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.     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).     

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.     

Altered synaptic marker abundance in the hippocampal stratum oriens of Ts65Dn mice is associated with exuberant expression of versican

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.     

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.     

Touchscreen learning deficits in Ube3a,Ts65Dn and Mecp2 mouse models of neurodevelopmental disorders with intellectual disabilities

Mutant mouse models of neurodevelopmental disorders with intellectual disabilities provide useful translational research tools, especially in cases where robust cognitive deficits are reproducibly detected. However, motor, sensory and/or health issues consequent to the mutation may introduce artifacts that preclude testing in some standard cognitive assays. Touchscreen learning and memory tasks in small operant chambers have the potential to circumvent these confounds. Here we use touchscreen visual discrimination learning to evaluate performance in the maternally derived Ube3a mouse model of Angelman syndrome, the Ts65Dn trisomy mouse model of Down syndrome, and the Mecp2^Bird mouse model of Rett syndrome. Significant deficits in acquisition of a 2‐choice visual discrimination task were detected in both Ube3a and Ts65Dn mice. Procedural control measures showed no genotype differences during pretraining phases or during acquisition. Mecp2 males did not survive long enough for touchscreen training, consistent with previous reports. Most Mecp2 females failed on pretraining criteria. Significant impairments on Morris water maze spatial learning were detected in both Ube3a and Ts65Dn, replicating previous findings. Abnormalities on rotarod in Ube3a, and on open field in Ts65Dn, replicating previous findings, may have contributed to the observed acquisition deficits and swim speed abnormalities during water maze performance. In contrast, these motor phenotypes do not appear to have affected touchscreen procedural abilities during pretraining or visual discrimination training. Our findings of slower touchscreen learning in 2 mouse models of neurodevelopmental disorders with intellectual disabilities indicate that operant tasks offer promising outcome measures for the preclinical discovery of effective pharmacological therapeutics.     

Temporal and brain region‐specific elevations of soluble Amyloid‐β40‐42 in the Ts65Dn mouse model of Down syndrome and Alzheimer’s disease

Down syndrome (DS) is a leading cause of intellectual disability that also results in hallmark Alzheimer''s disease (AD) pathologies such as amyloid beta (Aβ) plaques and hyperphosphorylated tau. The Ts65Dn mouse model is commonly used to study DS, as trisomic Ts65Dn mice carry 2/3 of the triplicated gene homologues as occur in human DS. The Ts65Dn strain also allows investigation of mechanisms common to DS and AD pathology, with many of these triplicated genes implicated in AD; for example, trisomic Ts65Dn mice overproduce amyloid precursor protein (APP), which is then processed into soluble Aβ_40‐42 fragments. Notably, Ts65Dn mice show alterations to the basal forebrain, which parallels the loss of function in this region observed in DS and AD patients early on in disease progression. However, a complete picture of soluble Aβ_40‐42 accumulation in a region‐, age‐, and sex‐specific manner has not yet been characterized in the Ts65Dn model. Here, we show that trisomic mice accumulate soluble Aβ_40‐42 in the basal forebrain, frontal cortex, hippocampus, and cerebellum in an age‐specific manner, with elevation in the frontal cortex and hippocampus as early as 4 months of age. Furthermore, we detected sex differences in accumulation of Aβ_40‐42 within the basal forebrain, with females having significantly higher Aβ_40‐42 at 7–8 months of age. Lastly, we show that APP expression in the basal forebrain and hippocampus inversely correlates with Aβ_40‐42 levels. This spatial and temporal characterization of soluble Aβ_40‐42 in the Ts65Dn model allows for further exploration of the role soluble Aβ plays in the progression of other AD‐like pathologies in these key brain regions.     

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.     

The GAD65 knock out mouse – a model for GABAergic processes in fear‐ and stress‐induced psychopathology

The γ‐amino butyric acid (GABA) synthetic enzyme glutamic acid decarboxylase (GAD)65 is critically involved in the activity‐dependent regulation of GABAergic inhibition in the central nervous system. It is also required for the maturation of the GABAergic system during adolescence, a phase that is critical for the development of several neuropsychiatric diseases. Mice bearing a null mutation of the GAD65 gene develop hyperexcitability of the amygdala and hippocampus, and a phenotype of increased anxiety and pathological fear memory reminiscent of posttraumatic stress disorder. Although genetic association of GAD65 in human has not yet been reported, these findings are in line with observations of reduced GABAergic function in these brain regions of anxiety disorder patients. The particular value of GAD65(?/?) mice thus lies in modeling the effects of reduced GABAergic function in the mature nervous system. The expression of GAD65 and a second GAD isozyme, GAD67, are differentially regulated in response to stress in limbic brain areas suggesting that by controlling GABAergic inhibition these enzymes determine the vulnerability for the development of pathological anxiety and other stress‐induced phenotypes. In fact, we could recently show that GAD65 haplodeficiency, which results in delayed postnatal increase of GABA levels, provides resilience to juvenile‐stress‐induced anxiety to GAD65(+/?) mice thus foiling the increased fear and anxiety in homozygous GAD65(?/?) mice.     

