Cholinergic Senescence in the Ts65Dn Mouse Model for Down Syndrome

Neurochemical Research - Down syndrome (DS) induces a variable phenotype including intellectual disabilities and early development of Alzheimer’s disease (AD). Moreover, individuals with DS...     

Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer's disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822?bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice.     

Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model

Individuals with Down syndrome develop β-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein ( App ) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPα and sAPPβ) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Aβ40 and Aβ42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain.     

Perinatal loss of Ts65Dn Down syndrome mice

Ts65Dn mice inherit a marker chromosome, T(17(16))65Dn, producing segmental trisomy for orthologs of about half of the genes on human chromosome 21. These mice display a number of phenotypes that are directly comparable to those in humans with trisomy 21 and are the most widely used animal model of Down syndrome (DS). However, the husbandry of Ts65Dn mice is complicated. Males are sterile, and only 20-40% of the offspring of Ts65Dn mothers are trisomic at weaning. The lower-than-expected frequency of trisomic offspring has been attributed to losses at meiosis, during gestation and at postnatal stages, but no systematic studies support any of these suppositions. We show that the T(17(16))65Dn marker chromosome is inherited at expected frequency and is fully compatible with development to midgestation. Disproportional loss of trisomic offspring occurs in late gestation and continues through birth to weaning. Different maternal H2 haplotypes are significantly associated with the frequency of trisomy at weaning in patterns different from those reported previously. The proportion of trisomic mice per litter decreases with age of the Ts65Dn mother. These results provide the first statistical and numerical evidence supporting the prenatal and perinatal pattern of loss in the Ts65Dn mouse model of DS.     

Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.     

Widespread impairment of cell proliferation in the neonate Ts65Dn mouse, a model for Down syndrome

Objectives: Among the many pathological aspects of Down syndrome, brain hypoplasia and mental retardation have been recently ascribed to defective proliferation of neural precursors during central nervous system development. By analogy, other features of Down syndrome, such as heart defects, gastrointestinal abnormalities, craniofacial dystrophy and reduced growth rate could be related, at least in theory, to similar proliferation impairment in peripheral tissues.
Materials and methods: In order to test this hypothesis, we evaluated cell proliferation in peripheral tissues of the Ts65Dn mouse, one of the animal models most commonly used to investigate Down syndrome.
Results: In fibroblast cultures from neonatal Ts65Dn mice, we found that cell proliferation was notably impaired. While length of the cell cycle was similar in fibroblasts from Ts65Dn and control mice, the number of actively proliferating cells was significantly smaller in Ts65Dn mice. Moreover, fibroblasts from Ts65Dn animals exhibited limited population-doubling capacity, decreased proliferative lifespan and premature senescence. Analysis of cell proliferation in the skin of neonates, in vivo , showed that in Ts65Dn mice, cell proliferation was significantly reduced compared to control mice.
Conclusions: Our results suggest that defective proliferation may be a generalized feature of trisomic mice. In view of the genetic and phenotypic similarities between Ts65Dn mice and individuals with Down syndrome, proliferation impairment in various organs may also occur in subjects with Down syndrome. Thus, perturbation of a basic developmental function, cell proliferation, may be a critical determinant that contributes to the many aspects of pathology of this condition.     

Normal protein composition of synapses in Ts65Dn mice: a mouse model of Down syndrome

Down syndrome (DS) is the most prevalent form of intellectual disability caused by the triplication of ∼230 genes on chromosome 21. Recent data in Ts65Dn mice, the foremost mouse model of DS, strongly suggest that cognitive impairment in individuals with DS is a consequence of reduced synaptic plasticity because of chronic over-inhibition. It remains unclear however whether changes in plasticity are tied to global molecular changes at synapses, or are due to regional changes in the functional properties of synaptic circuits. One interesting framework for evaluating the activity state of the DS brain comes from in vitro studies showing that chronic pharmacological silencing of neuronal excitability orchestrates stereotyped changes in the protein composition of synaptic junctions. In the present study, we use proteomic strategies to evaluate whether synapses from the Ts65Dn cerebrum carry signatures characteristic of inactive cortical neurons. Our data reveal that synaptic junctions do not exhibit overt alterations in protein composition. Only modest changes in the levels of synaptic proteins and in their phosphorylation are observed. This suggests that subtle changes in the functional properties of specific synaptic circuits rather than large-scale homeostatic shifts in the expression of synaptic molecules contribute to cognitive impairment in people with DS.     

