Yu-Wen Hwang

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 hyperphosphory‐lated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase‐3β (GSK‐3β). Dyrk1A‐induced tau phosphorylation inhibited taus 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. Liu, F., Liang, Z., Wegiel, J., Hwang, Y.‐W., Iqbal, K., Grundke‐Iqbal, I., Ramakrishna, N., Gong, C.‐X. Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome. FASEB J. 22, 3224–3233 (2008)

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 Aβ40 and 42 levels, and leading to brain β‐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.

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

Harmine is a β-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.

Mnb/Dyrk1A Phosphorylation Regulates the Interaction of Dynamin 1 with SH3 Domain-Containing Proteins†

Mnb/Dyrk1A is a proline-directed serine/threonine kinase implicated in Downs syndrome. Mnb/Dyrk1A was shown to phosphorylate dynamin 1 and alter its interactions with several SH3 domain-containing endocytic accessory proteins. To determine the mechanism of regulation, we mapped the Mnb/Dyrk1A phosphorylation sites in dynamin 1. Using a combination of deletion mutants and synthetic peptides, three potential Mnb/Dyrk1A phosphorylation sites (S778, S795, and S857) were first identified. Phosphorylation at S795 and S857 was confirmed in full-length dynamin 1, and S857 was subsequently determined to be the major Mnb/Dyrk1A phosphorylation site in vitro. Phosphorylation at S857 was demonstrated to be the basis for altering the binding of dynamin 1 to amphiphysin 1 and Grb 2 by site-directed mutants mimicking phosphorylation. Furthermore, S857 of dynamin 1 is phosphorylated by the endogenous kinase in brain extracts and in PC12 cells. In PC12 cells, the state of S857 phosphorylation is dependent on membrane potentials. These results suggest that S857 phosphorylation is a physiological event, which regulates the binding of dynamin 1 to SH3 domain-containing proteins. Since S857 is unique to dynamin 1xa isoforms, Mnb/Dyrk1A regulation of dynamin 1 is expected to be specific to these spliced variants.

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.

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.

The Dominant Negative Effects of H-Ras Harboring a Gly to Ala Mutation at Position 60

v-H-Ras harboring the Gly-60 to Ala mutation (G60A) lacks the ability to induce germinal vesicle breakdown in Xenopus oocytes. Moreover, this mutant is capable of inhibiting the activity of v-H-Ras to induce oocyte germinal vesicle breakdown when co-injected. The duration and the extent of inhibition depends on the molar ratio of v-H-Ras(G60A) to v-H-Ras. The inhibition is not due to a general toxicity of v-H-Ras(G60A) to oocytes because oocytes injected with v-H-Ras(G60A) can be readily induced to mature by other mitogenic agents, such as insulin, insulin-like growth factor 1, insulin-like growth factor 2, and phosphatidylcholine-specific phospholipase C. The dominant negative effect of v-H-Ras(G60A) requires proper membrane attachment of v-H-Ras(G60A). By using a competition assay, it was concluded that the dominant negative phenotype of v-H-Ras(G60A) resulted from sequestering H-Ras downstream effector(s). Raf-1 was identified as one of the sequestered targets.

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 β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [32P]-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 β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-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.

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.

Intracellular distribution of differentially phosphorylated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A): DYRK1A Phosphorylation and Distribution

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.