Building the Future Therapies for Down Syndrome: The Third International Conference of the T21 Research Society.

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.

Prenatal treatment with EGCG enriched green tea extract rescues GAD67 related developmental and cognitive defects in Down syndrome mouse models.

Down syndrome is a common genetic disorder caused by trisomy of chromosome 21. Brain development in affected foetuses might be improved through prenatal treatment. One potential target is DYRK1A, a multifunctional kinase encoded by chromosome 21 that, when overexpressed, alters neuronal excitation-inhibition balance and increases GAD67 interneuron density. We used a green tea extract enriched in EGCG to inhibit DYRK1A function only during gestation of transgenic mice overexpressing Dyrk1a (mBACtgDyrk1a). Adult mice treated prenatally displayed reduced levels of inhibitory markers, restored VGAT1/VGLUT1 balance, and rescued density of GAD67 interneurons. Similar results for gabaergic and glutamatergic markers and interneuron density were obtained in Dp(16)1Yey mice, trisomic for 140 chromosome 21 orthologs; thus, prenatal EGCG exhibits efficacy in a more complex DS model. Finally, cognitive and behaviour testing showed that adult Dp(16)1Yey mice treated prenatally had improved novel object recognition memory but do not show improvement with Y maze paradigm. These findings provide empirical support for a prenatal intervention that targets specific neural circuitries.

LPS-Induced Inflammation Abolishes the Effect of DYRK1A on IkB Stability in the Brain of Mice.

Down syndrome is characterized by premature aging and dementia with neurological features that mimic those found in Alzheimer's disease. This pathology in Down syndrome could be related to inflammation, which plays a role in other neurodegenerative diseases. We previously found a link between the NFkB pathway, long considered a prototypical proinflammatory signaling pathway, and the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is associated with early onset of Alzheimer's disease in Down syndrome patients. Here, we sought to determine the role of DYRK1A on regulation of the NFkB pathway in the mouse brain. We found that over-expression of Dyrk1A (on a C57BL/6J background) stabilizes IκBα protein levels by inhibition of calpain activity and increases cytoplasmic p65 sequestration in the mouse brain. In contrast, Dyrk1A-deficient mice (on a CD1 background) have decreased IκBα protein levels with an increased calpain activity and decreased cytoplasmic p65 sequestration in the brain. Taken together, our results demonstrate a role of DYRK1A in regulation of the NFkB pathway. However, decreased IκBα and DYRK1A protein levels associated with an increased calpain activity were found in the brains of mice over-expressing Dyrk1A after lipopolysaccharide treatment. Although inflammation induced by lipopolysaccharide treatment has a positive effect on calpastatin and a negative effect on DYRK1A protein level, a positive effect on microglial activation is maintained in the brains of mice over-expressing Dyrk1A.

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells.

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.

GABAergic over-inhibition, a promising hypothesis for cognitive deficits in Down syndrome.

Down syndrome (DS), also known as trisomy 21, is the most common genetic cause of intellectual disability. It is also a model human disease for exploring consequences of gene dosage imbalance on complex phenotypes. Learning and memory impairments linked to intellectual disabilities in DS could result from synaptic plasticity deficits and excitatory-inhibitory alterations leading to changes in neuronal circuitry in the brain of affected individuals. Increasing number of studies in mouse and cellular models converge towards the assumption that excitatory-inhibitory imbalance occurs in DS, likely early during development. Thus increased inhibition appears to be a common trend that could explain synaptic and circuit disorganization. Interestingly using several potent pharmacological tools, preclinical studies strongly demonstrated that cognitive deficits could be restored in mouse models of DS. Clinical trials have not yet provided robust data for therapeutic application and additional studies are needed. Here we review the literature and our own published work emphasizing the over-inhibition hypothesis in DS and their links with gene dosage imbalance paving the way for future basic and clinical research.

Overexpression of the DYRK1A Gene (Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A) Induces Alterations of the Serotoninergic and Dopaminergic Processing in Murine Brain Tissues.

