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.     

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.     

Mouse models of Down syndrome: gene content and consequences

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), is challenging to model in mice. Not only is it a contiguous gene syndrome spanning 35 Mb of the long arm of Hsa21, but orthologs of Hsa21 genes map to segments of three mouse chromosomes, Mmu16, Mmu17, and Mmu10. The Ts65Dn was the first viable segmental trisomy mouse model for DS; it is a partial trisomy currently popular in preclinical evaluations of drugs for cognition in DS. Limitations of the Ts65Dn are as follows: (i) it is trisomic for 125 human protein-coding orthologs, but only 90 of these are Hsa21 orthologs and (ii) it lacks trisomy for ~75 Hsa21 orthologs. In recent years, several additional mouse models of DS have been generated, each trisomic for a different subset of Hsa21 genes or their orthologs. To best exploit these models and interpret the results obtained with them, prior to proposing clinical trials, an understanding of their trisomic gene content, relative to full trisomy 21, is necessary. Here we first review the functional information on Hsa21 protein-coding genes and the more recent annotation of a large number of functional RNA genes. We then discuss the conservation and genomic distribution of Hsa21 orthologs in the mouse genome and the distribution of mouse-specific genes. Lastly, we consider the strengths and weaknesses of mouse models of DS based on the number and nature of the Hsa21 orthologs that are, and are not, trisomic in each, and discuss their validity for use in preclinical evaluations of drug responses.     

A neural crest deficit in Down syndrome mice is associated with deficient mitotic response to Sonic hedgehog

Trisomy 21 results in phenotypes collectively referred to as Down syndrome (DS) including characteristic facial dysmorphology. Ts65Dn mice are trisomic for orthologs of about half of the genes found on human chromosome 21 and exhibit DS-like craniofacial abnormalities, including a small dysmorphic mandible. Quantitative analysis of neural crest (NC) progenitors of the mandible revealed a paucity of NC and a smaller first pharyngeal arch (PA1) in Ts65Dn as compared to euploid embryos. Similar effects in PA2 suggest that trisomy causes a neurocristopathy in Ts65Dn mice (and by extension, DS). Further analyses demonstrated deficits in delamination, migration, and mitosis of trisomic NC. Addition of Sonic hedgehog (Shh) growth factor to trisomic cells from PA1 increased cell number to the same level as untreated control cells. Combined with previous demonstrations of a deficit in mitogenic response to Shh by trisomic cerebellar granule cell precursors, these results implicate common cellular and molecular bases of multiple DS phenotypes.     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I)

Summary.  Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this “gene dosage effect” hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18–19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis. Received June 27, 2002 Accepted July 19, 2002 Published online November 14, 2002 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AIRE, autoimmune regulator; DS, Down syndrome; HCS, holocarboxylase synthetase; Prep1, Pbx regulating protein 1; Tiam1, T-cell lymphoma invasion and metastasis 1     

Increased Skin Tumor Incidence and Keratinocyte Hyper-Proliferation in a Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic disorder caused by the presence of an extra copy of human chromosome 21 (Hsa21). People with DS display multiple clinical traits as a result of the dosage imbalance of several hundred genes. While many outcomes of trisomy are deleterious, epidemiological studies have shown a significant risk reduction for most solid tumors in DS. Reduced tumor incidence has also been demonstrated in functional studies using trisomic DS mouse models. Therefore, it was interesting to find that Ts1Rhr trisomic mice developed more papillomas than did their euploid littermates in a DMBA-TPA chemical carcinogenesis paradigm. Papillomas in Ts1Rhr mice also proliferated faster. The increased proliferation was likely caused by a stronger response of trisomy to TPA induction. Treatment with TPA caused hyperkeratosis to a greater degree in Ts1Rhr mice than in euploid, reminiscent of hyperkeratosis seen in people with DS. Cultured trisomic keratinocytes also showed increased TPA-induced proliferation compared to euploid controls. These outcomes suggest that altered gene expression in trisomy could elevate a proliferation signalling pathway. Gene expression analysis of cultured keratinocytes revealed upregulation of several trisomic and disomic genes may contribute to this hyperproliferation. The contributions of these genes to hyper-proliferation were further validated in a siRNA knockdown experiment. The unexpected findings reported here add a new aspect to our understanding of tumorigenesis with clinical implications for DS and demonstrates the complexity of the tumor repression phenotype in this frequent condition.     

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.     

Postnatal lethality and cardiac anomalies in the Ts65Dn Down Syndrome mouse model

The Ts65Dn mouse is a well-studied model for Down syndrome (DS). The presence of the translocation chromosome T17¹⁶ (referred to as T65Dn) produces a trisomic dosage imbalance for over 100 genes on the distal region of mouse Chromosome 16. This dosage imbalance, with more than half of the orthologs of human Chromosome 21 (Hsa21), causes several phenotypes in the trisomic mice that are reminiscent of DS. Careful examination of neonates in a newly established Ts65Dn colony indicated high rates of postnatal lethality. Although the transmission rate for the T65Dn chromosome has been previously reported as 20%–40%, genotyping of all progeny indicates transmission at birth is near the 50% expected with Mendelian transmission and survival. Remarkably, in litters with maternal care that allowed survival of some pups, postnatal lethality occurred primarily in pups that inherited the T65Dn marker chromosome. This selective loss within 48 h of birth reduced the transmission of the marker chromosome from 49% at birth to 34% at weaning. Gross morphologic examination revealed cardiovascular anomalies, i.e., right aortic arch accompanied by septal defects, in 8.3% of the trisomic newborn cadavers examined. This is an intriguing finding because the orthologs of the DiGeorge region of HSA22, which are posited to contribute to the aortic arch abnormalities seen in trisomy 16 mice, are not triplicated in Ts65Dn mice. These new observations suggest that the Ts65Dn mouse models DS not only in its previously described phenotypes but also with elevated postnatal lethality and congenital heart malformations that may contribute to mortality.     

Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction

Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.17¹⁶)]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre‐existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high‐resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12‐month‐old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal–exosomal pathway.     

Trisomic and allelic differences influence phenotypic variability during development of Down syndrome mice

Individuals with full or partial Trisomy 21 (Ts21) present with clinical features collectively referred to as Down syndrome (DS), although DS phenotypes vary in incidence and severity between individuals. Differing genetic and phenotypic content in individuals with DS as well as mouse models of DS facilitate the understanding of the correlation between specific genes and phenotypes associated with Ts21. The Ts1Rhr mouse model is trisomic for 33 genes (the "Down syndrome critical region" or DSCR) hypothesized to be responsible for many clinical DS features, including craniofacial dysmorphology with a small mandible. Experiments with Ts1Rhr mice showed that the DSCR was not sufficient to cause all DS phenotypes by identifying uncharacteristic craniofacial abnormalities not found in individuals with DS or other DS mouse models. We hypothesized that the origins of the larger, dysmorphic mandible observed in adult Ts1Rhr mice develop from larger embryonic craniofacial precursors. Because of phenotypic variability seen in subsequent studies with Ts1Rhr mice, we also hypothesized that genetic background differences would alter Ts1Rhr developmental phenotypes. Using Ts1Rhr offspring from two genetic backgrounds, we found differences in mandibular precursor volume as well as total embryonic volume and postnatal body size of Ts1Rhr and nontrisomic littermates. Additionally, we observed increased relative expression of Dyrk1a and differential expression of Ets2 on the basis of the genetic background in the Ts1Rhr mandibular precursor. Our results suggest that trisomic gene content and allelic differences in trisomic or nontrisomic genes influence variability in gene expression and developmental phenotypes associated with DS.     

The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17

The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype.     

SV40 T-antigen expression in cultured fibroblasts from patients with down syndrome and their parents

Expression of simian papovavirus 40 (SV40) T-antigen following in vitro infection was studied in skin fibroblasts from patients with Down syndrome (DS) and their parents to determine whether the increased susceptibility to SV40 infection reflected the cytogenetic defect or the leukemia risk associated with this syndrome. As a group, fibroblasts from patients with DS showed elevated T-antigen expression 72 hrs after infection compared to that of a healthy control population. However, among 24 patients tested, the cell lines of only 11 showed statistically significant increases in T-antigen expression. A cell line from a patient with concurrent DS and acute myelogenous leukemia had a normal value. T-antigen expression did not correlate with the percentage of cells trisomic for chromosome 21 in 18 cell lines examined or with the number of copies of this chromosome in disomic and trisomic cell strains cloned from three mosaic patients.Collectively, cell lines from parents of trisomy 21 patients also showed increased susceptibility to SV40 infection; however, in five families tested, a consistent pattern of genetic transmission of elevated T-antigen expression from parent to offspring was not observed. Q-banding of cell lines in one family showed that elevated T-antigen expression is not a marker of parental nondisjunction. Variation in susceptibility to human interferon, an antiviral agent, did not account for variation in T-antigen levels among these cell lines. Thus, the abnormalities of T-antigen expression in DS appear independent of the hyperdiploid state and are not a sensitive indicator of cancer risk.     

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.     

An extra human chromosome 21 reduces mlc-2a expression in chimeric mice and Down syndrome

An extra copy of human chromosome 21 (Chr 21) causes Down syndrome (DS), which is characterized by mental retardation and congenital heart disease (CHD). Chimeric mice containing Chr 21 also exhibit phenotypic traits of DS including CHD. In this study, to identify genes contributing to DS phenotypes, we compared the overall protein expression patterns in hearts of Chr 21 chimeras and wild type mice by two-dimensional electrophoresis. The endogenous mouse atrial specific isoform of myosin light chain-2 (mlc-2a) protein was remarkably downregulated in the hearts of chimeric mice. We also confirmed that the human MLC-2A protein level was significantly lower in a human DS neonate heart, as compared to that of a normal control. Since mouse mlc-2a is involved in heart morphogenesis, our data suggest that the downregulation of this gene plays a crucial role in the CHD observed in DS. The dosage imbalance of Chr 21 has a trans-acting effect which lowers the expression of other genes encoded elsewhere in the genome.     

A unique downregulation of h2-calponin gene expression in Down syndrome: a possible attenuation mechanism for fetal survival by methylation at the CpG island in the trisomic chromosome 21.

To understand the effect of trisomic chromosome 21 on the cause of Down syndrome (DS), DNA methylation in the CpG island, which regulates the expression of adjacent genes, was investigated with the DNAs of chromosome 21 isolated from DS patients and their parents. A methylation-sensitive enzyme, BssHII, was used to digest DNAs of chromosome 21, and the resulting DNA fragments were subjected to RLGS (restriction landmark genomic scanning). Surprisingly, the CpG island of the h2-calponin gene was shown to be specifically methylated by comparative studies with RLGS and Southern blot analysis. In association with this methylation, h2-calponin gene expression was attenuated to the normal level, although other genes in the DS region of chromosome 21 were expressed dose dependently at 1.5 times the normal level. These results and the high miscarriage rate associated with trisomy 21 embryos imply that the altered in vivo methylation that attenuates downstream gene expression, which is otherwise lethal, permits the generation of DS neonates. The h2-calponin gene detected by the RLGS procedure may be one such gene that is attenuated.     

Relevance of drug uptake,cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone

Sensitivity of human fibroblasts derived from Down's syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.