Embryonic statistical analyses reveal 2 growth phenotypes in mouse models of Down syndrome.

BACKGROUND: Down syndrome is associated with several comorbidities, including intellectual disability, growth restriction, and congenital heart defects. The prevalence of Down syndrome-associated comorbidities is highly variable, and intellectual disability, although fully penetrant, ranges from mild to severe. Understanding the basis of this interindividual variability might identify predictive biomarkers of in utero and postnatal outcomes that could be used as endpoints to test the efficacy of future therapeutic interventions. OBJECTIVE: The main objective of this study was to examine if antenatal interindividual variability exists in mouse models of Down syndrome and whether applying statistical approaches to clinically relevant measurements (ie, the weights of the embryo, placenta, and brain) could define cutoffs that discriminate between subgroups of trisomic embryos. STUDY DESIGN: Three commonly used mouse models of Down syndrome (Dp(16)1/Yey, Ts65Dn, and Ts1Cje) and a new model (Ts66Yah) were used in this study. Trisomic and euploid littermate embryos were used from each model with total numbers of 102 for Ts66Yah, 118 for Dp(16)1/Yey, 92 for Ts65Dn, and 126 for Ts1Cje. Placental, embryonic, and brain weights and volumes at embryonic day 18.5 were compared between genotypes in each model. K-mean clustering analysis was applied to embryonic and brain weights to identify severity classes in trisomic embryos, and brain and placental volumetric measurements were compared between genotypes and classes for each strain. In addition, Ts66Yah embryos were examined for malformations because embryonic phenotypes have never been examined in this model. RESULTS: Reduced body and brain weights were present in Ts66Yah, Dp(16)1/Yey, and Ts65Dn embyos. Cluster analysis identified 2 severity classes in trisomic embryos-mild and severe-in all 4 models that were distinguishable using a putative embryonic weight cutoff of <0.5 standard deviation below the mean. Ts66Yah trisomic embryos develop congenital anomalies that are also found in humans with Down syndrome, including congenital heart defects and renal pelvis dilation. CONCLUSION: Statistical approaches applied to clinically relevant measurements revealed 2 classes of phenotypic severity in trisomic mouse models of Down syndrome. Analysis of severely affected trisomic animals may facilitate the identification of biomarkers and endpoints that can be used to prenatally predict outcomes and the efficacy of treatments.

The Impact of Mmu17 Non-Hsa21 Orthologous Genes in the Ts65Dn Mouse Model of Down Syndrome: The Gold Standard Refuted.

BACKGROUND: Despite successful preclinical treatment studies to improve neurocognition in the Ts65Dn mouse model of Down syndrome, translation to humans has failed. This raises questions about the appropriateness of the Ts65Dn mouse as the gold standard. We used the novel Ts66Yah mouse that carries an extra chromosome and the identical segmental Mmu16 trisomy as Ts65Dn without the Mmu17 non-Hsa21 orthologous region. METHODS: Forebrains from embryonic day 18.5 Ts66Yah and Ts65Dn mice, along with euploid littermate controls, were used for gene expression and pathway analyses. Behavioral experiments were performed in neonatal and adult mice. Because male Ts66Yah mice are fertile, parent-of-origin transmission of the extra chromosome was studied. RESULTS: Forty-five protein-coding genes mapped to the Ts65Dn Mmu17 non-Hsa21 orthologous region; 71%-82% are expressed during forebrain development. Several of these genes are uniquely overexpressed in Ts65Dn embryonic forebrain, producing major differences in dysregulated genes and pathways. Despite these differences, the primary Mmu16 trisomic effects were highly conserved in both models, resulting in commonly dysregulated disomic genes and pathways. Delays in motor development, communication, and olfactory spatial memory were present in Ts66Yah but more pronounced in Ts65Dn neonates. Adult Ts66Yah mice showed milder working memory deficits and sex-specific effects in exploratory behavior and spatial hippocampal memory, while long-term memory was preserved. CONCLUSIONS: Our findings suggest that triplication of the non-Hsa21 orthologous Mmu17 genes significantly contributes to the phenotype of the Ts65Dn mouse and may explain why preclinical trials that used this model have unsuccessfully translated to human therapies.

Visual discrimination and inhibitory control deficits in mouse models of Down syndrome: A pilot study using rodent touchscreen technology.

