NEP effect on premature mortality in hAPP-J20 mice depends on the genetic background strain. Kaplan-Meier cumulative survival plots (top panels) and cumulative hazard plots (bottom panels) demonstrate premature mortality in hAPP-J20 and hAPP-J20/NEP mice, compared with NTG and NEP mice, on the C57BL/6J (A) and B6D2 (B) backgrounds (p < 0.001, Logrank test). Premature mortality was significantly greater in hAPP-J20/NEP mice than in hAPP-J20 mice on the C57BL/6J background (p < 0.01, Logrank test), but not on the B6D2 background. The incidence of premature mortality in hAPP-J20/NEP mice was also greater in the C57BL/6J background than in the B6D2 background (p < 0.01, Logrank test). Number of mice observed (C57BL/6J vs B6D2): NTG (142, 112), hAPP-J20 (102, 84), NEP (150, 95), and hAPP-J20/NEP (82, 66).

NEP overexpression has subtle effects on calcium- and synaptic activity-dependent markers in the dentate gyrus of hAPP mice. Horizontal (A, B) or coronal (C, D) brain sections were obtained from C57BL/6J mice of the indicated genotypes and numbers at 2–4 (A, C) or 5–6 (B, D) months of age and immunostained for calbindin (A, B) or Fos (C, D). A, B, For calbindin, a two-way ANOVA revealed a significant hAPP effect (A: F_(1,46) = 44.6, p < 0.001; B: F_(1,94) = 20.1, p < 0.001) and an hAPP x NEP interaction (A: F_(1,46) = 4.95, p < 0.05; B: F_(1,94) = 5.85, p < 0.05) for both ages. C, D, For Fos, a two-way ANOVA revealed only an hAPP effect (C: F_(1,22) = 16.8, p < 0.001; D: F_(1,55) = 47.4, p < 0.001), but no hAPP x NEP interaction. *p < 0.05 vs NTG; **p < 0.01, ***p < 0.001 vs NTG and NEP (ANOVA followed by Fisher’s PLSD posthoc test). Values are means ± SEM.

Overexpression of NEP prevents hyperactivity but does not alter elevated plus maze abnormalities in hAPP-J20 mice. Total activity in the periphery of an open field (A, B) and the proportional distance traveled in the open arms of an elevated plus maze (C, D) were compared among C57BL/6J (A, C) and B6D2 (B, D) mice of the indicated genotypes at 3–4 months of age. A two-way ANOVA revealed an hAPP effect (A: F_(1,98) = 13.0, p < 0.001; B: F_(1,116) = 12.9, p < 0.001; C: F_(1,98) = 46.5, p < 0.001; D: F_(1,116) = 10.8, p < 0.01) and an hAPP x NEP interaction (A: F_(1,98) = 8.3, p < 0.01; B: F_(1,116) = 5.22, p < 0.05). **p < 0.01 vs NTG mice, ***p < 0.001 vs all other groups; ⁺⁺⁺p < 0.001 vs NTG and NEP mice (Fisher’s PLSD test). Values are means ± SEM.

Overexpression of NEP does not reduce levels of Aβ*56 or Aβ trimers. Different Aβ assemblies were measured in 5–6-month-old hAPP_FAD TG C57BL/6J mice from line J20 and from the lower expresser line J9. A, Representative western blot of membrane-enriched fractions from combined hippocampal and cortex-plus lysates probed with the anti-Aβ antibody 6E10, which recognizes Aβ*56 and hAPP, as indicated on right. B, Aβ trimers in soluble fractions of combined hippocampal and cortex-plus lysates were immunoprecipitated with the anti-Aβ antibody 4G8 and detected by western blotting with the anti-Aβ antibody 3D6. C, D, Relative levels of Aβ*56 (C) and Aβ trimers (D) were determined by densitometric analysis of western blot signals. E, Representative western blot of TBS and guanidine fractions isolated from hemibrains of 3–17-month-old hAPP-J20 mice. Aβ monomers and dimers were immunoprecipitated with the 6E10 antibody and detected by western blotting with the 3D6 antibody. Note that dimers were detected only in 17-month-old mice. Similar results were obtained with TBST fractions (data not shown). **p < 0.01, ***p < 0.001 vs hAPP-J20 and hAPP-J20/NEP mice (Tukey’s test). Values are means ± SEM.

