A high-performance liquid chromatography assay for Dyrk1a,a Down syndrome-associated kinase

Down syndrome is the most common aneuploidy. It is caused by the presence of an extra copy of chromosome 21. Several studies indicate that aberrant expression of the kinase Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is implicated in Down syndrome, in particular in the onset of mental retardation. Moreover, elevated Dyrk1a activity may also be a risk factor for other neurodegenerative disorders such as Alzheimer’s disease. Over the past years, Dyrk1a has appeared as a potential drug target. Availability of sensitive and quantitative enzyme assays is of prime importance to understand the role of Dyrk1a and to develop specific inhibitors. Here, we describe a new method to measure Dyrk1a activity based on the separation and quantification of specific fluorescent peptides (substrate and phosphorylated product) by high-performance liquid chromatography (HPLC). Kinetic and mechanistic analyses using well-known inhibitors of Dyrk1a confirmed the reliability of this approach. In addition, this assay was further validated using brain extracts of mice models expressing different copies of the Dyrk1a gene. Our results indicate that this novel Dyrk1a assay is simple, sensitive, and specific. It avoids the use of radioactivity-based approaches that, until now, have been widely employed to measure Dyrk1a activity.     

Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-regulated alternative splicing of tau in Down syndrome

Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS.     

Overexpression of Dyrk1A Is Implicated in Several Cognitive,Electrophysiological and Neuromorphological Alterations Found in a Mouse Model of Down Syndrome

Down syndrome (DS) phenotypes result from the overexpression of several dosage-sensitive genes. The DYRK1A (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A) gene, which has been implicated in the behavioral and neuronal alterations that are characteristic of DS, plays a role in neuronal progenitor proliferation, neuronal differentiation and long-term potentiation (LTP) mechanisms that contribute to the cognitive deficits found in DS. The purpose of this study was to evaluate the effect of Dyrk1A overexpression on the behavioral and cognitive alterations in the Ts65Dn (TS) mouse model, which is the most commonly utilized mouse model of DS, as well as on several neuromorphological and electrophysiological properties proposed to underlie these deficits. In this study, we analyzed the phenotypic differences in the progeny obtained from crosses of TS females and heterozygous Dyrk1A (+/−) male mice. Our results revealed that normalization of the Dyrk1A copy number in TS mice improved working and reference memory based on the Morris water maze and contextual conditioning based on the fear conditioning test and rescued hippocampal LTP. Concomitant with these functional improvements, normalization of the Dyrk1A expression level in TS mice restored the proliferation and differentiation of hippocampal cells in the adult dentate gyrus (DG) and the density of GABAergic and glutamatergic synapse markers in the molecular layer of the hippocampus. However, normalization of the Dyrk1A gene dosage did not affect other structural (e.g., the density of mature hippocampal granule cells, the DG volume and the subgranular zone area) or behavioral (i.e., hyperactivity/attention) alterations found in the TS mouse. These results suggest that Dyrk1A overexpression is involved in some of the cognitive, electrophysiological and neuromorphological alterations, but not in the structural alterations found in DS, and suggest that pharmacological strategies targeting this gene may improve the treatment of DS-associated learning disabilities.     

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.     

The App-Runx1 region is critical for birth defects and electrocardiographic dysfunctions observed in a Down syndrome mouse model

Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality (Coq7, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, and Vwf) and heart function (Tfb1m, Adam19, Slc8a1/Ncx1, and Rcan1). In addition cardiac connexins (Cx40, Cx43) and sodium channel sub-units (Scn5a, Scn1b, Scn10a) were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people.     

Dysregulation of gene expression in mouse trisomy 16, an animal model of Down syndrome.

In humans, trisomy 21 results in a specific phenotype known as Down syndrome (DS). The mechanism by which an extra copy of normal genes leads to the DS phenotype is unknown. Most studies in DS and other aneuploid organisms have shown that gene dose is proportional to gene expression. To date, most genes examined have encoded either metabolic enzymes or constitutively expressed products. In the trisomy 16 mouse, an animal model of DS, we found marked dysregulation of two developmentally regulated genes, App and Prn-p. Dysregulation varied from tissue to tissue and during development in the same tissue. We conclude that abnormal phenotypes seen in aneuploid conditions may result in part from disordered expression of developmentally regulated genes.     

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.     

Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome

Converging clinical data suggest that peripheral inflammation is likely involved in the pathogenesis of the neuropsychiatric symptoms associated with metabolic syndrome (MetS). However, the question arises as to whether the increased prevalence of behavioral alterations in MetS is also associated with central inflammation, i.e. cytokine activation, in brain areas particularly involved in controlling behavior. To answer this question, we measured in a mouse model of MetS, namely the diabetic and obese db/db mice, and in their healthy db/+ littermates emotional behaviors and memory performances, as well as plasma levels and brain expression (hippocampus; hypothalamus) of inflammatory cytokines. Our results shows that db/db mice displayed increased anxiety-like behaviors in the open-field and the elevated plus-maze (i.e. reduced percent of time spent in anxiogenic areas of each device), but not depressive-like behaviors as assessed by immobility time in the forced swim and tail suspension tests. Moreover, db/db mice displayed impaired spatial recognition memory (hippocampus-dependent task), but unaltered object recognition memory (hippocampus-independent task). In agreement with the well-established role of the hippocampus in anxiety-like behavior and spatial memory, behavioral alterations of db/db mice were associated with increased inflammatory cytokines (interleukin-1β, tumor necrosis factor-α and interleukin-6) and reduced expression of brain-derived neurotrophic factor (BDNF) in the hippocampus but not the hypothalamus. These results strongly point to interactions between cytokines and central processes involving the hippocampus as important contributing factor to the behavioral alterations of db/db mice. These findings may prove valuable for introducing novel approaches to treat neuropsychiatric complications associated with MetS.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Transduced Tat-glyoxalase protein attenuates streptozotocin-induced diabetes in a mouse model

Diabetes mellitus (DM) is characterized by hyperglycemia. Glyoxalase 1 (GLO) has considerable potential as a possible therapeutic agent for DM. However, the precise action of GLO remains unclear in DM. In this study, we examined the protective effects of GLO protein in a streptozotocin (STZ)-induced diabetes animal model using cell-permeable Tat-GLO protein. Purified Tat-GLO protein was efficiently transduced into RINm5F cells in a time- and dose-dependent manner and protected cells against sodium nitroprusside (SNP)-induced cell death and DNA fragmentation. Furthermore, Tat-GLO protein significantly inhibited blood glucose levels and altered the serum biochemical parameters in STZ-induced diabetic mice. These results demonstrate that transduced Tat-GLO protein protects pancreatic cells by the inhibition of STZ-mediated toxicity. Therefore, Tat-GLO protein could be useful as a therapeutic agent against DM.     

Thrombin-induced autoinhibitory factor, Down syndrome critical region-1, attenuates NFAT-dependent vascular cell adhesion molecule-1 expression and inflammation in the endothelium

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.     

Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis

Background

Viral bronchiolitis is the leading cause of hospitalization in young infants. It is associated with the development of childhood asthma and contributes to morbidity and mortality in the elderly. Currently no therapies effectively attenuate inflammation during the acute viral infection, or prevent the risk of post-viral asthma. We hypothesized that early treatment of a paramyxoviral bronchiolitis with azithromycin would attenuate acute and chronic airway inflammation.

Methods

Mice were inoculated with parainfluenza type 1, Sendai Virus (SeV), and treated daily with PBS or azithromycin for 7 days post-inoculation. On day 8 and 21 we assessed airway inflammation in lung tissue, and quantified immune cells and inflammatory mediators in bronchoalveolar lavage (BAL).

Results

Compared to treatment with PBS, azithromycin significantly attenuated post-viral weight loss. During the peak of acute inflammation (day 8), azithromycin decreased total leukocyte accumulation in the lung tissue and BAL, with the largest fold-reduction in BAL neutrophils. This decreased inflammation was independent of changes in viral load. Azithromycin significantly attenuated the concentration of BAL inflammatory mediators and enhanced resolution of chronic airway inflammation evident by decreased BAL inflammatory mediators on day 21.

Conclusions

In this mouse model of paramyxoviral bronchiolitis, azithromycin attenuated acute and chronic airway inflammation. These findings demonstrate anti-inflammatory effects of azithromycin that are not related to anti-viral activity. Our findings support the rationale for future prospective randomized clinical trials that will evaluate the effects of macrolides on acute viral bronchiolitis and their long-term consequences.     

Lung proteome alterations in a mouse model for nonallergic asthma

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.     

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

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

CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.     

Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. Endothelial cell activation is mediated by many different extra-cellular signals, which result in overlapping yet distinct patterns of gene expression. Here we show, in DNA microarray analyses, that vascular endothelial growth factor (VEGF) and thrombin result in dramatic and rapid upregulation of Down syndrome critical region (DSCR)-1 gene encoding exons 4-7, a negative feedback regulator of calcium-calcineurin-NF-AT signaling. VEGF- and thrombin-mediated induction of DSCR-1 involves the cooperative binding of NF-ATc and GATA-2/3 to neighboring consensus motifs in the upstream promoter. Constitutive expression of DSCR-1 in endothelial cells markedly impaired NF-ATc nuclear localization, proliferation, and tube formation. Under in vivo conditions, overexpression of DSCR-1 reduced vascular density in matrigel plugs and melanoma tumor growth in mice. Taken together, these findings support a model in which VEGF- and thrombin-mediated induction of endothelial cell proliferation triggers a negative feedback loop consisting of DSCR-1 gene induction and secondary inhibition of NF-AT signaling. As a natural brake in the angiogenic process, this negative pathway may lend itself to therapeutic manipulation in pathological states.