DYRK1A, a Novel Determinant of the Methionine-Homocysteine Cycle in Different Mouse Models Overexpressing this Down-Syndrome-Associated Kinase

BACKGROUND:Hyperhomocysteinemia, characterized by increased plasma homocysteine level, is associated with an increased risk of atherosclerosis. On the contrary, patients with Down syndrome appear to be protected from the development of atherosclerosis. We previously found a deleterious effect of hyperhomocysteinemia on expression of DYRK1A, a Down-syndrome-associated kinase. As increased expression of DYRK1A and low plasma homocysteine level have been associated with Down syndrome, we aimed to analyze the effect of its over-expression on homocysteine metabolism in mice. METHODOLOGY/PRINCIPAL FINDINGS:Effects of DYRK1A over-expression were examined by biochemical analysis of methionine metabolites, real-time quantitative reverse-transcription polymerase chain reaction, and enzyme activities. We found that over-expression of Dyrk1a increased the hepatic NAD(P)H:quinone oxidoreductase and S-adenosylhomocysteine hydrolase activities, concomitant with decreased level of plasma homocysteine in three mice models overexpressing Dyrk1a. Moreover, these effects were abolished by treatment with harmine, the most potent and specific inhibitor of Dyrk1a. The increased NAD(P)H:quinone oxidoreductase and S-adenosylhomocysteine hydrolase activities were also found in lymphoblastoid cell lines from patients with Down syndrome. CONCLUSIONS/SIGNIFICANCE:Our results might give clues to understand the protective effect of Down syndrome against vascular defect through a decrease of homocysteine level by DYRK1A over-expression. They reveal a link between the Dyrk1a signaling pathway and the homocysteine cycle

Molecular signatures of cardiac defects in down syndrome lymphoblastoid cell lines suggest altered ciliome and hedgehog pathways

Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD) and less frequently a ventricular septal defect (VSD) or atrial septal defect (ASD). Lymphoblastoid cell lines (LCLs) were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD-; n = 22) were compared with those of LCLs from patients with cardiac malformations (CHD+; n = 21). After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD) carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD- and AVSD and CHD- and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset). Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21. © 2012 Ripoll et al

Effects of harmine on hepatic SAHH and NQO1 activities, and on plasma Hcy levels in Tg 189N3 mice.

<p>Hepatic SAHH (A) and NQO1 (B) activities are presented as percent of untreated (Vehicle) non-transgenic (Tg –) mice activities. (C) Plasma Hcy level. Data correspond to means ± SEM and the statistical analysis was done with one-way ANOVA followed by Student's unpaired <i>t</i>-test. n = number of mice.</p

Relative expression of DYRK1A, SAHH and NQO1 and SAHH and NQO1 activities obtained from lymphoblastoid cell lines.

<p>The values of lymphoblastoid cell lines (LCLs) from patients with DS (T21) were normalized to the mean lymphoblastoid cell lines from control individuals (control). Data correspond to means ± SEM and the statistical analysis was done by Student's unpaired <i>t</i>-tests. n = number of LCLs. * <i>p</i><0.05; <b>^†</b><i>p</i><0.006.</p

Relative expression of SAHH, BHMT, MS and MTHFR gene based upon Q-PCR data obtained from non-transgenic and Tg189N3 mice.

<p>The values of Tg189N3 were normalized to the mean Tg – mice. Data correspond to means ± SEM and the statistical analysis was done by Student's unpaired <i>t</i>-tests. n = number of mice. *<i>p</i> = 0.068; <b>^†</b><i>p</i>< 0.03.</p

Hepatic DYRK1A mRNA and protein expression in liver of transgenic mice.

<p>Relative expression of DYRK1A gene was based on Q-PCR data and protein expression was determined by normalization of the density of images from DYRK1A with that of β-actin of the same blot. The values of Tg 152F7, Tg189N3 and Ts65Dn were normalized to the mean Tg – mice from each lines. The blots are representative of three independent experiments. Data correspond to means ± SEM and the statistical analysis was done by Student's unpaired <i>t</i>-tests. n = number of mice.</p

Transcriptome signature of heart defects.

<p>Comparison of ratios for DE genes up- or down-regulated in the AVSD or ASD+VSD group between (1) array ratio in the Ts21 CHD⁻ vs 2N, (2) array ratio in CHD⁺ vs CHD⁻, (3) q-PCR ratio from different experiments in CHD⁺ vs CHD⁻ using additional samples (total number in parentheses). Gene nomenclature, <i>AUTS2</i>: autism susceptibility candidate 2; <i>CNN2</i>: calponin 2; <i>DSCR3</i>: Down syndrome critical region gene 3; <i>DYNLT3</i>: dynein, light chain, Tctex-type 3; <i>NQO1</i>: NAD(P)H dehydrogenase, quinone 1; <i>OFD1</i>: oral-facial-digital syndrome 1; <i>PDIA4</i>: protein disulfide isomerase family A, member 4; <i>PIGP</i>: phosphatidylinositol glycan anchor biosynthesis, class P; <i>TTC3</i>: tetratricopeptide repeat domain 3; <i>TUBB2B</i>: tubulin, beta 2B; <i>ACTG1</i>: actin, gamma 1; <i>ALDOC</i>: aldolase C, fructose-bisphosphate; <i>CACYBP</i>: calcyclin binding protein; <i>DTYMK</i>: deoxythymidylate kinase (thymidylate kinase); <i>ENO2</i>: enolase 2 (gamma, neuronal); <i>GART</i>: phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase; <i>IFNAR1</i>: interferon (alpha, beta, and omega) receptor 1; <i>PLTP</i>: phospholipid transfer protein; <i>RPL10A</i>: ribosomal protein L10a; <i>TNFAIP2</i>: tumor necrosis factor, alpha-induced protein 2.</p

Hepatic SAHH activity is increased in Tg 189N3 and Ts65Dn transgenic mice.

<p>SAHH activity in (A) Tg 189N3 and (B) Ts65Dn transgenic mice. The values were normalized to the mean of Tg – mice from each line. Data correspond to means ± SEM and the statistical analysis was done by Student's unpaired <i>t</i>-test. n = number of mice.</p

DYRK1A protein expression in liver of CBS-deficient mice crossbred with 152F7 transgenic mice.

<p>(A) Western immunoblots showing DYRK1A expression in liver of wild type mice (<i>Cbs</i>^+/+ Tg -), heterozygous mice (<i>Cbs</i>^+/− Tg -), 152F7 transgenic mice (<i>Cbs</i>^+/+ Tg 152F7), and heterozygous mice crossbred with 152F7 transgenic mice (<i>Cbs</i>^+/− Tg 152F7). Proteins were subjected to immunoblot analysis using antibodies specific to DYRK1A (85.5 kDa). After stripping, the membranes were reprobed with anti-β-actin antibody (41.7 kDa) for the control. (B) Relative protein expression was determined by normalization of the density of images from DYRK1A with that of β-actin of the same blot. The values of <i>Cbs</i>^+/− Tg -, <i>Cbs</i>^+/+ Tg 152F7, or <i>Cbs</i>^+/− Tg 152F7 were normalized to the mean of <i>Cbs</i>^+/+ Tg - mice. The blots are representative of three independent experiments. Data correspond to means ± SEM and the statistical analysis was done with one-way ANOVA followed by Student's unpaired <i>t</i>-tests. n = number of mice.</p