ApoE4 Accelerates Early Seeding of Amyloid Pathology Chia-Chen Liu, Na Zhao, Yuan Fu, Na Wang, Cynthia Linares, Chih-Wei Tsai, Guojun Bu  Neuron  Volume 96, Issue 5, Pages 1024-1032.e3 (December 2017) DOI: 10.1016/j.neuron.2017.11.013 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of Cell-Type-Specific and Inducible ApoE Mice (A) The structure of the ROSA-targeting vector with the Tet-off regulation cassette for APOE and eGFP expression. The resultant mice were named iE3 and iE4. Breeding iE3 or iE4 mice with GFAP-Cre mice, which removed the loxP-flanked Neor gene, led to expression of apoE3 or apoE4 in astrocytes. (B and C) ApoE levels in the cortex of iE3 and iE4 mice at 3 months of age with or without GFAP-Cre were analyzed by western blotting (B) and ELISA (C). (D) Immunohistochemical analysis of GFP for the cortical region of iE3 mice in the presence or absence of Cre. (E) The iE3 mice at 3 months of age were fed with regular (−Dox) or Dox-containing chow for 2 weeks. ApoE in the cortex was analyzed by ELISA. Data represent mean ± SEM. ∗∗p < 0.01; N.S., not significant. (F) Comparison of apoE levels in the cortex of apoE inducible amyloid model mice with apoE-TR mice at 3–4 months of age examined by ELISA. Data represent mean ± SEM. ∗∗p < 0.01. (G) Cortex and hippocampus (HPC) from apoE3 inducible (Cre+) amyloid model mice were co-immunostained for GFP, which represents apoE distribution (green) and astrocyte-specific (anti-GFAP; red) or neuron-specific (anti-NeuN; red) markers. Scale bar, 100 μm. See also Figure S1. Neuron 2017 96, 1024-1032.e3DOI: (10.1016/j.neuron.2017.11.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Astrocytic ApoE4 Increases Insoluble Aβ and Amyloid Pathology When Expressed Early (A) Illustration of apoE induction paradigms at different stages of amyloid pathology. (B and C) Insoluble apoE levels in the cortex of APP/PS1 mice expressing (B) apoE3 (APP/iE3; n = 17–18/group) throughout 9 months (0–9 m On) or (C) apoE4 (APP/iE4; n = 13–16/group) throughout 9 months, during 0–6 months (0–6 m On; n = 6–8/group), or during 6–9 months (6–9 m On; n = 8–9/group) were examined by ELISA. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01. (D–F) Insoluble Aβ40 and Aβ42 levels in the cortex of APP/PS1 mice expressing apoE4 (APP/iE4; n = 13–16/group) throughout the entire 9 months (D), during 0–6 months (n = 6–8/group) (E), or during 6–9 months (n = 8–9/group) (F) were examined by specific Aβ ELISA. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; N.S., not significant. (G–J) Brain sections from 9-month-old APP/PS1 mice expressing apoE3 (APP/iE3; n = 12–14/group) (G), or apoE4 (APP/iE4) throughout the entire 9 months (n = 11/group) (H), during 0–6 months (n = 9/group) (I), or during 6–9 months (n = 7–8/group) (J) were immunostained with a pan-Aβ antibody. Representative images of Aβ staining in the cortical and hippocampal regions are shown. Scale bar, 1 mm. Open circles are females; closed circles are males. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; N.S., not significant. See also Figures S1–S3. Neuron 2017 96, 1024-1032.e3DOI: (10.1016/j.neuron.2017.11.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Induced ApoE4 Expression in Astrocytes Impairs ISF Aβ Clearance in the Hippocampus of APP/PS1 Mice The ISF Aβ40 levels in APP/PS1 mice expressing apoE3 (A and B; APP/iE3; n = 8/group) or apoE4 (C and D; APP/iE4; n = 4/group) at the age of 3–4 months were analyzed. To assess Aβ40 half-life, the mice were treated with a γ-secretase inhibitor, and the hippocampal ISF Aβ40 levels were monitored. The slope from the individual linear regressions from log (percent [%] ISF Aβ40) versus time for each mouse was used to calculate the mean half-life (t1/2) of elimination for Aβ from the ISF (B and D). Data represent mean ± SEM. ∗p < 0.05; N.S., not significant. Neuron 2017 96, 1024-1032.e3DOI: (10.1016/j.neuron.2017.11.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 ApoE Isoform-Specific Effects on Aβ-Associated Gliosis (A–D) Brain sections from 9-month-old APP/PS1 mice expressing apoE3 (APP/iE3) or apoE4 (APP/iE4) in the astrocytes throughout the entire 9 months were immunostained with GFAP antibody (A and B) or Iba1 antibody (C and D). Scale bar, 100 μm. The immunoreactivities of GFAP or Iba1 in cortex (n = 11–13/group) and hippocampus (n = 9–12/group) were quantified. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; N.S., not significant. (E and F) The levels of GFAP, presynaptic marker synaptophysin (Syp), and postsynaptic marker PSD-95 in the cortex (n = 16–18/group) of APP/PS1 mice expressing apoE3 (APP/iE3) or apoE4 (APP/iE4) in the astrocytes throughout the entire 9 months were examined by western blotting. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; N.S., not significant. See also Figure S4. Neuron 2017 96, 1024-1032.e3DOI: (10.1016/j.neuron.2017.11.013) Copyright © 2017 Elsevier Inc. Terms and Conditions