Identification and characterization of a new Down syndrome model, Ts[Rb(12.1716)]2Cje, resulting from a spontaneous Robertsonian fusion between T(1716)65Dn and mouseChromosome 12

The segmental trisomy model, Ts65Dn, has been a valuable resource for the study of the molecular and developmental processes associated with the pathogenesis of Down syndrome. However, male infertility and poor transmission of the small marker chromosome, T(17¹⁶)65Dn, carrying the distal end of mouse Chromosome 16 (MMU16) are limiting factors in the efficient production of these animals for experimental purposes. We describe here the identification and preliminary characterization of mice, designated Ts[Rb(12.17¹⁶)]2Cje, carrying a chromosomal rearrangement of the Ts65Dn genome whereby the marker chromosome has been translocated to Chromosome 12 (MMU12) forming a Robertsonian chromosome. This stable rearrangement confers fertility in males and increases the frequency of transmitted segmental trisomy through the female germline. We confirm retention of a dosage imbalance of human Chromosome 21 (HSA21)-homologous genes from App to the telomere and expression levels similar to Ts65Dn within the triplicated region. In addition, we characterized the dendritic morphology of granule cells in the fascia dentata in Ts[Rb(12.17¹⁶)]2Cje and 2N control mice. Quantitative confocal microscopy revealed decreased spine density on the dendrites of dentate granule cells and significantly enlarged dendritic spines affecting the entire population in Ts[Rb(12.17¹⁶)]2Cje as compared to 2N controls. These findings document that the structural dendritic spine abnormalities are similar to those previously observed in Ts65Dn mice. We conclude that this new model of Down syndrome offers reproductive advantages without sacrificing the integrity of the Ts65Dn model.     

Reactive oxygen intermediates during programmed cell death induced in the thymus of the Ts(1716)65Dn mouse, a murine model for human Down's syndrome.

Down's syndrome (DS) is one of the most frequent genetic disorders in humans. It has been suggested that overexpression of copper-zinc superoxide dismutase (SOD-1) in DS may be involved in some of the abnormalities observed, mainly neurodegenerative and immunopathological processes. One of the consequences is early thymic involution. Recently, Ts(1716)65Dn mice (Ts65Dn mice), made segmentally trisomic for a chromosome 16 segment, fulfill the criteria for a DS model. To study the possible role of SOD-1 overexpression in thymocyte biology, we analyzed the role of reactive oxygen intermediates during in vivo and in vitro programmed cell death (PCD) induced in the thymus of Ts65Dn mice. Our main findings can be summarized as follows. Ts65Dn thymuses exhibit greater PCD activity than controls, as ascertained by a combination of morphological, histochemical, and ultrastructural procedures. Ts65Dn thymocytes were highly susceptible to PCD induced by both LPS (in vivo) and dexamethasone, a synthetic glucocorticoid agonist (both in vivo and in vitro). Thymus abnormalities were probably caused by SOD-1 hyperexpression in Ts65Dn cells, in that reactive oxygen intermediate generation (specifically H2O2 production) is enhanced in thymocytes and clearly correlates with apoptosis. Similarly, oxidative injury correlated with the formation of lipid peroxidation by-products and antioxidants which partly inhibit PCD in thymocytes.     