Overexpression of NEP in hAPP-J20 mice does not prevent learning and memory deficits in the Morris water maze. C57BL/6J (A, C, E, G, I) and B6D2 (B, D, F, H, J) mice (n = 9–11 mice per genotype and background) were tested at 3–5 months of age. These groups of mice had no significant differences in swim speeds during the hidden platform training (data not shown). A, B, Compared with NTG and NEP mice, hAPP-J20 and hAPP-J20/NEP mice had significant and comparable learning impairments. In both strain backgrounds, a two-way repeated measures ANOVA revealed a significant main effect of hAPP on hidden platform performance (F_(1,36) ≥ 40.4; p < 0.001), but no NEP effect or hAPP x NEP interaction. A significant main effect of hAPP (F_(1,36) = 11.0; p < 0.01) was found in the visible platform task only on the C57BL/6J background (A). C–J, Probe trials at the beginning of the fourth day of training (C–F) and 16–18h after the last day of hidden platform training (G–J) revealed comparable impairments in hAPP-J20 and hAPP-J20/NEP mice relative to NTG and NEP mice. Target, adjacent (Adj) left (L) or right (R), and opposite (Opp) refer to quadrants (C, D, G, and H) or to the target platform and corresponding locations in the respective non-target quadrants (E, F, I and J). A two-way ANOVA identified a significant main effect of hAPP for the percent time spent in the target quadrant (C, D, H: F_(1,36) ≥ 9.7, p < 0.01; G: F_(1,36) = 6.3, p < 0.05) and for the number of target crossings (E, F, I: F_(1,36) ≥ 17.7, p < 0.001; J: F_(1,36) = 7.0, p < 0.05). There was no NEP effect or hAPP x NEP interaction. *p < 0.05, **p < 0.01, ***p < 0.001 vs NTG controls; ⁺p < 0.05 vs NTG and NEP controls (Fisher’s PLSD test). Values are means ± SEM.

NEP overexpression reduces Aβ levels and prevents early plaque deposition without altering levels of C-terminal APP fragments. A,B Neprilysin overexpression in NEP and hAPP-J20/NEP mice was confirmed by western blot analysis of membrane fractions isolated from cortex-plus tissues (see Methods) of 2–4-month-old mice. Tubulin was used as a loading control. A, Representative western blot. B, Densitometric quantitation of western blot signals revealed that NEP levels were 11-fold higher in NEP and hAPP-J20/NEP mice than in NTG controls and that endogenous NEP levels in hAPP-J20 mice and NTG controls were similar. Numbers in bars represent numbers of mice analyzed per group. C, Particulate membrane fractions were isolated from cortex-plus tissues of 5–6-month-old hAPP-J20 and hAPP-J20/NEP mice and analyzed by western blotting with an antibody (CT-15) against hAPP C-terminal fragments (CTFs). Tubulin was used as a loading control. Labels on right identify bands putatively representing phosphorylated or unphosphorylated C83, C89, or C99. The rightmost lane contains a sample from a TG mouse of line I5, which expresses wildtype hAPP (Mucke et al., 2000); it illustrates that this assay was sensitive enough to detect the effect of the Swedish mutation in hAPP-J20 on CTF production. D, Aβ levels in hippocampal (Hip) and cortex-plus (Ctx) tissues of 2–4-month-old hAPP-J20 and hAPP-J20/NEP mice without plaques were determined by ELISA. E,F Levels of Aβ monomers in 6–7-month-old hAPP-J20 and hAPP-J20/NEP mice were compared by western blot analysis of TBST fractions isolated from whole hemibrains. E, Representative western blot. F, Densitometric quantitation of western blot signals. G,H Aβ deposits in hippocampal sections of 4–6-month-old hAPP-J20 and hAPP-J20/NEP mice were detected by immunostaining with the 3D6 antibody. I,J, Quantitation of percent area occupied by Aβ-immunoreactive deposits (I) and number of deposits (J). *p < 0.05, **p < 0.01, ***p < 0.001 vs hAPP mice (Student’s t test). ###p < 0.001 vs hAPP and NTG mice (Tukey’s test). Values are mean ± SEM from mice on the C57BL/6J background.