Several non-verbal cognitive and behavioral tests have been developed to assess learning deficits in humans with Down syndrome (DS). Here we used rodent touchscreen paradigms in adult male mice to investigate visual discrimination (VD) learning and inhibitory control in the Dp(16)1/Yey (C57BL/6J genetic background), Ts65Dn (mixed B6 X C3H genetic background) and Ts1Cje (C57BL/6J genetic background) mouse models of DS. Dp(16)1/Yey and Ts1Cje models did not exhibit motivation or learning deficits during early pre-training, however, Ts1Cje mice showed a significant learning delay after the introduction of the incorrect stimulus (late pre-training), suggesting prefrontal cortex defects in this model. Dp(16)1/Yey and Ts1Cje mice display learning deficits in VD but these deficits were more pronounced in the Dp(16)1/Yey model. Both models also exhibited compulsive behavior and abnormal cortical inhibitory control during Extinction compared to WT littermates. Finally, Ts65Dn mice outperformed WT littermates in pre-training stages by initiating a significantly higher number of trials due to their hyperactive behavior. Both Ts65Dn and WT littermates showed poor performance during late pre-training and were not tested in VD. These studies demonstrate significant learning deficits and compulsive behavior in the Ts1Cje and Dp(16)1/Yey mouse models of DS. They also demonstrate that the mouse genetic background (C57BL/6J vs. mixed B6 X C3H) and the absence of hyperactive behavior are key determinants of successful learning in touchscreen behavioral testing. These data will be used to select the mouse model that best mimics cognitive deficits in humans with DS and evaluate the effects of future therapeutic interventions.

Novel insights from fetal and placental phenotyping in 3 mouse models of Down syndrome.

BACKGROUND: In human fetuses with Down syndrome, placental pathology, structural anomalies and growth restriction are present. There is currently a significant lack of information regarding the early life span in mouse models of Down syndrome. OBJECTIVE: The objective of this study was to examine embryonic day 18.5 and placental phenotype in the 3 most common mouse models of Down syndrome (Ts65Dn, Dp(16)1/Yey, Ts1Cje). Based on prenatal and placental phenotyping in 3 mouse models of Down syndrome, we hypothesized that one or more of them would have a similar phenotype to human fetuses with trisomy 21, which would make it the most suitable for in utero treatment studies. STUDY DESIGN: Here, C57BL6J/6 female mice were mated to Dp(16)1/Yey and Ts1Cje male mice and Ts65Dn female mice to C57BL/B6Eic3Sn.BLiAF1/J male mice. At embryonic day 18.5, dams were euthanized. Embryos and placentas were examined blindly for weight and size. Embryos were characterized as euploid or trisomic, male or female by polymerase chain reaction. A subset of embryos (34 euploid and 34 trisomic) were examined for malformations. RESULTS: The Ts65Dn mouse model showed the largest differences in fetal growth, brain development, and placental development when comparing euploid and trisomic embryos. For the Dp(16)1/Yey mouse model, genotype did not impact fetal growth, but there were differences in brain and placental development. For the Ts1Cje mouse model, no significant association was found between genotype and fetal growth, brain development, or placental development. Euploid mouse embryos had no congenital anomalies; however, 1 mouse embryo died. Hepatic necrosis was seen in 6 of 12 Dp(16)1/Yey (50%) and 1 of 12 Ts1Cje (8%) mouse embryos; hepatic congestion or inflammation was observed in 3 of 10 Ts65Dn mouse embryos (30%). Renal pelvis dilation was seen in 5 of 12 Dp(16)1/Yey (42%), 5 of 10 Ts65Dn (50%), and 3 of 12 Ts1Cje (25%) mouse embryos. In addition, 1 Ts65Dn mouse embryo and 1 Dp(16)1/Yey mouse embryo had an aortic outflow abnormality. Furthermore, 2 Ts1Cje mouse embryos had ventricular septal defects. Ts65Dn mouse placentas had increased spongiotrophoblast necrosis. CONCLUSION: Fetal and placental growth showed varying trends across strains. Congenital anomalies were primarily seen in trisomic embryos. The presence of liver abnormalities in all 3 mouse models of Down syndrome (10 of 34 cases) is a novel finding. Renal pelvis dilation was also common (13 of 34 cases). Future research will examine human autopsy material to determine if these findings are relevant to infants with Down syndrome. Differences in placental histology were also observed among strains.

