Down syndrome (DS), trisomy of the long arm of human chromosome 21 (Hsa21), is the most common genetic cause of intellectual disability (ID). Currently, there are no effective pharmacotherapies. The success of clinical trials to improve cognition depends in part on the design of preclinical evaluations in mouse models. To broaden understanding of the common limitations of experiments in learning and memory, we report performance in context fear conditioning (CFC) in three mouse models of DS, the Dp(16)1Yey, Dp(17)1Yey and Dp(10)1Yey (abbreviated Dp16, Dp17 and Dp10), separately trisomic for the human Hsa21 orthologs mapping to mouse chromosomes 16, 17 and 10, respectively. We examined female and male mice of the three lines on the standard C57BL/6J background at 3 months of age and Dp17 and Dp10 at 18 months of age. We also examined female and male mice of Dp17 and Dp10 at 3 months of age as F1 hybrids obtained from a cross with the DBA/2J background. Results indicate that genotype, sex, age and genetic background affect CFC performance. These data support the need to use both female and male mice, trisomy of sets of all Hsa21 orthologs, and additional ages and genetic backgrounds to improve the reliability of preclinical evaluations of drugs for ID in DS.

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), is the most common genetic cause of intellectual disability (ID). There are no treatments for the cognitive deficits. The Ts65Dn is a partial trisomy mouse model of DS that shows learning and memory (LM) impairments and other abnormalities relevant to those seen in DS. Many drugs and small molecules have been shown to rescue the LM deficits, but little is known about the associated molecular responses. Here, patterns of protein expression are described in hippocampus of Ts65Dn and euploid littermate controls exposed to a battery of LM and behavior tests with and without chronic treatment with the GABAA receptor α5 subunit-selective negative allosteric modulator, RO4938581, that rescued LM deficits. Levels of 91 proteins/protein modifications, selected for relevance to LM and synaptic plasticity, were measured: 44 of 52 abnormalities present in vehicle-treated Ts65Dn were corrected by RO4938581. Superimposing protein data onto the molecular pathway defining long-term potentiation (LTP) shows that profiles are consistent with both abnormal LTP in vehicle-treated Ts65Dn and its observed rescue by RO4938581. Lastly, comparing these results with those from Ts65Dn treated, using a different protocol, with the NMDA receptor antagonist, memantine, that also rescues LM impairments, identifies common and divergent responses to the two drugs. Expansion of this approach to include additional drugs and DS models would aid in determining critical protein abnormalities and in identifying cocktails of drugs and/or new drug targets that would be effective in clinical trials for ID in DS.

The Ts65Dn is a popular mouse model of Down syndrome (DS). It displays DS-relevant features of learning/memory deficits and age-related loss of functional markers in basal forebrain cholinergic neurons. Here we describe protein expression abnormalities in brain regions of 12-month-old male Ts65Dn mice. We show that the magnitudes of abnormalities of human chromosome 21 and nonehuman chromosome 21 orthologous proteins are greater at 12 months than at w6 months. Age-related exacerbations involve the number of components affected in the mechanistic target of rapamycin pathway, the levels of components of the mitogen-activated protein kinase pathway, and proteins associated with Alzheimer's disease. Among brain regions, the number of abnormalities in cerebellum decreased while the number in cortex greatly increased with age. The Ts65Dn is being used in preclinical evaluations of drugs for cognition in DS. Most commonly, drug evaluations are tested in similar to 4- to 6- month-old mice. Data on age-related changes in magnitude and specificity of protein perturbations can be used to understand the molecular basis of changes in cognitive ability and to predict potential age-related specificities in drug efficacies. (C) 2017 Elsevier Inc. All rights reserved.

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials.

While many sex differences in structure and function of the mammalian brain have been described, the molecular correlates of these differences are not broadly known. Also unknown is how sex differences at the protein level are perturbed by mutations that lead to intellectual disability (ID). Down syndrome (DS) is the most common genetic cause of ID and is due to trisomy of human chromosome 21 (Hsa21) and the resulting increased expression of Hsa21-encoded genes. The Dp(10)1Yey mouse model (Dp10) of DS is trisomic for orthologs of 39 Hsa21 protein-coding genes that map to mouse chromosome 10 (Mmu10), including four genes with known sex differences in functional properties. How these genes contribute to the DS cognitive phenotype is not known.

Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein responses and interactions following normal learning.