Trisomy 21 (T21) or Down syndrome (DS) is the most common genetic disorder associated with intellectual disability and affects around 5 million persons worldwide. Neuroanatomical phenotypes associated with T21 include slight reduction of brain size and weight, abnormalities in several brain areas including spines dysgenesis, dendritic morphogenesis, and early neuroanatomical characteristics of Alzheimer's disease. Monoamine neurotransmitters are involved in dendrites development, functioning of synapses, memory consolidation, and their levels measured in the cerebrospinal fluid, blood, or brain areas that are modified in individuals with T21. DYRK1A is one of the recognized key genes that could explain some of the deficits present in individuals with T21. We investigated by high-performance liquid chromatography with electrochemical detection the contents and processing of monoamines neurotransmitters in four brain areas of female and male transgenic mice for the Dyrk1a gene (mBactgDyrk1a). DYRK1A overexpression induced dramatic deficits in the serotonin contents of the four brain areas tested and major deficits in dopamine and adrenaline contents especially in the hypothalamus. These results suggest that DYRK1A overexpression might be associated with the modification of monoamines content found in individuals with T21 and reinforce the interest to target the level of DYRK1A expression as a therapeutic approach for persons with T21.

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.

Safety and efficacy of cognitive training plus epigallocatechin-3-gallate in young adults with Down's syndrome (TESDAD): a double-blind, randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Early cognitive intervention is the only routine therapeutic approach used for amelioration of intellectual deficits in individuals with Down's syndrome, but its effects are limited. We hypothesised that administration of a green tea extract containing epigallocatechin-3-gallate (EGCG) would improve the effects of non-pharmacological cognitive rehabilitation in young adults with Down's syndrome. METHODS: We enrolled adults (aged 16-34 years) with Down's syndrome from outpatient settings in Catalonia, Spain, with any of the Down's syndrome genetic variations (trisomy 21, partial trisomy, mosaic, or translocation) in a double-blind, placebo-controlled, phase 2, single centre trial (TESDAD). Participants were randomly assigned at the IMIM-Hospital del Mar Medical Research Institute to receive EGCG (9 mg/kg per day) or placebo and cognitive training for 12 months. We followed up participants for 6 months after treatment discontinuation. We randomly assigned participants using random-number tables and balanced allocation by sex and intellectual quotient. Participants, families, and researchers assessing the participants were masked to treatment allocation. The primary endpoint was cognitive improvement assessed by neuropsychologists with a battery of cognitive tests for episodic memory, executive function, and functional measurements. Analysis was on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT01699711. FINDINGS: The study was done between June 5, 2012, and June 6, 2014. 84 of 87 participants with Down's syndrome were included in the intention-to-treat analysis at 12 months (43 in the EGCG and cognitive training group and 41 in the placebo and cognitive training group). Differences between the groups were not significant on 13 of 15 tests in the TESDAD battery and eight of nine adaptive skills in the Adaptive Behavior Assessment System II (ABAS-II). At 12 months, participants treated with EGCG and cognitive training had significantly higher scores in visual recognition memory (Pattern Recognition Memory test immediate recall, adjusted mean difference: 6·23 percentage points [95% CI 0·31 to 12·14], p=0·039; d 0·4 [0·05 to 0·84]), inhibitory control (Cats and Dogs total score, adjusted mean difference: 0·48 [0·02 to 0·93], p=0·041; d 0·28 [0·19 to 0·74]; Cats and Dogs total response time, adjusted mean difference: -4·58 s [-8·54 to -0·62], p=0·024; d -0·27 [-0·72 to -0·20]), and adaptive behaviour (ABAS-II functional academics score, adjusted mean difference: 5·49 [2·13 to 8·86], p=0·002; d 0·39 [-0·06 to 0·84]). No differences were noted in adverse effects between the two treatment groups. INTERPRETATION: EGCG and cognitive training for 12 months was significantly more effective than placebo and cognitive training at improving visual recognition memory, inhibitory control, and adaptive behaviour. Phase 3 trials with a larger population of individuals with Down's syndrome will be needed to assess and confirm the long-term efficacy of EGCG and cognitive training. FUNDING: Jérôme Lejeune Foundation, Instituto de Salud Carlos III FEDER, MINECO, Generalitat de Catalunya.

Impact of Dyrk1A level on alcohol metabolism.

Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.

New Perspectives for the Rescue of Cognitive Disability in Down Syndrome.

Down syndrome (DS) is a relatively common genetic condition caused by the triplication of human chromosome 21. No therapies currently exist for the rescue of neurocognitive impairment in DS. This review presents exciting findings showing that it is possible to restore brain development and cognitive performance in mouse models of DS with therapies that can also apply to humans. This knowledge provides a potential breakthrough for the prevention of intellectual disability in DS.

Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia.

Hyperhomocysteinemia results from hepatic metabolism dysfunction and is characterized by a high plasma homocysteine level, which is also an independent risk factor for cardiovascular disease. Elevated levels of homocysteine in plasma lead to hepatic lesions and abnormal lipid metabolism. Therefore, lowering homocysteine levels might offer therapeutic benefits. Recently, we were able to lower plasma homocysteine levels in mice with moderate hyperhomocysteinemia using an adenoviral construct designed to restrict the expression of DYRK1A, a serine/threonine kinase involved in methionine metabolism (and therefore homocysteine production), to hepatocytes. Here, we aimed to extend our previous findings by analyzing the effect of hepatocyte-specific Dyrk1a gene transfer on intermediate hyperhomocysteinemia and its associated hepatic toxicity and liver dysfunction. Commensurate with decreased plasma homocysteine and alanine aminotransferase levels, targeted hepatic expression of DYRK1A in mice with intermediate hyperhomocysteinemia resulted in elevated plasma paraoxonase-1 and lecithin:cholesterol acyltransferase activities and apolipoprotein A-I levels. It also rescued hepatic apolipoprotein E, J, and D levels. Further, Akt/GSK3/cyclin D1 signaling pathways in the liver of treated mice were altered, which may help prevent homocysteine-induced cell cycle dysfunction. DYRK1A gene therapy could be useful in the treatment of hyperhomocysteinemia in populations, such as end-stage renal disease patients, who are unresponsive to B-complex vitamin therapy.

DYRK1A BAC transgenic mouse: a new model of thyroid dysgenesis in Down syndrome.

The most common thyroid abnormality among Down syndrome (DS) children corresponds to a mildly elevated TSH, with T4 decreased or in the normal range and thyroid hypoplasia, from the neonatal period onward, which aggravate their mental impairment. Transgenic Dyrk1A mice, obtained by bacterial artificial chromosome engineering (mBACTgDyrk1A), have 3 copies of the Dyrk1A gene. The objective is to determine whether this transgenic Dyrk1A (Dyrk1A(+/++)) mouse is an adequate murine model for the study of thyroid dysgenesis in DS. Embryonic thyroid development from embryonic day 13.5 (E13.5) to E17.5 was analyzed in wild-type (WT) and Dyrk1A(+/++) mice by immunofluorescence with anti-Nkx2-1, anti-thyroglobulin, and anti-T4 antibodies, markers of early thyroid development, hormonogenesis, and final differentiation, respectively. The expression of transcription factors Nkx2-1, Pax8, and Foxe1 involved in thyroidogenesis were studied by quantitative RT-PCR at the same embryonic stages. We then compared the adult phenotype at 8 to 12 weeks in Dyrk1A(+/++) and WT mice for T4 and TSH levels, thyroidal weight, and histological analysis. Regarding thyroidal development, at E15.5, Dyrk1A(+/++) thyroid lobes are double the size of WT thyroids (P = .01), but the thyroglobulin stained surface in Dyrk1A(+/++) thyroids is less than a third as large at E17.5 (P = .04) and their differentiated follicular surface half the size (P = .004). We also observed a significant increase in Nkx2-1, Foxe1, and Pax8 RNA levels in E13.5 and E17.5 Dyrk1A(+/++) embryonic thyroids. Dyrk1A(+/++) young adult mice have significantly lower plasma T4 (2.4 ng/mL versus WT, 3.7 ng/mL; P = 0.019) and nonsignificantly higher plasma TSH (114 mUI/L versus WT, 73mUI/L; P = .09). In addition, their thyroids are significantly heavier (P = .04) and exhibit large disorganized regions. Dyrk1A overexpression directly leads to thyroidal embryogenetic, functional and morphological impairment. The young adult thyroid phenotype is probably a result of embryogenetic impairment. The Dyrk1A(+/++) mouse can be considered a suitable study model for thyroid dysgenesis in DS.

Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise.