Apigenin as a Candidate Prenatal Treatment for Trisomy 21: Effects in Human Amniocytes and the Ts1Cje Mouse Model.

Human fetuses with trisomy 21 (T21) have atypical brain development that is apparent sonographically in the second trimester. We hypothesize that by analyzing and integrating dysregulated gene expression and pathways common to humans with Down syndrome (DS) and mouse models we can discover novel targets for prenatal therapy. Here, we tested the safety and efficacy of apigenin, identified with this approach, in both human amniocytes from fetuses with T21 and in the Ts1Cje mouse model. In vitro, T21 cells cultured with apigenin had significantly reduced oxidative stress and improved antioxidant defense response. In vivo, apigenin treatment mixed with chow was administered prenatally to the dams and fed to the pups over their lifetimes. There was no significant increase in birth defects or pup deaths resulting from prenatal apigenin treatment. Apigenin significantly improved several developmental milestones and spatial olfactory memory in Ts1Cje neonates. In addition, we noted sex-specific effects on exploratory behavior and long-term hippocampal memory in adult mice, and males showed significantly more improvement than females. We demonstrated that the therapeutic effects of apigenin are pleiotropic, resulting in decreased oxidative stress, activation of pro-proliferative and pro-neurogenic genes (KI67, Nestin, Sox2, and PAX6), reduction of the pro-inflammatory cytokines INFG, IL1A, and IL12P70 through the inhibition of NFκB signaling, increase of the anti-inflammatory cytokines IL10 and IL12P40, and increased expression of the angiogenic and neurotrophic factors VEGFA and IL7. These studies provide proof of principle that apigenin has multiple therapeutic targets in preclinical models of DS.

Placental development and function in trisomy 21 and mouse models of Down syndrome: Clues for studying mechanisms underlying atypical development.

Down syndrome (DS) is the most common genetic disorder leading to developmental disability. The phenotypes associated with DS are complex and vary between affected individuals. Placental abnormalities in DS include differences in cytotrophoblast fusion that affect subsequent conversion to syncytiotrophoblast, atypical oxidative stress/antioxidant balance, and increased expression of genes that are also upregulated in the brains of individuals with Alzheimer's disease. Placentas in DS are prematurely senescent, showing atypical evidence of mineralization. Fetuses with DS are especially susceptible to adverse obstetric outcomes, including early in utero demise, stillbirth and growth restriction, all of which are related to placental function. The placenta, therefore, may provide key insights towards understanding the phenotypic variability observed in individuals with DS and aid in identifying biomarkers that can be used to evaluate phenotypic severity and prenatal treatments in real time. To address these issues, many different mouse models of DS have been generated to identify the mechanisms underlying developmental changes in many organ systems. Little is known, however, regarding placental development in the currently available mouse models of DS. Based upon the relative paucity of data on placental development in preclinical mouse models of DS, we recommend that future evaluation of new and existing models routinely include histologic and functional assessments of the placenta. In this paper we summarize studies performed in the placentas of both humans and mouse models with DS, highlighting gaps in knowledge and suggesting directions for future research.

Challenges and Opportunities for Translation of Therapies to Improve Cognition in Down Syndrome.

While preclinical studies have reported improvement of behavioral deficits in the Ts65Dn mouse model of Down syndrome (DS), translation to human clinical trials to improve cognition in individuals with DS has had a poor success record. Timing of the intervention, choice of animal models, strategy for drug selection, and lack of translational endpoints between animals and humans contributed to prior failures of human clinical trials. Here, we focus on in vitro cell models from humans with DS to identify the molecular mechanisms underlying the brain phenotype associated with DS. We emphasize the importance of using these cell models to screen for therapeutic molecules, followed by validating them in the most suitable animal models prior to initiating human clinical trials.

Significant Effects of Maternal Diet During Pregnancy on the Murine Fetal Brain Transcriptome and Offspring Behavior.

