Development of Novel Fluorinated Polyphenols as Selective Inhibitors of DYRK1A/B Kinase for Treatment of Neuroinflammatory Diseases including Parkinson's Disease.

Natural polyphenol derivatives such as those found in green tea have been known for a long time for their useful therapeutic activity. Starting from EGCG, we have discovered a new fluorinated polyphenol derivative (1c) characterized by improved inhibitory activity against DYRK1A/B enzymes and by considerably improved bioavailability and selectivity. DYRK1A is an enzyme that has been implicated as an important drug target in various therapeutic areas, including neurological disorders (Down syndrome and Alzheimer's disease), oncology, and type 2 diabetes (pancreatic ß-cell expansion). Systematic structure-activity relationship (SAR) on trans-GCG led to the discovery that the introduction of a fluoro atom in the D ring and methylation of the hydroxy group from para to the fluoro atom provide a molecule (1c) with more desirable drug-like properties. Owing to its good ADMET properties, compound 1c showed excellent activity in two in vivo models, namely the lipopolysaccharide (LPS)-induced inflammation model and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model for Parkinson's disease.

Design, synthesis, and biological evaluation of polyphenol derivatives as DYRK1A inhibitors. The discovery of a potentially promising treatment for Multiple Sclerosis.

Green tea and its natural components are known for their usefulness against a variety of diseases. In particular, the activity of main catechin Epigallocatechin gallate (EGCG) against Dual-specificity tyrosine-(Y)-phosphorylation Regulated Kinase-1A (DYRK1A) has been reported; here we are showing a structure-activity relationship (SAR) for EGCG against this molecular target. We have studied the influence of all four rings on the activity and the nature of its absolute geometry. This work has led to the identification of the more potent and stable trans fluoro-catechin derivative 1f (IC50 = 35 nM). This molecule together with a novel delivery method showed good efficacy in vivo when tested in a validated model of multiple sclerosis (EAE).

Abnormalities of DYRK1A-Cytoskeleton Complexes in the Blood Cells as Potential Biomarkers of Alzheimer's Disease.

BACKGROUND: DYRK1A is implicated in mental retardation and Alzheimer's disease (AD) dementia of Down syndrome (DS) individuals. The protein is associated with cytoskeleton and altered expression has been shown to impair the cytoskeletal network via dosage effect. OBJECTIVE: Our original observations of marked reduction of cytoskeletal proteins associated with DYRK1A in brains and lymphoblastoid cell lines from DS and AD prompted an investigation whether cytoskeleton abnormalities could potentially be used as biomarkers of AD. METHODS: Our assay relied on quantification of co-immunoprecipitated cytoskeletal proteins with DYRK1A (co-IP assay) and analysis of the profile of G- and F-actin fractions obtained by high-speed centrifugations (spin-down assay). RESULTS: In co-IP assay, both DS and AD samples displayed reduced abundance of associated cytoskeletal proteins. In spin-down assay, G-actin fractions of controls displayed two closely spaced bands of actin in SDS-PAGE; while in AD and DS, only the upper band of the doublet was present. In both assays, alterations of actin cytoskeleton were present in DS, sporadic and familial AD cases, and in asymptomatic persons who later progressed to confirmed AD, but not in non-AD donors. In blind testing involving six AD and six controls, the above tests positively identified ten cases. Analysis of blood samples revealed the diversity of mild cognitive impairment (MCI) cases regarding the presence of the AD biomarker allowing distinction between likely prodromal AD and non-AD MCI cases. CONCLUSIONS: Both brain tissue and lymphocytes from DS and AD displayed similar semi-quantitative and qualitative alterations of actin cytoskeleton. Their specificity for AD-type dementia and the presence before clinical onset of the disease make them suitable biomarker candidates for early and definite diagnosis of AD. The presence of alterations in peripheral tissue points to systemic underlying mechanisms and suggests that early dysfunction of cytoskeleton may be a predisposing factor in the development of AD.

An ELISA DYRK1A non-radioactive kinase assay suitable for the characterization of inhibitors.

The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer's disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC 50s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods.  Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors.

Intracellular distribution of differentially phosphorylated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A).

