Figure 5  Aβ ELISA Confirms Arrest of Progression without Clearance of Peptide in Mice with Preexisting Aggregates
Aβ levels in untreated 6- and 9-mo-old tTA/APP line 107 mice (shown in Figure 4) were compared to those in 9- and 12-mo-old animals treated with dox from the age of 6 mo. Single transgenic APP samples were included as negative controls. Cortical homogenates were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black.
(A and B) Most Aβ in the brains of plaque-bearing mice is extracted into the FA and SDS fractions. Consistent with amyloid burden (Figures 4 and Figure S3), SDS- and FA-extracted Aβ levels in untreated 9-mo-old mice were significantly higher than in untreated 6-mo-old mice (Tukey post-hoc test applied to significant effect of group ANOVA for SDS and FA fractions F 3,18 = 4.72–12.92, p < 0.02). In contrast, 3 or 6 mo of transgene suppression held Aβ at levels equivalent to those harbored when treatment was started (p > 0.2 compared to 6 mo untreated mice, Tukey post-hoc test). *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test. Significance for APP versus 9-mo-old untreated mice is based on Student's t-test.
(C) The PBS fraction represents less than 0.1% of total Aβ (note the change in y-axis from [A] and [B]), but only here do Aβ levels in the dox-treated mice differ from those in younger untreated mice. Although both peptides appear elevated in the treated groups compared to the untreated 6-mo-old mice, only Aβ40 reaches statistical significance (p < 0.05, Tukey post-hoc test applied to significant effect of group ANOVA for Aβ40 F 3,18 = 4.60, p < 0.02). A similar trend was seen for Aβ42, where ANOVA yielded a significant effect of group for PBS-soluble Aβ42 (F 3,18 = 3.75, p < 0.03), however this was due only to differences between the untreated 6- and 9-mo-old groups. •, p < 0.05 versus 6-mo-old untreated mice, Tukey post-hoc test; •••, p < 0.001 versus 6-mo-old untreated mice, Student's t-test.