Vaccination had no significant effect on the levels of APP or CTFs.

<p>(A) Western blot showing bands for APP and CTFs in brain samples from 2N and Ts65Dn mice. Tubulin was used as internal reference. The lanes are: 2N-vehicle (2, 6, 10, 13); Ts65Dn-vehicle (4, 8); 2N-DS-01 (1, 5, 9, 12, 15); Ts65Dn-DS-01 (3, 7, 11, 14). (B) Quantification of APP showed a higher level in Ts65Dn mice (although here it reached only borderline significance, <i>p</i> = 0.07). Following treatment with DS-01, no significant difference was observed in APP relative to the vehicle for either genotype (2N, vehicle vs DS-01, <i>p</i> = 0.9; Ts65Dn; vehicle vs DS-01, <i>p</i> = 0.4). (C) Quantitation of CTFs revealed significantly higher levels in T65Dn brains in both vehicle-treated and vaccine-treated mice (2N vehicle vs Ts65Dn vehicle, <i>p</i> = 0.01; 2N DS-01 vs Ts65Dn DS-01, <i>p</i> = 0.008). Following DS-01 treatment, no significant difference was observed in CTFs (2N, vehicle vs DS-01, <i>p</i> = 0.7; Ts65Dn; vehicle vs DS-01, <i>p</i> = 0.2). The number of mice used for APP and CTFs was: 2N- vehicle/Ts65Dn- vehicle/2N-DS-01/Ts65Dn-DS-01 = 7/5/8/8. (D) Quantification of α-CTF and (E) β-CTF levels in vehicle-treated and immunized mice. There was no significant effect of vaccine-treatment (α-CTFs: 2N, vehicle vs DS-01 <i>p</i> = 0.9; Ts65Dn, vehicle vs DS-01 <i>p =</i> 0.8.; β-CTF: 2N, vehicle vs DS-01 <i>p</i> = 0.9; Ts65Dn, vehicle vs DS-01 <i>p =</i> 0.9). The number of mice used was: 2N- vehicle/Ts65Dn- vehicle/2N-DS-01/Ts65Dn-DS-01 = 4/5/5/7. Error bars, SEM. All statistical analyses were performed using two-tailed Student T test #, <i>p</i> = 0.07, ns- non-significant, *—<i>p</i> < 0.05, **—<i>p</i> < 0.01.</p

Immunization with DS-01 prevented the atrophy of cholinergic neurons.

<p>(A) The area of ChAT+ cell bodies was significantly larger in Ts65Dn-DS-01 relative to Ts65Dn-vehicle treated mice (<i>p</i> = 0.03). (B) Number and <b>c</b> optical density of ChAT+ cells in medial septum were similar in DS-01-treated and vehicle-treated 2N and Ts65Dn mice. Two-tailed Student T test, *—<i>p</i> < 0.05. Error bars, SEM. The number of mice used was as follows: 2N- vehicle/Ts65Dn- vehicle/2N-DS-01/Ts65Dn-DS-01 = 4/4/4/4.</p

Characterization of vaccine-induced plasma immunoreactivity.

<p>(A) Assessment of immunoreactivity against human and mouse Aβ. Different quantities of mouse or human Aβ were blotted with dilutions of plasma (1:100 and 1:1000). The vaccine-induced antibodies were specific to mouse Aβ. (B) Western blots of two homogenates from Ts65Dn (lane 1) and 2N brains (lane 2) comparing vaccine-induced plasma (green signals) and a commercial anti-Aβ antibody to the C-terminus of APP (red signals). Only the commercial APP C-terminal antibody allowed the detection of APP and CTF. Unidentified bands were also detected using each of the antibodies, but no overlapping bands were observed, best appreciated in the right panel at higher magnification. The brain samples loaded were: vehicle-treated Ts65Dn (lane 1), vehicle-treated 2N (lane 2), synthetic mouse Aβ (lane 3). (C) Western blots of homogenates from CHO or PC12 cells using vaccine-induced plasma and a commercial anti-Aβ antibody. (Left panel) Lysates of wild type CHO cells (lanes 1 and 3), or CHO cells transfected with APP (lanes 2 and 4), were probed with plasma (1:1000) (lanes 1 and 2) or with the APP C-terminal antibody (1:1000) (lanes 3 and 4). (Right panel) The lysates of PC12 cells transfected with GFP alone were probed with plasma (lanes 1 and 2), with the APP C-terminal antibody (lanes 5 and 6) or with anti-GFP antibody (lanes 9 and 10). The lysates of PC12 cells expressing C99/GFP probed with plasma (lanes 3 and 4), with the APP C-terminal antibody (lanes 7 and 8), or with anti-GFP antibody (lanes 11 and 12). There was no cross-reactivity of vaccine-induced plasma with full length APP or CTFs. (D) Varying amounts of recombinant C99 were blotted with the vaccine-induced plasma (green bands) or with a commercial anti-APP antibody (red band). Vaccine-induced plasma demonstrated sensitivity at least 30-fold less than the APP C-terminal antibody.</p

Behavioral evaluation and memory function following DS-01 immunization.

