Down Syndrome Cognitive Phenotypes Modeled in Mice Trisomic for All HSA 21 Homologues

<div><p>Down syndrome (DS), trisomy for chromosome 21, is the most common genetic cause of intellectual disability. The genomic regions on human chromosome 21 (HSA21) are syntenically conserved with regions on mouse chromosomes 10, 16, and 17 (Mmu10, Mmu16, and Mmu17). Recently, we created a genetic model of DS which carries engineered duplications of all three mouse syntenic regions homologous to HSA21. This ‘triple trisomic’ or TTS model thus represents the most complete and accurate murine model currently available for experimental studies of genotype-phenotype relationships in DS. Here we extended our initial studies of TTS mice. Locomotor activity, stereotypic and repetitive behavior, anxiety, working memory, long-term memory, and synaptic plasticity in the dentate gyrus were examined in the TTS and wild-type (WT) control mice. Changes in locomotor activity were most remarkable for a significant increase in ambulatory time and a reduction in average velocity of TTS mice. No changes were detected in repetitive and stereotypic behavior and in measures of anxiety. Working memory showed no changes when tested in Y-maze, but deficiency in a more challenging T-maze test was detected. Furthermore, long-term object recognition memory was significantly reduced in the TTS mice. These changes were accompanied by deficient long-term potentiation in the dentate gyrus, which was restored to the WT levels following blockade of GABAA receptors with picrotoxin (100 μM). TTS mice thus demonstrated a number of phenotypes characteristic of DS and may serve as a new standard by which to evaluate and direct findings in other less complete models of DS.</p></div

Working memory.

<p>A: Alternation rate in Y-maze; B: Number of arm entries in Y-maze; C: Alternation rate in T-maze; D: Averaged time spent on T-maze test. Mean ± SEM. Number of tested animals WT = 22; TTS = 12. **p < 0.01 and ***p<0.001, significantly different from WT mice.</p

Spontaneous locomotor activity.

<p>A-C: Ambulatory distance; D-F: Ambulatory time; G-I: Resting time; J-K: Average velocity. The values averaged for 2-min sequential periods (A, D, G, J) and for the total 10-min testing period (B, E, H, K) are shown. Also, the percentage of distance and time on the arena periphery (C, F, I) are shown. L: Number of pellets dropped during the test. All results are mean ± SEM. Number of mice examined: WT = 22; TTS = 12. *p < 0.05, **p < 0.01, and ***p<0.001, significantly different from WT mice.</p

Mouse genetic models of DS and breeding schema of TTS mice.

<p><b>A:</b> Mouse genetic models. <i>Left</i>: Correspondence of syntenic genomic regions on human chromosome 21 (HSA21) to mouse chromosomes (Mmu) 10, 16, and 17. <i>Right</i>: Schema of the triplicated genomic regions in several notable mouse models of DS. Mouse and human genomic regions are shown in green and blue respectively. Triplicated genomic regions not present on HSA21 and, therefore, not triplicated in DS, are shown in black. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134861#pone.0134861.t001" target="_blank">Table 1</a> for details regarding the size and boundaries of the triplicated regions. <b>B:</b> Breeding schema of TTS mice. First, Dp(10)1Yey/+, Dp(16)1Yey/+, and Dp(17)1Yey/+ mice (green rectangles) were generated by triplications of the corresponding syntenic regions. Next, compound Dp(10)1Yey/+;Dp(16)1Yey/+ mice (black rectangle) were generated by crossing Dp(10)1Yey/+ and Dp(16)1Yey/+ mutant mice. Finally, TTS mice (red rectangle) were generated by crossing Dp(10)1Yey/+;Dp(16)1Yey/+ and Dp(17)1Yey/+ mice.</p

Major behavioral and physiological phenotypes in mouse genetic models of Down syndrome.

<p>‘☑’–Results of this study;</p><p>‘↔’—No change;</p><p>‘↑’–Increased;</p><p>‘↓’—Reduced;</p><p>‘─’–Data not published.</p><p>Major behavioral and physiological phenotypes in mouse genetic models of Down syndrome.</p

Novel object recognition.

<p>A: The time spent on investigation of the objects during the testing phase of the experiment; B: Discrimination index. Mean ± SEM; *p < 0.05, significantly different from WT mice.</p

Synaptic efficiency and plasticity in the dentate gyrus.

<p>A: Input-output relationship in the DG. Evoked responses were similar in MML of WT and TTS DG (blue and red markers respectively). B: Time course of the averaged initial slope of field EPSPs during the experiment. Tetanization (arrow) evoked stable LTP in WT, but not in TTS mice (blue and red markers respectively). The results are mean ± SEM. The number of slices/mice examined: WT = 9/4; TTS = 8/4. C: Suppression of inhibition by picrotoxin allowed for induction of normal LTP in the TTS slices. The results are mean ± SEM. The number of slices/mice examined: WT = 6/3; TTS = 7/3.</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

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

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