Onset of radial-arm water maze deficit occurs between 16 and 18 months for outcrossed AβPP/PS1 mice and as early as 8 months in inbred C57BL/6 AβPP/PS1 mice. (A) Diagram of radial-arm water maze. Maze contained six arms with large visual cues positioned on the walls of the room adjacent to the distal end of each arm. A hidden platform (HP) was submerged at the end of one arm. (B) Controls assessing escape motivation, swimming ability, and vision across trials in outcrossed WT and AβPP/PS1 mice at 12, 16, and 18 months and inbred C57BL/6 WT and AβPP/PS1 mice at 8 months. Symbols represent mean seconds per excursion during first five trials ± SE. Two-factor ANOVAs (genotype × group): no interaction, F < 1. Errors made across trials by WT and AβPP/PS1 mice at (C-E) 12, 16, and 18 months in outcrossed mice and (F) 8 months in inbred C57BL/6 mice. Symbols represent mean errors made per trial during groups of five trials ± SE. Two-factor ANOVAs (genotype × trial) with repeated measures (trial): 12 months outcrossed: main effect of trial, F_(5,27) = 22.08, p < 0.001; 16 months outcrossed: main effect of trial, F_(5,10) = 15.63, p < 0.001; 18 months outcrossed: interaction, F_(5,14) = 4.35, p < 0.01; 8 months inbred C57BL/6: interaction, F_(5,19) = 4.63, p < 0.001. Post hoc Student’s t-tests, *p < 0.05, ***p < 0.001. Excursion time and water maze analyses: 12 months outcrossed: WT, n = 15 mice; AβPP/PS1, n = 14 mice; 16 months outcrossed: WT, n = 5 mice; AβPP/PS1, n = 7 mice; 18 months outcrossed: WT, n= 8 mice; AβPP/PS1, n = 8 mice; 8 months inbred C57BL/6: WT, n = 11 mice; AβPP/PS1, n = 10 mice. (G-H) Performance in radial-arm water maze reversal does not differ between WT and AβPP/PS1 mice at 12 months. (G) Total errors and (H) perseverant errors made per trial by WT and AβPP/PS1 mice during reversal task at 12 months. Symbols represent mean errors made per trial during groups of four trials ± SE. Two-factor ANOVAs (genotype × trial) with repeated measures (trial): total errors: main effect of trial, F_(2,27) = 29.36, p < 0.001; perseverant errors: main effect of trial, F_(2,27) = 15.09, p < 0.001. Water maze reversal analyses: 12 months: WT, n = 15 mice; AβPP/PS1, n = 14 mice.

Novel object recognition does not differ between outcrossed WT and AβPP/PS1 mice at 18 months while inbred C57BL/6 AβPP/PS1 mice have defects in this task at 8 months. (A) Diagram of object placement during sample and choice phases. L = left object, R = right object, F = familiar object, N = novel object. Object exploration times by outcrossed WT and AβPP/PS1 mice at 18 months during the (B) sample phase and (C) choice phase. Bars represent mean seconds spent exploring object ± SE. Two-factor ANOVAs (genotype × object): sample phase: no interaction, F < 1; choice phase: interaction, F_(1,14) = 6.05, p < 0.05. Post hoc Student’s t-tests, **p < 0.01, ***p < 0.001. Object exploration times by inbred C57BL/6 WT and AβPP/PS1 mice at 8 months during the (D) sample phase and (E) choice phase. Bars represent mean seconds spent exploring object ± SE. Two-factor ANOVAs (genotype × object): sample phase: no interaction, F < 1; choice phase: interaction, F_(1,19) = 3.89, p < 0.05. Post hoc Student’s t-tests, ***p < 0.001. Novel object recognition analyses: outcrossed mice: WT, n= 8 mice; AβPP/PS1, n = 8 mice; inbred C57BL/6: WT, n = 11 mice; AβPP/PS1, n = 10 mice.