BACKGROUND: Maternal over- and undernutrition in pregnancy plays a critical role in fetal brain development and function. The effects of different maternal diet compositions on intrauterine programing of the fetal brain is a lesser-explored area. The goal of this study was to investigate the impact of two chowmaternal diets on fetal brain gene expression signatures, fetal/neonatal growth, and neonatal and adult behavior in a mouse model. METHODS: Throughout pregnancy and lactation, female C57Bl/6J mice were fed one of two standard, commercially available chow diets (pellet versus powder). The powdered chow diet was relatively deficient in micronutrients and enriched for carbohydrates and n-3 long-chain polyunsaturated fatty acids compared to the pelleted chow. RNA was extracted from embryonic day 15.5 forebrains and hybridized to whole genome expression microarrays (N = 5/maternal diet group). Functional analyses of significantly differentially expressed fetal brain genes were performed using Ingenuity Pathways Analysis and Gene Set Enrichment Analysis. Neonatal behavior was assessed using a validated scale (N = 62 pellet-exposed and 31 powder-exposed). Hippocampal learning, locomotor behavior, and motor coordination were assessed in a subset of adults using fear conditioning, open field testing, and Rotarod tests (N = 16 pellet-exposed, 14 powder-exposed). RESULTS: Comparing powdered to pelleted chow diets, neither maternal weight trajectory in pregnancy nor embryo size differed. Maternal powdered chow diet was associated with 1647 differentially expressed fetal brain genes. Functional analyses identified significant upregulation of canonical pathways and upstream regulators involved in cell cycle regulation, synaptic plasticity, and sensory nervous system development in the fetal brain, and significant downregulation of pathways related to cell and embryo death. Pathways related to DNA damage response, brain immune response, amino acid and fatty acid transport, and dopaminergic signaling were significantly dysregulated. Powdered chow-exposed neonates were significantly longer but not heavier than pelleted chow-exposed counterparts. On neonatal behavioral testing, powdered chow-exposed neonates achieved coordination- and strength-related milestones significantly earlier, but sensory maturation reflexes significantly later. On adult behavioral testing, powdered chow-exposed offspring exhibited hyperactivity and hippocampal learning deficits. CONCLUSION: In wild-type offspring, two diets that differed primarily with respect to micronutrient composition had significant effects on the fetal brain transcriptome, neonatal and adult behavior. These effects did not appear to be mediated by alterations in gross maternal nutritional status nor fetal/neonatal weight. Maternal dietary content is an important variable to consider for investigators evaluating fetal brain development and offspring behavior.

Lifespan analysis of brain development, gene expression and behavioral phenotypes in the Ts1Cje, Ts65Dn and Dp(16)1/Yey mouse models of Down syndrome.

Down syndrome (DS) results from triplication of human chromosome 21. Neuropathological hallmarks of DS include atypical central nervous system development that manifests prenatally and extends throughout life. As a result, individuals with DS exhibit cognitive and motor deficits, and have delays in achieving developmental milestones. To determine whether different mouse models of DS recapitulate the human prenatal and postnatal phenotypes, here, we directly compared brain histogenesis, gene expression and behavior over the lifespan of three cytogenetically distinct mouse models of DS: Ts1Cje, Ts65Dn and Dp(16)1/Yey. Histological data indicated that Ts65Dn mice were the most consistently affected with respect to somatic growth, neurogenesis and brain morphogenesis. Embryonic and adult gene expression results showed that Ts1Cje and Ts65Dn brains had considerably more differentially expressed (DEX) genes compared with Dp(16)1/Yey mice, despite the larger number of triplicated genes in the latter model. In addition, DEX genes showed little overlap in identity and chromosomal distribution in the three models, leading to dissimilarities in affected functional pathways. Perinatal and adult behavioral testing also highlighted differences among the models in their abilities to achieve various developmental milestones and perform hippocampal- and motor-based tasks. Interestingly, Dp(16)1/Yey mice showed no abnormalities in prenatal brain phenotypes, yet they manifested behavioral deficits starting at postnatal day 15 that continued through adulthood. In contrast, Ts1Cje mice showed mildly abnormal embryonic brain phenotypes, but only select behavioral deficits as neonates and adults. Altogether, our data showed widespread and unexpected fundamental differences in behavioral, gene expression and brain development phenotypes between these three mouse models. Our findings illustrate unique limitations of each model when studying aspects of brain development and function in DS. This work helps to inform model selection in future studies investigating how observed neurodevelopmental abnormalities arise, how they contribute to cognitive impairment, and when testing therapeutic molecules to ameliorate the intellectual disability associated with DS.This article has an associated First Person interview with the first author of the paper.