The gene encoding dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age-associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell-compartment-specific functions are associated with DYRK1A posttranslational modifications. Fractionation showed that, in both human and mouse brain, almost 80% of DYRK1A was associated with the cytoskeleton, and the remaining DYRK1A was present in the cytosolic and nuclear fractions. Coimmunoprecipitation revealed that DYRK1A in the brain cytoskeleton fraction forms complexes with filamentous actin, neurofilaments, and tubulin. Two-dimensional gel analysis of the fractions revealed DYRK1A with distinct isoelectric points: 5.5-6.5 in the nucleus, 7.2-8.2 in the cytoskeleton, and 8.7 in the cytosol. Phosphate-affinity gel electrophoresis demonstrated several bands of DYRK1A with different mobility shifts for nuclear, cytoskeletal, and cytosolic DYRK1A, indicating modification by phosphorylation. Mass spectrometry analysis disclosed one phosphorylated site in the cytosolic DYRK1A and multiple phosphorylated residues in the cytoskeletal DYRK1A, including two not previously described. This study supports the hypothesis that intracellular distribution and compartment-specific functions of DYRK1A may depend on its phosphorylation pattern.

Gene dosage-dependent association of DYRK1A with the cytoskeleton in the brain and lymphocytes of down syndrome patients.

The triplication of the DYRK1A gene encoding proline-directed serine/threonine kinase and located in the critical region of Down syndrome (DS) has been implicated in cognitive deficits and intellectual disability of individuals with DS. We investigated the effect of abnormal levels of this kinase on the cytoskeleton in brain and peripheral tissues of DS subjects. In DS tissues, the predictable approximately equal to 1.5-fold enhancement of the levels of DYRK1A protein was demonstrated. An association of DYRK1A with all 3 major cytoskeleton networks was identified using immunoprecipitation. We concentrated on the actin cytoskeleton because its association with DYRK1A was the most affected by the enzyme levels. As measured by coimmunoprecipitation in DS tissues, but not in fragile X lymphocytes, actin association with DYRK1A was reduced. This reduced association was dependent on the state of phosphorylation of cytoskeletal proteins and was present only in cells overproducing DYRK1A kinase; therefore, the effect was attributable to the DYRK1A gene dosage. Alterations of DYRK1A-actin assemblies were detected in newborn and infant groups, thereby linking DYRK1A overexpression with abnormal brain development of DS children. The identification of the actin cytoskeleton as one of cellular targets of DYRK1A action provides new insights into a gene dosage-sensitive mechanism by which DYRK1A could contribute to the pathogenesis of DS. In addition, the presence of this DS-specific cytoskeleton anomaly in lymphocytes attests to the systemic nature of some features of DS. To our knowledge, this is the first study conducted in human tissue that shows DYRK1A association with the cytoskeleton.

Activity-dependent phosphorylation of dynamin 1 at serine 857.

Dynamin 1 is thought to mediate synaptic transmission through interactions with multiple endocytic accessory proteins in a phosphorylation-dependent manner. Previously, we have shown that DYRK1A, a chromosome 21-encoded kinase implicated in the mental retardation of Down syndrome, phosphorylates primarily serine 857 (S857) in the proline-rich domain, found only in 1xa, one of the alternative C-terminal splicing isoforms of dynamin 1. Dynamin 1xa and 1xb isoforms are able to assemble into heterologous complexes and are coregulated by DYRK1A phosphorylation in binding to amphiphysin in vitro. To help in assessing the physiological significance of S857 phosphorylation, we developed a semiquantitative method for measuring the cellular level of phospho-S857 (pS857). Dynamin 1xa is highly phosphorylated at S857 in resting hippocampal neurons and in a hippocampal cell line, with >60% of all endogenous protein phosphorylated at this residue. In the hippocampus, the level of pS857 is dynamically controlled by synaptic stimulations with the involvement of Ca(2+)/calcineurin and AMPA/kainate receptors. Immunofluorescence staining shows that pS857 is found in the soma and throughout the entire length of apical dendrites in resting pyramidal neurons. Neuronal stimulation in the Schaffer collateral pathway promotes pS857 dephosphorylation in distal areas of apical dendrites, the region forming synapses with the impinging axons of Schaffer collateral. In summary, our results support the conclusion that S857 phosphorylation is a physiological event and its level is modulated by neuronal activity in nerve terminals.

Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation.

Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and ß-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and ß-adaptins led to dissociation of the AP2 complex, and released only ß-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and ß-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.

Effect of DYRK1A activity inhibition on development of neuronal progenitors isolated from Ts65Dn mice.