Substrain specific behavioral responses in male C57BL/6N and C57BL/6J mice to a shortened 21-hour day and high-fat diet

ABSTRACT

Altered circadian rhythms have negative consequences on health and behavior. Emerging evidence suggests genetics influences the physiological and behavioral responses to circadian disruption. We investigated the effects of a 21 h day (T = 21 cycle), with high-fat diet consumption, on locomotor activity, explorative behaviors, and health in male C57BL/6J and C57BL/6N mice. Mice were exposed to either a T = 24 or T = 21 cycle and given standard rodent chow (RC) or a 60% high-fat diet (HFD) followed by behavioral assays and physiological measures. We uncovered numerous strain differences within the behavioral and physiological assays, mainly that C57BL/6J mice exhibit reduced susceptibility to the obesogenic effects of (HFD) and anxiety-like behavior as well as increased circadian and novelty-induced locomotor activity compared to C57BL/6N mice. There were also substrain-specific differences in behavioral responses to the T = 21 cycle, including exploratory behaviors and circadian locomotor activity. Under the 21-h day, mice consuming RC displayed entrainment, while mice exposed to HFD exhibited a lengthening of activity rhythms. In the open-field and light-dark box, mice exposed to the T = 21 cycle had increased novelty-induced locomotor activity with no further effects of diet, suggesting daylength may affect mood-related behaviors. These results indicate that different circadian cycles impact metabolic and behavioral responses depending on genetic background, and despite circadian entrainment.     

Effects of individual and combined exposure to sodium arsenite and sodium fluoride on tissue oxidative stress, arsenic and fluoride levels in male mice

Arsenic and fluoride are potent toxicants, widely distributed through drinking water and food and often result in adverse health effects. The present study examined the effects of sodium meta-arsenite (100 mg/l in drinking water) and sodium fluoride (5 mg/kg, oral, once daily), administered either alone or in combination for 8 weeks, on various biochemical variables indicative of tissue oxidative stress and cell injury in Swiss albino male mice. A separate group was first exposed to arsenic for 4 weeks followed by 4 weeks of fluoride exposure. Exposure to arsenic or fluoride led to a significant depletion of blood delta-aminolevulinic acid dehydratase (ALAD) activity and glutathione (GSH) level. These changes were accompanied by increased level of blood and tissues reactive oxygen species (ROS) level. An increase in the level of liver and kidney thiobarbituric acid reactive substance (TBARS) along with a concomitant decrease in the activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) and reduced GSH content were observed in both arsenic and fluoride administered mice. The changes were significantly more pronounced in arsenic exposed animals than in fluoride. It was interesting to observe that during combined exposure the toxic effects were less pronounced compared to the effects of arsenic or fluoride alone. In some cases antagonistic effects were noted following co-exposure to arsenic and fluoride. Arsenic and fluoride concentration increased significantly on exposure. Interestingly, their concentration decreased significantly on concomitant exposure for 8 weeks. However, the group which was administered arsenic for 4 weeks followed by 4 weeks of fluoride administration showed no such protection suggesting that the antagonistic effect of fluoride on arsenic or vice versa is possible only during interaction at the gastro intestinal sites. These results are new and interesting and require further exploration.     

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.     

Neural responses to territorial challenge and nonsocial stress in male song sparrows: segregation, integration, and modulation by a vasopressin V1 antagonist

The present experiments were conducted to determine (1) which basal forebrain regions and/or their peptidergic components are responsive to social challenge and nonsocial stress, and (2) the influence of an arginine vasopressin V(1) antagonist (AVPa) on these responses. Experiments were conducted in wild-caught male song sparrows (Melospiza melodia) that were housed on seminatural territories (field-based flight cages). Subjects were each fitted with a chronic guide cannula directed at the lateral ventricle and exposed to one of five conditions before sacrifice and histochemistry: saline + simulated territorial intrusion (STI; consisting of song playback and presentation of a caged conspecific male), AVPa + STI, saline + empty cage, AVPa + empty cage, unhandled. Two tissue series were prepared and immunofluorescently double-labeled for ZENK (egr-1) protein and either arginine vasotocin (AVT; avian homologue of AVP) or corticotropin releasing factor (CRF). The results indicate that the neuronal populations that are sensitive to nonsocial stress (capture, handling and infusion) and STI are at least partially segregated. Increases in ZENK-immunoreactive (-ir) nuclei following handling and infusion were observed in a large number of areas, whereas neural responses that were specific to STI were more limited. However, multiple areas showed responses to both handling and STI. AVPa infusions significantly reduced or eliminated most experimental increases in ZENK-ir, suggesting a broad role for endogenous AVT in the modulation of baseline activity and/or stress responsivity, and a much more limited role in the specific response to social challenge. Particular attention is given to the numerous zones of the lateral septum (LS), which are differentially responsive to handling, STI, and V(1)-like receptor blockade. These data suggest that septal AVT modulates neural responses to general stressors, not social stimuli specifically. Thus, species differences in septal AVT function (as previously described in songbirds) likely reflect differences in the relationship of stress or anxiety to species-specific behaviors, or to behavior in species-typical contexts.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.