Amyloid plaque burden increases significantly with age in outcrossed and inbred C57BL/6 AβPP/PS1 mouse hippocampus and is highest in 12-month inbred C57BL/6 mice. (A) Diagram of mouse hippocampus showing regions scored for plaque deposition, adapted from (. CA1 = CA1 region, CA3 = CA3 region, Rad = stratum radiatum, Or = stratum oriens, DG = dentate gyrus, Mol = molecular layer. Scale bar = 200 µm. (B) Plaque burden in the AβPP/PS1 hippocampus in outcrossed mice at 12 and 18 months and in inbred C57BL/6 mice at 8 and 12 months. Bars represent means ± SE. Kruskal-Wallis test: plaque burden: p < 0.01. Post hoc Mann-Whitney, *p < 0.05, **p < 0.01. Plaque deposition analyses: 12 months outcrossed, n = 5 mice (6 sections per mouse); 18 months outcrossed, n = 8 mice (6 sections per mouse); 8 months inbred C57BL/6, n = 3 mice (6 sections per mouse); 12 months inbred C57BL/6, n = 6 mice (4–6 sections per mouse). (C-F) Representative fluorescent images of Thioflavin S stained sections from the AβPP/PS1 hippocampus at (C) 12 months in outcrossed mice, (D) 18 months in outcrossed mice, (E) 8 months in inbred C57BL/6 mice, and (F) 12 months in inbred C57BL/6 mice. Bright puncta indicate Thioflavin S -reactive amyloid deposits. Scale bars = 200 µm.

Amyloid plaque burden increases significantly with age in outcrossed and inbred C57BL/6 mouse temporal cortex and is higher in inbred C57BL/6 mice than outcrossed mice at 12 months. (A) Diagram of mouse cortex showing region scored for plaque deposition, adapted from (). TeA = temporal cortex, Ent = entorhinal cortex. Scale bar = 200 µm. (B) Plaque burden in the temporal cortex in 12- and 18-month outcrossed AβPP/PS1 mice and 8- and 12-month inbred C57BL/6 mice. Bars represent means ± SE. Kruskal-Wallis test: plaque burden: p < 0.01. Post hoc Mann-Whitney tests of plaque burden, *p < 0.05**, p < 0.01. Plaque deposition analyses: 12 months outcrossed, n = 5 mice (6 sections per mouse); 18 months outcrossed, n = 8 mice (6 sections per mouse); 8 months inbred C57BL/6, n = 3 mice (6 sections per mouse); 12 months inbred C57BL/6, n = 6 mice (4–6 sections per mouse). (C-F) Representative fluorescent images of Thioflavin S -stained sections from the AβPP/PS1 cortex at (C) 12 months in outcrossed mice, (D) 18 months in outcrossed mice, (E) 8 months in inbred C57BL/6 mice, and (F) 12 months in inbred C57BL/6 mice. Bright puncta indicate Thioflavin S -reactive amyloid deposits. Scale bars = 200 µm.

Measurements of animal survival, PS1 levels, AβPP levels, and AβPP processing. (A) Kaplan-Meier curve showing cumulative survival percentage for outcrossed WT and AβPP/PS1 mice up to 18 months. Symbols represent cumulative percent of animals surviving to each month. Mantel-Haenszel log-rank test: p = 0.61. Animal survival analysis: WT, n = 83 mice (9 deaths total); AβPP/PS1, n = 81 mice (11 deaths total). Immunoblots of (B) full length (FL) AβPP, (C) soluble AβPPα (sAβPPα), (D) AβPP β-C-terminal fragment (β-CTF) and presenilin-1 (PS1), (E) soluble Aβ (sAβ), and (F) insoluble Aβ levels in outcrossed WT and AβPP/PS1 mice on outcrossed at 12 and 18 months and inbred C57BL/6WT and AβPP/PS1 at 8 and 12 months. HSP70 and actin levels are shown as loading controls. Numbers on left of immunoblots represent approximate molecular weights (kDa). (G) Quantification of non-amyloidogenic pathway including full length AβPP and sAβPPα levels normalized to respective loading controls. Bars represent means ± SE. Kruskal-Wallis test: Full length AβPP: p < 0.001; sAβPPα: no significance, p > 0.05. Post hoc Mann-Whitney tests, **p < 0.01, ***p < 0.001. Full length AβPP analysis: n = 3 mice/group. sAβPPα analysis: outcrossed mice: 12M, n= 9 mice; 18M, n = 3 mice; inbred C57BL/6: 8M, n = 3 mice; 12M, n = 16 mice. (H) Quantification of amyloidogenic pathway, including β-CTF, PS1, sAβ, and insoluble Aβ normalized to respective loading controls, when possible. Bars represent means ± SE. Kruskal-Wallis test: PS1: no significance, p > 0.05; β-CTF: no significance, p > 0.05; sAβ: p < 0.05; insoluble Aβ: p < 0.05. Post hoc Mann-Whitney tests, *p < 0.05, **p < 0.01. PS1, β-CTF, sAβ, and insoluble Aβ analysis: n = 3 mice/group.