Overexpression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A), encoded by a gene located in the Down syndrome (DS) critical region, is considered a major contributor to developmental abnormalities in DS. DYRK1A regulates numerous genes involved in neuronal commitment, differentiation, maturation, and apoptosis. Because alterations of neurogenesis could lead to impaired brain development and mental retardation in individuals with DS, pharmacological normalization of DYRK1A activity has been postulated as DS therapy. We tested the effect of harmine, a specific DYRK1A inhibitor, on the development of neuronal progenitor cells (NPCs) isolated from the periventricular zone of newborn mice with segmental trisomy 16 (Ts65Dn mice), a mouse model for DS that overexpresses Dyrk1A by 1.5-fold. Trisomy did not affect the ability of NPCs to expand in culture. Twenty-four hours after stimulation of migration and neuronal differentiation, NPCs showed increased expression of Dyrk1A, particularly in the trisomic cultures. After 7 days, NPCs developed into a heterogeneous population of differentiating neurons and astrocytes that expressed Dyrk1A in the nuclei. In comparison with disomic cells, NPCs with trisomy showed premature neuronal differentiation and enhanced γ-aminobutyric acid (GABA)-ergic differentiation, but astrocyte development was unchanged. Harmine prevented premature neuronal maturation of trisomic NPCs but not acceleration of GABA-ergic development. In control NPCs, harmine treatment caused altered neuronal development of NPCs, similar to that in trisomic NPCs with Dyrk1A overexpression. This study suggests that pharmacological normalization of DYRK1A activity may have a potential role in DS therapy.

Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

Harmine is a ß-carboline alkaloid. The compound is a potent inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A), a kinase implicated in Down syndrome. In this study, we show that harmine functions as an ATP-competitive inhibitor against Dyrk1A. Our conclusion is supported by kinetic analysis of harmine inhibition as well as by the characterization of a Dyrk1A mutation conferring significant resistance to harmine. The mutation, V306A, is located next to the highly conserved D307 residue in kinases known to coordinate the phosphate groups of ATP through a Mg²+ ion. The V306A mutation offers harmine resistance by differentially altering Dyrk1A affinity for harmine and ATP. The V306A mutation causes no apparent alteration to Dyrk1A activity except for the reduction in ATP affinity. This deficiency could be fully compensated by supplying ATP with a concentration in the physiological range. Our results reveal that harmine inhibits Dyrk1A activity by interacting with residues in the ATP-binding pocket and displacing ATP. Our results also suggest that harmine will be a good lead compound for further designing of selective ATP-competitive Dyrk1A inhibitors through exploration of the ATP-binding pocket of Dyrk1A.

The role of DYRK1A in neurodegenerative diseases.

Recent studies indicate that the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene, which is located on chromosome 21q22.2 and is overexpressed in Down syndrome (DS), may play a significant role in developmental brain defects and in early onset neurodegeneration, neuronal loss and dementia in DS. The identification of hundreds of genes deregulated by DYRK1A overexpression and numerous cytosolic, cytoskeletal and nuclear proteins, including transcription factors, phosphorylated by DYRK1A, indicates that DYRK1A overexpression is central for the deregulation of multiple pathways in the developing and aging DS brain, with structural and functional alterations including mental retardation and dementia. DYRK1A overexpression in DS brains may contribute to early onset neurofibrillary degeneration directly through hyperphosphorylation of tau and indirectly through phosphorylation of alternative splicing factor, leading to an imbalance between 3R-tau and 4R-tau. The several-fold increases in the number of DYRK1A-positive and 3R-tau-positive neurofibrillary tangles in DS support this hypothesis. Moreover, the enhanced phosphorylation of amyloid precursor protein by overexpressed DYRK1A facilitates amyloidogenic amyloid precursor protein cleavage elevating Aß40 and 42 levels, and leading to brain ß-amyloidosis. Therefore, inhibiting DYRK1A activity in DS may serve to counteract the phenotypic effects of its overexpression and is a potential method of treatment of developmental defects and the prevention of age-associated neurodegeneration, including Alzheimer-type pathology.

Link between DYRK1A overexpression and several-fold enhancement of neurofibrillary degeneration with 3-repeat tau protein in Down syndrome.

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 × of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.

Dyrk1A binds to multiple endocytic proteins required for formation of clathrin-coated vesicles.

In spite of a nuclear targeting sequence, a substantial amount of dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk1A) is located within the cytoplasm of neurons. Analysis of fractionated rat brains revealed that the majority of Dyrk1A was in the postnuclear precipitate. The kinase in this fraction was resistant to high salt and Triton X-100 extraction at pH 6.5. Hypothesizing that Dyrk1A binds tightly with cell constituents, we searched for Dyrk1A binding proteins in the Triton X-100-insoluble fraction extracted with urea and fractionated by column chromatography. An overlay assay using the recombinant kinase revealed that multiple proteins are capable of binding to Dyrk1A. Among them, we identified clathrin heavy chain and dynamin 1 as potential candidates. An overlay assay using purified and partially purified proteins showed the binding of Dyrk1A with both proteins. Under native conditions, Dyrk1A precipitated with newly formed clathrin cages and with dynamin via the GST-amphiphysin SH3 domain. We also identified another endocytic protein, endophilin 1, as an additional Dyrk1A binding protein. We then tested whether the clathrin-coated vesicle (CCV)-associated proteins could be phosphorylated by Dyrk1A. Multiple proteins apparently distinctive from the known substrates were phosphorylated in the brain CCV. Our findings suggest a role for Dyrk1A in controlling synaptic vesicle recycling processes.