<p>(A) The difference in spontaneous locomotor activity between 2N and Ts65Dn mice was unaffected by immunization. (B) In comparison to mice treated with vehicle, both 2N and Ts65Dn immunized with DS-01 showed significantly enhanced discrimination index (DI) in the novel object recognition test (two-tailed Student T test, <i>p</i> = 0.03). The number of mice used was as follows: 2N- vehicle/Ts65Dn- vehicle/2N-DS-01/Ts65Dn-DS-01 = 18/11/20/13. (C) Positive correlation between the level of anti-Aβ40 IgG and the DI (Spearman r correlation 0.4, <i>p</i> = 0.002). Mice having no titers and spreading over the entire range of DI are those immunized with vehicle. The two top performing (highest DI%) are vehicle-treated 2N mice and the two worth performing (lowest DI%) are vehicle-treated Ts65Dn mice. Data from both 2N and Ts65Dn mice immunized with DS-01 are spread in a cloud (solid circle) above DI of 70% while the majority of vehicle-immunized mice had a lower DI value (dashed circle). (D) In the fear conditioning test, during the contextual session, vehicle-treated Ts65Dn mice showed significantly less freezing versus 2N vehicle-treated mice (two-tailed Student T test, <i>p</i> = 0.004). In vaccinated Ts65Dn mice, freezing was significantly different from vehicle-treated Ts65Dn (two-tailed Student T test <i>p</i> = 0.05) and not significantly different from that in 2N vaccinated mice (two-tailed Student T test <i>p</i> = 0.3). *—<i>p</i> < 0.05, **—<i>p</i> < 0.01; Error bars, SEM. The number of mice used was as follows: 2N- vehicle/Ts65Dn- vehicle/2N-DS-01/Ts65Dn-DS-01 = 18/11/20/12.</p

Anti-mouse Aβ antibody levels in 2N and Ts65Dn mice immunized with either vehicle or DS-01.

<p>(A) Anti-mouse-Aβ40 and (B) Aβ42 IgG titers were detected in plasma of DS-01 immunized mice following the 2^nd and the 4^th injection. Significant titers remained as late as 40 days after the 6^th injection. There was no significant difference between titers in 2N versus Ts65Dn mice. (C to F) Analysis of anti-mouse-Aβ40 IgG isotypes following the 4^th immunization. (G) Anti mouse Aβ40 IgM titers were lower in Ts65Dn mice. (H to M) Analysis of anti-mouse-Aβ42 IgG isotypes following the 4^th immunization. (N) Anti mouse Aβ42 IgM titers were lower in Ts65Dn mice. One-way ANOVA, Bonferroni's multiple comparison test *—<i>p</i> < 0.05; **—<i>p</i> < 0.01; ***—<i>p</i> < 0.001. Error bars, SEM. The number of mice was: 2N-vehicle/Ts65Dn-vehicle/2N-DS-01/Ts65Dn-DS-01 = 18/11/20/15.</p

APP expression in the brain of postnatal Ts65Dn mice.

<p>(A) APP mRNA was significantly increased in the Ts65Dn brain relative to the 2N control (<i>p</i> = 0.006). The data are mean ± SD, n (pooled samples) = 6. (B) Full length APP, CTFs and Aβ40 were significantly increased in the Ts65Dn brain as compared to 2N controls (<i>p</i> = 0.00001, <i>p</i> = 0.002 and <i>p</i> = 0.002 respectively). The values are mean ± SEM: the number of samples (N for mice, n for pooled samples) was: full length APP: 2N, N = 5; Ts65Dn, N = 5. APP-CTF’s: 2N, N = 5; Ts65Dn, N = 5. Aβ 40: 2N, n = 3; Ts65Dn, n = 3. ***—<i>p</i> < 0.001, ****—<i>p</i> < 0.0001. Error bars, SEM. <i>p</i> values were calculated using two-tailed Student T test.</p