Perinatal Natural History of the Ts1Cje Mouse Model of Down Syndrome: Growth Restriction, Early Mortality, Heart Defects, and Delayed Development.

BACKGROUND: The Ts1Cje model of Down syndrome is of particular interest for perinatal studies because affected males are fertile. This permits affected pups to be carried in wild-type females, which is similar to human pregnancies. Here we describe the early natural history and growth profiles of Ts1Cje embryos and neonates and determine if heart defects are present in this strain. METHODS: Pups were studied either on embryonic (E) day 15.5, or from postnatal (P) day 3 through weaning on P21. PCR amplification targeting the neomycin cassette (present in Ts1Cje) and Sry (present in males) was used to analyze pup genotypes and sex ratios. Body weights and lengths, as well as developmental milestones, were recorded in Ts1Cje mice and compared to their wild-type (WT) littermates. Histological evaluations were performed at E15.5 to investigate the presence or absence of heart defects. Pups were divided into two groups: Ts1Cje-I pups survived past weaning and Ts1Cje-II pups died at some point before P21. RESULTS: Ts1Cje mouse embryos showed expected Mendelian ratios (45.8%, n = 66 for Ts1Cje embryos; 54.2%, n = 78 for WT embryos). Histological analysis revealed the presence of ventricular septal defects (VSDs) in 21% of Ts1Cje E15.5 embryos. After weaning, only 28.2% of pups were Ts1Cje (185 Ts1Cje out of 656 total pups generated), with males predominating (male:female ratio of 1.4:1). Among the recovered dead pups (n = 207), Ts1Cje (63.3%, n = 131, p<0.01) genotype was found significantly more often than WT (36.7%, n = 76). Retrospective analysis of Ts1Cje-II (pre-weaning deceased) pups showed that they were growth restricted compared to Ts1Cje-I pups (post-weaning survivors). Growth restriction correlated with statistically significant delays in achieving several neonatal milestones between P3 and P21 compared to Ts1Cje-I (post-weaning survivors) neonates and WT littermates. CONCLUSIONS: Ts1Cje genotype is not associated with increased early in utero mortality. Cardiac defects, specifically VSDs, are part of the phenotype in this strain. There is increased neonatal mortality in Ts1Cje pups, with sex differences observed. Ts1Cje mice that died in the neonatal period were more likely to be growth restricted and delayed in achieving neonatal developmental milestones.

Changing Paradigms in Down Syndrome: The First International Conference of the Trisomy 21 Research Society.

Down syndrome (DS) is the most common genetic cause of intellectual disability (ID) in humans with an incidence of ∼1:1,000 live births worldwide. It is caused by the presence of an extra copy of all or a segment of the long arm of human chromosome 21 (trisomy 21). People with DS present with a constellation of phenotypic alterations involving most organs and organ systems. ID is present in all people with DS, albeit with variable severity. DS is also the most frequent genetic cause of Alzheimer's disease (AD), and ∼50% of those with DS will develop AD-related dementia. In the last few years, significant progress has been made in understanding the crucial genotype-phenotype relationships in DS, in identifying the alterations in molecular pathways leading to the various clinical conditions present in DS, and in preclinical evaluations of potential therapies to improve the overall health and well-being of individuals with DS. In June 2015, 230 scientists, advocates, patients, and family members met in Paris for the 1st International Conference of the Trisomy 21 Research Society. Here, we report some of the most relevant presentations that took place during the meeting.

An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome.

Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice.

Males are from Mars, and females are from Venus: sex-specific fetal brain gene expression signatures in a mouse model of maternal diet-induced obesity.