Hyperhomocysteinemia resulting from cystathionine beta synthase (CBS) deficiency can produce cognitive dysfunction. We recently found that CBS-deficient mice exhibit increased expression of the serine/threonine kinase dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) in the brain. When dysregulated, DYRK1A contributes to the neurodegeneration, neuronal death, and loss of function observed in neurodegenerative diseases. However, brain plasticity can be improved by interventions like enriched environment combined with voluntary exercise (EE/VE). The present study sought to assess the effects of EE/VE on molecular mechanisms linked to DYRK1A overexpression in the brain of CBS-deficient mice. EE/VE was applied to 3-month-old female CBS-deficient mice for 1 month. Without intervention, CBS-deficient mice exhibited increased DYRK1A and decreased brain-derived neurotrophic factor (BDNF) levels in the cortex and hippocampus. However, EE/VE rescued these altered DYRK1A and BDNF levels in the hippocampus of CBS-deficient mice. We conclude that exercise combined with enriched environment can restore the altered molecular mechanisms in the brain of CBS-deficient mice.

Dyrk1A induces pancreatic ß cell mass expansion and improves glucose tolerance.

Type 2 diabetes is caused by a limited capacity of insulin-producing pancreatic ß cells to increase their mass and function in response to insulin resistance. The signaling pathways that positively regulate functional ß cell mass have not been fully elucidated. DYRK1A (also called minibrain/MNB) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. A significant amount of data implicates DYRK1A in brain growth and Down syndrome, and recent data indicate that Dyrk1A haploinsufficient mice have a low functional ß cell mass. Here we ask whether Dyrk1A upregulation could be a way to increase functional ß cell mass. We used mice overexpressing Dyrk1A under the control of its own regulatory sequences (mBACTgDyrk1A). These mice exhibit decreased glucose levels and hyperinsulinemia in the fasting state. Improved glucose tolerance is observed in these mice as early as 4 weeks of age. Upregulation of Dyrk1A in ß cells induces expansion of ß cell mass through increased proliferation and cell size. Importantly, mBACTgDyrk1A mice are protected against high-fat-diet-induced ß cell failure through increase in ß cell mass and insulin sensitivity. These studies show the crucial role of the DYRK1A pathway in the regulation of ß cell mass and carbohydrate metabolism in vivo. Activating the DYRK1A pathway could thus represent an innovative way to increase functional ß cell mass.

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.

Dyrk1a haploinsufficiency induces diabetes in mice through decreased pancreatic beta cell mass.

AIMS/HYPOTHESIS: Growth factors and nutrients are important regulators of pancreatic beta cell mass and function. However, the signalling pathways by which these factors modulate these processes have not yet been fully elucidated. DYRK1A (also named minibrain/MNB) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family that has been conserved across evolution. A significant amount of data implicates DYRK1A in brain growth and function, as well as in neurodegenerative processes in Alzheimer's disease and Down's syndrome. We investigated here whether DYRK1A would be an attractive candidate for beta cell growth modulation. METHODS: To study the role of DYRK1A in beta cell growth, we used Dyrk1a-deficient mice. RESULTS: We show that DYRK1A is expressed in pancreatic islets and provide evidence that changes in Dyrk1a gene dosage in mice strongly modulate glycaemia and circulating insulin levels. Specifically, Dyrk1a-haploinsufficient mice show severe glucose intolerance, reduced beta cell mass and decreased beta cell proliferation. CONCLUSIONS/INTERPRETATION: Taken together, our data indicate that DYRK1A is a critical kinase for beta cell growth as Dyrk1a-haploinsufficient mice show a diabetic profile.

One-carbon cycle alterations induced by Dyrk1a dosage.

Hyperhomocysteinemia due to cystathionine beta synthase deficiency confers diverse clinical manifestations. It is characterized by elevated plasma homocysteine levels, a common amino acid metabolized by remethylation to methionine or transsulfuration to cysteine. We recently found a relationship between hepatic Dyrk1A protein expression, a serine/threonine kinase involved in signal transduction in biological processes, hepatic S-adenosylhomocysteine activity, and plasma homocysteine levels. We aimed to study whether there is also a relationship between Dyrk1a and cystathionine beta synthase activity. We used different murine models carrying altered gene coy numbers for Dyrk1a, and found a decreased cystathionine beta synthase activity in the liver of mice under-expressing Dyrk1a, and an increased in liver of mice over-expressing Dyrk1a. For each model, a positive correlation was found between cystathionine beta synthase activity and Dyrk1a protein expression in the liver of mice, which was confirmed in a non-modified genetic context. The positive correlation found between liver Dyrk1a protein expression and CBS activity in modified and non-modified genetic context strengthens the role of this kinase in one carbon metabolism.

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.