The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome.

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

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 contributes to neurofibrillary degeneration in Down syndrome.

Adults with Down syndrome (DS) develop Alzheimer neurofibrillary degeneration in the brain, but the underlying molecular mechanism is unknown. Here, we report that the presence of an extra copy of the dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) gene due to trisomy 21 resulted in overexpression of Dyrk1A and elevated kinase activity in DS brain. Dyrk1A phosphorylated tau at several sites, and these sites were hyperphosphorylated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase-3beta (GSK-3beta). Dyrk1A-induced tau phosphorylation inhibited tau's biological activity and promoted its self-aggregation. In Ts65Dn mouse brain, an extra copy of the Dyrk1A gene caused increased expression and activity of Dyrk1A and resulted in increased tau phosphorylation. These findings strongly suggest a novel mechanism by which the overexpression of Dyrk1A in DS brain causes neurofibrillary degeneration via hyperphosphorylating tau.

Promotion of neuronal plasticity by (-)-epigallocatechin-3-gallate.

The consumption of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic compound found in green tea, has been associated with various neurological benefits including cognitive improvement. The physiological basis for this effect is unknown. In this study, we used synaptic transmission between the CA3 and CA1 regions (Schaffer collateral) of the mouse hippocampus to examine the effects of EGCG on neuronal plasticity. We found that the level of high frequency stimulation-evoked long-term potentiation (LTP) was significantly enhanced when hippocampal slices were pre-incubated with 10 microM EGCG for 1 h prior to the experiment. EGCG incubation also enabled hippocampal slices prepared from Ts65Dn mice, a Down syndrome mouse model deficient in LTP, to express LTP to a level comparable to the normal controls. EGCG treatment did not alter the degree of pair-pulse inhibition; therefore, the enhancement effect of EGCG is unlikely to involve the attenuation of this inhibitory mechanism.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro.

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is localized in human chromosome 21, and its overexpression has been associated with the learning and memory deficits of Down syndrome. DYRK1A contains a Y319XY321 motif shared by all members of the DYRK protein kinase family. Residue Y321 in the motif is phosphorylated in DYRK1A prepared from Escherichia coli and from eukaryotic cells. It has been proposed that the YXY motif is an equivalent of the TXY motif, the activation loop, of mitogen-activated protein kinase and that phosphorylation at the motif is required for DYRK activity. In this study, the role of tyrosine phosphorylation in the activity of DYRK1A was investigated in detail. Wild-type DYRK1A with a reduced level of phosphotyrosine (pY) was prepared by treating E. coli-produced DYRK1A with two different protein tyrosine phosphatases. The resulting pY-depleted DYRK1A could not regain pY during autophosphorylation but was as active as the untreated control. These findings were further supported by the observation that DYRK1A retained significant enzymatic activity when both tyrosine residues in the YXY motif were replaced with either histidine or glutamine. Together, we conclude that tyrosine phosphorylation and tyrosine residues in the YXY motif are not directly involved in DYRK1A enzymatic activity in vitro.

Trisomy-driven overexpression of DYRK1A kinase in the brain of subjects with Down syndrome.

Down syndrome (DS) is the most common genetic disorder associated with mental retardation (MR). It is believed that many of the phenotypic features of DS stem from enhanced expression of a set of genes located within the triplicated region on chromosome 21. Among those genes is DYRK1A encoding dual-specificity proline-directed serine/treonine kinase, which, as documented by animal studies, can potentially contribute to cognitive deficits in DS. Whether this contribution can be exerted through elevated levels of DYRK1A protein in the brain of DS subjects was the main goal of the present study. The levels of DYRK1A protein were measured by Western blotting in six brain structures that included cerebral and cerebellar cortices and white matter. The study involved large cohorts of DS subjects and age-matched controls representing infants and adults of different age, gender and ethnicity. Trisomic Ts65Dn mice, an animal model of DS, were also included in the study. Both in trisomic mice and in DS subjects, the brain levels of DYRK1A protein were increased approximately 1.5-fold, indicating that this protein is overexpressed in gene dosage-dependent manner. The exception was an infant group, in which there was no enhancement suggesting the existence of a developmentally regulated mechanism. We found DYRK1A to be present in every analyzed structure irrespective of age. This widespread occurrence and constitutive expression of DYRK1A in adult brain suggest an important, but diverse from developmental role played by this kinase in adult central nervous system. It also implies that overexpression of DYRK1A in DS may be potentially relevant to MR status of these individuals during their entire life span.

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins.

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.