BACKGROUND: Maternal obesity is associated with adverse neurodevelopmental outcomes in children, including autism spectrum disorders, developmental delay, and attention-deficit hyperactivity disorder. The underlying mechanisms remain unclear. We previously identified second-trimester amniotic fluid and term cord blood gene expression patterns suggesting dysregulated brain development in fetuses of obese compared with lean women. OBJECTIVE: We sought to investigate the biological significance of these findings in a mouse model of maternal diet-induced obesity. We evaluated sex-specific differences in fetal growth, brain gene expression signatures, and associated pathways. STUDY DESIGN: Female C57BL/6J mice were fed a 60% high-fat diet or 10% fat control diet for 12-14 weeks prior to mating. During pregnancy, obese dams continued on the high-fat diet or transitioned to the control diet. Lean dams stayed on the control diet. On embryonic day 17.5, embryos were weighed and fetal brains were snap frozen. RNA was extracted from male and female forebrains (10 per diet group per sex) and hybridized to whole-genome expression arrays. Significantly differentially expressed genes were identified using a Welch's t test with the Benjamini-Hochberg correction. Functional analyses were performed using ingenuity pathways analysis and gene set enrichment analysis. RESULTS: Embryos of dams on the high-fat diet were significantly smaller than controls, with males more severely affected than females (P = .01). Maternal obesity and maternal obesity with dietary change in pregnancy resulted in significantly more dysregulated genes in male vs female fetal brains (386 vs 66, P < .001). Maternal obesity with and without dietary change in pregnancy was associated with unique brain gene expression signatures for each sex, with an overlap of only 1 gene. Changing obese dams to a control diet in pregnancy resulted in more differentially expressed genes in the fetal brain than maternal obesity alone. Functional analyses identified common dysregulated pathways in both sexes, but maternal obesity and maternal dietary change affected different aspects of brain development in males compared with females. CONCLUSION: Maternal obesity is associated with sex-specific differences in fetal size and fetal brain gene expression signatures. Male fetal growth and brain gene expression may be more sensitive to environmental influences during pregnancy. Maternal diet during pregnancy has a significant impact on the embryonic brain transcriptome. It is important to consider both fetal sex and maternal diet when evaluating the effects of maternal obesity on fetal neurodevelopment.

Absence of Prenatal Forebrain Defects in the Dp(16)1Yey/+ Mouse Model of Down Syndrome.

Studies in humans with Down syndrome (DS) show that alterations in fetal brain development are followed by postnatal deficits in neuronal numbers, synaptic plasticity, and cognitive and motor function. This same progression is replicated in several mouse models of DS. Dp(16)1Yey/+ (hereafter called Dp16) is a recently developed mouse model of DS in which the entire region of mouse chromosome 16 that is homologous to human chromosome 21 has been triplicated. As such, Dp16 mice may more closely reproduce neurodevelopmental changes occurring in humans with DS. Here, we present the first comprehensive cellular and behavioral study of the Dp16 forebrain from embryonic to adult stages. Unexpectedly, our results demonstrate that Dp16 mice do not have prenatal brain defects previously reported in human fetal neocortex and in the developing forebrains of other mouse models, including microcephaly, reduced neurogenesis, and abnormal cell proliferation. Nevertheless, we found impairments in postnatal developmental milestones, fewer inhibitory forebrain neurons, and deficits in motor and cognitive performance in Dp16 mice. Therefore, although this new model does not express prenatal morphological phenotypes associated with DS, abnormalities in the postnatal period appear sufficient to produce significant cognitive deficits in Dp16.

Pharmacological correction of excitation/inhibition imbalance in Down syndrome mouse models.

Cognitive impairment in Down syndrome (DS) has been linked to increased synaptic inhibition. The underlying mechanisms remain unknown, but memory deficits are rescued in DS mouse models by drugs targeting GABA receptors. Similarly, administration of epigallocatechin gallate (EGCG)-containing extracts rescues cognitive phenotypes in Ts65Dn mice, potentially through GABA pathway. Some developmental and cognitive alterations have been traced to increased expression of the serine-threonine kinase DYRK1A on Hsa21. To better understand excitation/inhibition balance in DS, we investigated the consequences of long-term (1-month) treatment with EGCG-containing extracts in adult mBACtgDyrk1a mice that overexpress Dyrk1a. Administration of POL60 rescued components of GABAergic and glutamatergic pathways in cortex and hippocampus but not cerebellum. An intermediate dose (60 mg/kg) of decaffeinated green tea extract (MGTE) acted on components of both GABAergic and glutamatergic pathways and rescued behavioral deficits as demonstrated on the alternating paradigm, but did not rescue protein level of GABA-synthesizing GAD67. These results indicate that excessive synaptic inhibition in people with DS may be attributable, in large part, to increased DYRK1A dosage. Thus, controlling the level of active DYRK1A is a clear issue for DS therapy. This study also defines a panel of synaptic markers for further characterization of DS treatments in murine models.

