Extended Data Fig. 9. AAV-mediated liver expression of apoE4 enhances amyloid pathology and related toxicity.

a, Schematic illustration of the experimental paradigm. 5xFAD amyloid mice at 1-1.5 month of age were transduced with AAV-Alb-apoE3 or AAV-Alb-apoE4 virus via intravenous injection. b, c, The amyloid deposition in the brain of experimental mice at 4 months of age was examined by immunostaining for Aβ. Scale bar, 1 mm. The amyloid plaque burdens in the cortex and hippocampus (E3, n=14; E4, n=16) were quantified. **, P = 0.007. d, e, ApoE levels in the plasma and brain of experimental mice (E3, n=15; E4, n=16) were measured by ELISAs. **, P = 0.0098. f, TBSX-soluble and -insoluble (guanidine; GDN) Aβ40 and Aβ42 levels in the cortex of 4-month-old 5xFAD mice transduced with AAV-Alb-apoE3 (n=15) or AAV-Alb-apoE4 (n=16) were examined by specific Aβ ELISA. TBSX-Aβ40 (*, P = 0.012); TBSX-Aβ42 (*, P = 0.031). GDN-Aβ40 (*, P = 0.049); GDN-Aβ42 (*, P = 0.033). g, Thio S-positive plaques in the cortex of experimental mice were shown and quantified. Scale bar, 100 μm. h, i, Brain sections from experimental mice (n=12/genotype) were immunostained with GFAP or Iba1 antibody. Scale bar, 100 μm. The immunoreactivity of GFAP and Iba1 in cortex and hippocampus were quantified. j, Representative images of plaque-associated microglia in mice expressing apoE3 or apoE4 in the liver are shown. Scale bar, 50 μm. The number of Iba1-positive microglia (green) surrounding Aβ plaque (red) between 50-300 μm² plaque sizes were quantified. Each dot represents the average value from an individual mouse (E3, n=10; E4, n=11). *, P = 0.049. k, Co-immunofluorescence staining of LAMP1 (green) and Aβ plaques (red) was used to examine plaque-associated neuritic dystrophy. Scale bar, 50 μm. The LAMP1 immunoreactivity was quantified. *, P = 0.027. l, LAMP1 immunoreactivity was positively correlated with Thio S-positive fibrillar plaques. c-l, Data represent mean ± s.e.m., two-tailed Student's t-test.