Fig 2: Treatment of 5XFAD mice with riluzole rescues gene expression changes in the hippocampus.a Venn diagram illustrating the overlap of 247 genes that were changed in 5XFAD compared to wild type and in 5XFAD mice treated with riluzole compare to untreated mice. b Scatter plot illustrating the 247 overlapping genes showing fold change by 5XFAD (x-axis) against fold change with riluzole treatment on 5XFAD mice (y-axis). 86% of overlapping 5XFAD genes is reversed by riluzole treatment. The upper left quadrant represents 26 genes that had decreased expression with 5XFAD and increased expression after riluzole treatment. Conversely, the lower-right quadrant illustrates 189 genes that were increased with 5XFAD and decreased by riluzole. c The mRNA expression of Mouse APP from RNA-Seq data revealed no significant difference among WT, 5XFAD, and 5XFAD-Riluzole groups. d Histograms illustrating significantly enriched pathways based on genes differentially expressed by overlapping gene. (enrichment score > 1.3). e, f Histograms illustrating significantly enriched pathways with the highest sum enrichment scores across opposite conditions (Increased in 5XFAD and Decreased with riluzole treatment or vice-versa) based on differentially expressed genes (enrichment score > 1.3). Similar pathways and enrichment scores were observed when comparing genes decreased by 5XFAD and increased by riluzole, as well as for genes increased with age and decreased by riluzole. The RNA-Seq data is based on hippocampal tissue pooled into three replicate sequencing libraries/group from WT, n = 10 (pooled 3, 3, 4 mice); 5XFAD, n = 7 (pooled 2, 2, 3 mice); and 5XFAD-Riluzole, n = 8 (pooled 2, 3, 3 mice)

Fig 3: Crystal structures of BACE1 ectodomain in complex with peptide inhibitors. The molecular surface of the BACE1 ectodomain is displayed in yellow, its flap region (amino acids 131–136) indicated pink. Peptide inhibitors are shown in sticks representation. (a) BACE1 in complex with the cholesterol conjugated Pep#3 (cyan). The cholesterol is not resolved in the electron density map. In order to visualize the mobility of the cholesterol lipid anchor several representative conformers are depicted (black). (b) BACE1 in complex with the exosite Pep#6 (green) and the active site inhibitor GRL-8234 (omitted for clarity). Superimposed in cyan is the APP derived active site inhibitor P10-P4' StatVal (Turner et al., 2005). (c) BACE1 in complex with the dual, active and exosite binding peptide inhibitor Pep#16. The functionalities of the peptide from N- to C-terminus are sortase recognition motif (grey), exosite binding (green), linker (magenta), active site binding (cyan) and lipid anchor attachment (orange).

Fig 4: Full effector antibody A16 significantly promotes microglial engulfment of synapses in APP/PS1 mice at 48 h post-treatment. a Representative images of the engulfed PSD95 (red) and Aβ (magenta) puncta within Iba-1+ (green) microglial cells in the brains of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. Scale bar, 3 μm. b Representative images of the engulfed PSD95 (red) and C1q (cyan) puncta within Iba-1+ (green) microglial cells in the brains of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. Scale bar, 3 μm. c Representative images of the engulfed PSD95 (red) and IgG (cyan) puncta within Iba-1+ (green) microglial cells in the brains of APP/PS1 mice treated with A16 or PBS, at 48 h post-injection. Scale bar, 3 μm. d Quantification of the colocalized PSD95 and Aβ puncta, and PSD95 and C1q puncta, per Iba-1+ microglial cell (n = 5 mice). e Quantification of PSD95, Aβ, and C1q puncta per Iba-1+ microglial cell (n = 5 mice). f Representative images of Iba-1 (green)-stained microglial cells in the brains of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. Scale bar, 10 μm. Skeleton analysis (g, h) and Sholl analysis (i) of microglial cells in f (n = 5 mice). Arrows indicate the engulfed inputs in microglia in (a–c). Data are expressed as mean ± s.e.m. (d, e, i) or shown as boxplots (g, h). For boxplots, the central band displays the median, the boxes depict values between lower and upper quartile, and the whiskers represent the minimum and maximum values. One-way ANOVA with Tukey’s test was performed to determine significance. *P < 0.05, ****P < 0.0001; ns not significant

Fig 5: Full effector antibody A16 reduces Aβ burden, but enhances inflammation in the brains of APP/PS1 mice at 48 h post-treatment. a Detection of Aβ plaques by 4G8 immunolabeling in the brains of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. Scale bar, 200 μm. b Quantification of 4G8-labeled area in a (n = 5 mice). Levels of insoluble Aβ42 (c), soluble Aβ42 (d), insoluble Aβ40 (e), and soluble Aβ40 (f) in brain lysates of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection (n = 5 mice). g Detection of microglia and astrocytes by immunolabeling Iba-1 and GFAP in the brains of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. Scale bar, 200 μm. h Quantification of Iba-1-labeled area in g (n = 5 mice). i Quantification of GFAP-labeled area in g (n = 5 mice). j Western blot analysis of Iba-1 and GFAP in the cortex and hippocampus of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection. k Quantitation of Iba-1 and GFAP expression in j (n = 5 mice). l The levels of IL-1β, IL-6, and TNF-α in the brain lysates of APP/PS1 or WT mice treated with A16, Fab16, isotype control antibody, or PBS, at 48 h post-injection (n = 5 mice). m The expression signatures of inflammation-related gene (CD68, Clec7a), complement-related gene (C1q, C3, CD11b), and inflammatory factor-related gene (IL-6, TNF-α, IL-1β, iNOS) in microglial cells of APP/PS1 mice were analyzed by qPCR (n = 6 mice). Data are expressed as mean ± s.e.m. and were analyzed by one-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns not significant