The fetal brain transcriptome and neonatal behavioral phenotype in the Ts1Cje mouse model of Down syndrome.

Human fetuses with Down syndrome demonstrate abnormal brain growth and reduced neurogenesis. Despite the prenatal onset of the phenotype, most therapeutic trials have been conducted in adults. Here, we present evidence for fetal brain molecular and neonatal behavioral alterations in the Ts1Cje mouse model of Down syndrome. Embryonic day 15.5 brain hemisphere RNA from Ts1Cje embryos (n = 5) and wild type littermates (n = 5) was processed and hybridized to mouse gene 1.0 ST arrays. Bioinformatic analyses were implemented to identify differential gene and pathway regulation during Ts1Cje fetal brain development. In separate experiments, the Fox scale, ultrasonic vocalization and homing tests were used to investigate behavioral deficits in Ts1Cje pups (n = 29) versus WT littermates (n = 64) at postnatal days 3-21. Ts1Cje fetal brains displayed more differentially regulated genes (n = 71) than adult (n = 31) when compared to their age-matched euploid brains. Ts1Cje embryonic brains showed up-regulation of cell cycle markers and down-regulation of the solute-carrier amino acid transporters. Several cellular processes were dysregulated at both stages, including apoptosis, inflammation, Jak/Stat signaling, G-protein signaling, and oxidoreductase activity. In addition, early behavioral deficits in surface righting, cliff aversion, negative geotaxis, forelimb grasp, ultrasonic vocalization, and the homing tests were observed. The Ts1Cje mouse model exhibits abnormal gene expression during fetal brain development, and significant neonatal behavioral deficits in the pre-weaning period. In combination with human studies, this suggests that the Down syndrome phenotype manifests prenatally and provides a rationale for prenatal therapy to improve perinatal brain development and postnatal neurocognition.

Analysis of adult cerebral cortex and hippocampus transcriptomes reveals unique molecular changes in the Ts1Cje mouse model of down syndrome.

We investigated gene expression and functional differences between Ts1Cje mice and wild-type (WT) littermates in adult cerebral cortex and hippocampus. These two brain regions are affected in people with Down syndrome, but have not been previously molecularly characterized in Ts1Cje mice. Total RNA was prepared from the brains of 8-10-week-old Ts1Cje mice (n = 6) and WT littermates (n = 5) and hybridized to Affymetrix 1.0 ST gene mouse arrays. Differentially regulated genes were identified and used to perform in silico functional analyses to better characterize dysregulated pathways in both brain regions. Hippocampus had more significantly differentially expressed genes compared with cortex (30 vs. 7 at a Benjamini-Hochberg false discovery rate of 20%). We identified novel genes that were differentially regulated in adult brains, including Cyb5r1, Fsbp, Vmn2r110, Snd1 and Zhx2. Functional analyses in Ts1Cje mice highlighted the importance of NFAT signaling, oxidative stress, neuroinflammation and olfactory perception via G-protein signaling. In a comparison of adult Ts1Cje and WT brains, we identified new genes and pathway differences in the cortex and hippocampus. Our analyses identified physiologically relevant pathways that can serve as targets for the development of future treatments to improve neurocognition in Down syndrome.

Excitation/inhibition balance and learning are modified by Dyrk1a gene dosage.