Altered signaling pathways underlying abnormal hippocampal synaptic plasticity in the Ts65Dn mouse model of Down syndrome

The Ts65Dn mouse model of Down syndrome (DS) has an extra segment of chromosome (Chr.) 16 exhibits abnormal behavior, synaptic plasticity and altered function of several signaling molecules. We have further investigated signaling pathways that may be responsible for the impaired hippocampal plasticity in the Ts65Dn mouse. Here we report that calcium/calmodulin-dependent protein kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (ERK), protein kinase A (PKA) and protein kinase C (PKC), all of which have been shown to be involved in synaptic plasticity, are altered in the Ts65Dn hippocampus. We found that the phosphorylation of CaMKII and protein kinase Akt was increased, whereas ERK was decreased. Activities of PKA and PKC were decreased. Furthermore, abnormal PKC activity and an absence of the increase in Akt phosphorylation were demonstrated in the Ts65Dn hippocampus after high-frequency stimulation that induces long-term potentiation. Our findings suggest that abnormal synaptic plasticity in the Ts65Dn hippocampus is the result of compensatory alterations involving the glutamate receptor subunit GluR1 in either one or more of these signaling cascades caused by the expression of genes located on the extra segment of Chr. 16.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

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.     

Ts65Dn Mouse,a Down Syndrome Model,Exhibits Elevated myo-Inositol in Selected Brain Regions and Peripheral Tissues

myo-Inositol is elevated in the Down syndrome (DS; trisomy 21) brain and may play a role in mental retardation. In the present study, we examined brain regions and peripheral tissues of Ts65Dn mouse, a recently characterized genetic model of DS, for abnormal myo-inositol accumulation. A GC/MS technique was used to quantitate myo-inositol and other polyol species (ribitol, arabitol, xylitol, and 1,5-anhydrosorbitol) in tissues from the Ts65Dn mice and control diploid mice. myo-Inositol was found to be elevated in frontal cortex, hippocampus, and brain stem but not in cerebellum of the Ts65Dn mouse. Among peripheral organs examined, liver and skeletal muscle were found to excessively accumulate myo-inositol. In all tissues, concentrations of polyol internal controls were normal. The Ts65Dn mouse is useful to study the possible effect of elevated myo-inositol on cellular processes.     

Loss of correlations among proteins in brains of the Ts65Dn mouse model of down syndrome

The Ts65Dn mouse model of Down syndrome (DS) is trisomic for orthologs of 88 of 161 classical protein coding genes present on human chromosome 21 (HSA21). Ts65Dn mice display learning and memory impairments and neuroanatomical, electrophysiological, and cellular abnormalities that are relevant to phenotypic features seen in DS; however, little is known about the molecular perturbations underlying the abnormalities. Here we have used reverse phase protein arrays to profile 64 proteins in the cortex, hippocampus, and cerebellum of Ts65Dn mice and littermate controls. Proteins were chosen to sample a variety of pathways and processes and include orthologs of HSA21 proteins and phosphorylation-dependent and -independent forms of non-HSA21 proteins. Protein profiles overall show remarkable stability to the effects of trisomy, with fewer than 30% of proteins altered in any brain region. However, phospho-proteins are less resistant to trisomy than their phospho-independent forms, and Ts65Dn display abnormalities in some key proteins. Importantly, we demonstrate that Ts65Dn mice have lost correlations seen in control mice among levels of functionally related proteins, including components of the MAP kinase pathway and subunits of the NMDA receptor. Loss of normal patterns of correlations may compromise molecular responses to stimulation and underlie deficits in learning and memory.     

Monoacylglycerol Lipase Inhibitor JZL184 Improves Behavior and Neural Properties in Ts65Dn Mice,a Model of Down Syndrome

Genetic alterations or pharmacological treatments affecting endocannabinoid signaling have profound effects on synaptic and neuronal properties and, under certain conditions, may improve higher brain functions. Down syndrome (DS), a developmental disorder caused by triplication of chromosome 21, is characterized by deficient cognition and inevitable development of the Alzheimer disease (AD) type pathology during aging. Here we used JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL), to examine the effects of chronic MAGL inhibition on the behavioral, biochemical, and synaptic properties of aged Ts65Dn mice, a genetic model of DS. In both Ts65Dn mice and their normosomic (2N) controls, JZL184-treatment increased brain levels of 2-arachidonoylglycerol (2-AG) and decreased levels of its metabolites such as arachidonic acid, prostaglandins PGD2, PGE2, PGFα, and PGJ2. Enhanced spontaneous locomotor activity of Ts65Dn mice was reduced by the JZL184-treatement to the levels observed in 2N animals. Deficient long-term memory was also improved, while short-term and working types of memory were unaffected. Furthermore, reduced hippocampal long-term potentiation (LTP) was increased in the JZL184-treated Ts65Dn mice to the levels observed in 2N mice. Interestingly, changes in synaptic plasticity and behavior were not observed in the JZL184-treated 2N mice suggesting that the treatment specifically attenuated the defects in the trisomic animals. The JZL184-treatment also reduced the levels of Aβ40 and Aβ42, but had no effect on the levels of full length APP and BACE1 in both Ts65Dn and 2N mice. These data show that chronic MAGL inhibition improves the behavior and brain functions in a DS model suggesting that pharmacological targeting of MAGL may be considered as a perspective new approach for improving cognition in DS.     

Effects of neonatal neural progenitor cell implantation on adult neuroanatomy and cognition in the Ts65Dn model of Down syndrome

As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model.