuz. SDS-PAGE immunoblot demonstrated that RNAi of Psn abolished Aβ generation, whereas RNAi of Kuz increased it (left). Both CTFs accumulate after Psn knockdown, whereas alphaCTF is specifically reduced after Kuz knockdown. An Aβ immunoassay of conditioned media confirmed the Aβ effects (right). MW, Molecular weight. C, Graph of mean Aβ₄₀ z-scores plotted against mean fLuc z-scores from the genome-wide RNAi screen. Each dot represents a single gene. Multiple controls were included on every plate: negative controls, GFP (light blue) and human BACE1 (dark blue); positive controls, Psn (dark red), Kuz (green), βCOP (β-coatomer protein) (orange), and DIAP (red). βCOP is required for general secretion and viability and DIAP (Drosophila inhibitor of apoptosis protein) is a gene essential for viability. The negative controls GFP and human BACE1 varied around a z-score of 0 for both Aβ₄₀ and fLuc. Psn controls showed very high Aβ₄₀ z-scores, corresponding to strong reductions in Aβ secretion but no effect on fLuc, with a z-score for fLuc varying at ∼0. The dashed lines indicate the significance thresholds: genes were considered as hits if the Aβ₄₀ z-score was >2 or less than −2 and fLuc z-score was between −1.5 and 1.5. The vast majority (∼99%) of Drosophila genes (Probe; gray dots) varied around a z-score of 0 for both Aβ₄₀ and fLuc, in a similar range to the negative controls. However, some showed specific increases in Aβ (in the direction of Kuz) or specific reductions in Aβ (in the direction of Psn). The positions of the four γ-secretase complex components (Psn, Nct, Aph-1, and Pen-2) and Paf-AHα are labeled. D, S2 cells after treatment with dsRNAs against GFP or Paf-AHα. In the media, Aβ was reduced by 50%. This was a specific effect because there was no change in the secretion of a Gaussia luciferase secretion reporter (sGLuc) or in cellular fLuc activity. E, Protein sequence homology between Drosophila Paf-AHα and human PAFAH1B2 demonstrates a highly conserved protein. Green boxes highlight the 46% of amino acids that are identical, and the red box highlights the active serine (48) in the human protein. Bar graphs show mean and SEM of six individual experiments (**p < 0.01, ***p < 0.001, unpaired Student's t test).