Cognitive deficits in Down syndrome (DS) have been linked to increased synaptic inhibition, leading to an imbalance of excitation/inhibition (E/I). Various mouse models and studies from human brains have implicated an HSA21 gene, the serine/threonine kinase DYRK1A, as a candidate for inducing cognitive dysfunction. Here, consequences of alterations in Dyrk1a dosage were assessed in mouse models with varying copy numbers of Dyrk1a: mBACtgDyrk1a, Ts65Dn and Dp(16)1Yey (with 3 gene copies) and Dyrk1a(+/-) (one functional copy). Molecular (i.e. immunoblotting/immunohistochemistry) and behavioral analyses (e.g., rotarod, Morris water maze, Y-maze) were performed in mBACtgDyrk1a mice. Increased expression of DYRK1A in mBACtgDyrk1a induced molecular alterations in synaptic plasticity pathways, particularly expression changes in GABAergic and glutaminergic related proteins. Similar alterations were observed in models with partial trisomy of MMU16, Ts65Dn and Dp(16)1Yey, and were reversed in the Dyrk1a(+/-) model. Dyrk1a overexpression produced an increased number and signal intensity of GAD67 positive neurons, indicating enhanced inhibition pathways in three different models: mBACtgDyrk1a, hYACtgDyrk1a and Dp(16)1Yey. Functionally, Dyrk1a overexpression protected mice from PTZ-induced seizures related to GABAergic neuron plasticity. Our study shows that DYRK1A overexpression affects pathways involved in synaptogenesis and synaptic plasticity and influences E/I balance toward inhibition. Inhibition of DYRK1A activity offers a therapeutic target for DS, but its inhibition/activation may also be relevant for other psychiatric diseases with E/I balance alterations.

Prenatal treatment of Down syndrome: a reality?

PURPOSE OF REVIEW: Down syndrome affects more than 5 million people globally. During the last 10 years, there has been a dramatic increase in the research efforts focused on therapeutic interventions to improve learning and memory in Down syndrome. RECENT FINDINGS: This review summarizes the different functional abnormalities targeted by researchers in mouse models of Down syndrome. Three main strategies have been used: neural stem cell implantation; environmental enrichment and physical exercise; and pharmacotherapy. Pharmacological targets include the choline pathway, GABA and NMDA receptors, DYRK1A protein, oxidative stress and pathways involved in development and neurogenesis. Many strategies have improved learning and memory as well as electrophysiological and molecular alterations in affected animals. To date, eight molecules have been tested in human adult clinical trials. No studies have yet been performed on infants. However, compelling studies reveal that permanent brain alterations originate during fetal life in Down syndrome. Early prenatal diagnosis offers a 28 weeks window to positively impact brain development and improve postnatal cognitive outcome in affected individuals. Only a few approaches (Epigallocatechine gallate, NAP/SAL, fluoxetine, and apigenin) have been used to treat mice in utero; these showed therapeutic effects that persisted to adulthood. SUMMARY: In this article, we discuss the challenges, recent progress, and lessons learned that pave the way for new therapeutic approaches in Down syndrome.

Prefrontal deficits in a murine model overexpressing the down syndrome candidate gene dyrk1a.

The gene Dyrk1a is the mammalian ortholog of Drosophila minibrain. Dyrk1a localizes in the Down syndrome (DS) critical region of chromosome 21q22.2 and is a major candidate for the behavioral and neuronal abnormalities associated with DS. PFC malfunctions are a common denominator in several neuropsychiatric diseases, including DS, but the contribution of DYRK1A in PFC dysfunctions, in particular the synaptic basis for impairments of executive functions reported in DS patients, remains obscure. We quantified synaptic plasticity, biochemical synaptic markers, and dendritic morphology of deep layer pyramidal PFC neurons in adult mBACtgDyrk1a transgenic mice that overexpress Dyrk1a under the control of its own regulatory sequences. We found that overexpression of Dyrk1a largely increased the number of spines on oblique dendrites of pyramidal neurons, as evidenced by augmented spine density, higher PSD95 protein levels, and larger miniature EPSCs. The dendritic alterations were associated with anomalous NMDAR-mediated long-term potentiation and accompanied by a marked reduction in the pCaMKII/CaMKII ratio in mBACtgDyrk1a mice. Retrograde endocannabinoid-mediated long-term depression (eCB-LTD) was ablated in mBACtgDyrk1a mice. Administration of green tea extracts containing epigallocatechin 3-gallate, a potent DYRK1A inhibitor, to adult mBACtgDyrk1a mice normalized long-term potentiation and spine anomalies but not eCB-LTD. However, inhibition of the eCB deactivating enzyme monoacylglycerol lipase normalized eCB-LTD in mBACtgDyrk1a mice. These data shed light on previously undisclosed participation of DYRK1A in adult PFC dendritic structures and synaptic plasticity. Furthermore, they suggest its involvement in DS-related endophenotypes and identify new potential therapeutic strategies.