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

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

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