Dyrk1A overexpression in immortalized hippocampal cells produces the neuropathological features of Down syndrome

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 36 (2007) 270 – 279

Dyrk1A overexpression in immortalized hippocampal cells produces the neuropathological features of Down syndrome Joongkyu Park, a,1 Eun Jin Yang, b,1 Joo Heon Yoon, c and Kwang Chul Chung a,⁎ a

Department of Biology, College of Science, Yonsei University, Seoul 120-749, South Korea Department of Medical Science, Yonsei University College of Medicine, Seoul 120-752, South Korea c Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 120-752, South Korea b

Received 21 March 2007; revised 11 July 2007; accepted 17 July 2007 Available online 24 July 2007

Down syndrome (DS) is the most common genetic disorder, characterized by mental retardation, congenital heart abnormalities, and susceptibility to Alzheimer's disease (AD). Brain development of DS patients is associated with elevated apoptosis and abnormal neuronal differentiation. Those key features are closely associated with many genes mapped within Down syndrome critical region (DSCR) on human chromosome 21. Proline-directed serine/threonine kinase, Dyrk1A, is mapped within DSCR, and involved in the control of cell growth and postembryonic neurogenesis. Despite the potential involvement of Dyrk1A in neurodegeneration, its links to AD susceptibility and the neuropathology of DS patients are not yet clearly understood. Here, we report evidence supporting the correlation between Dyrk1A and neuropathology of DS. Our results show that Dyrk1A interacts with and directly phosphorylates tau and amyloid precursor protein in immortalized hippocampal progenitor H19-7 cells. In addition, the formation of tau inclusion and the enhanced generation of β-amyloid fragment were detected in H19-7 cells that overexpressed Dyrk1A. Furthermore, these cells show a marked increase in apoptotic cell death under conditions of serum deprivation and also exhibit defects in neuronal differentiation. These results suggest that up-regulation of Dyrk1A may cause AD-like pathogenesis and abnormal neurobiological features in DS patients. © 2007 Elsevier Inc. All rights reserved. Keywords: Dyrk1A; Down syndrome; Alzheimer's disease; Amyloid precursor protein; Amyloid-β; Tau; Apoptosis; Neuronal differentiation

Introduction Down syndrome (DS) is the most common genetic disorder. Individuals with DS possess an extra copy of all or part of chromosome 21 (Antonarakis et al., 2004). In addition to characteristic ⁎ Corresponding author. Department of Biology, College of Science, Yonsei University, Shinchon-dong 134, Seodaemun-gu, Seoul 120-749, South Korea. Fax: +82 2 313 1894. E-mail address: [email protected] (K.C. Chung). 1 These authors contributed equally to this work. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.07.007

physical features, DS individuals have congenital heart defects, gastrointestinal malformations, immune and endocrine system defects, a high incidence of leukemia, and mild to severe mental retardation. The neuropathology of DS is also complex; DS individuals have decreased brain weight, decreased neuronal number, and abnormal neuronal differentiation (Wisniewski et al., 1984). Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) is a member of an evolutionary conserved protein kinase family that is involved in the control of growth and development (Song et al., 1996; Guimera et al., 1996; Shindoh et al., 1996; Becker and Joost, 1999; Raich et al., 2003). Human DYRK1A is mapped to the Down syndrome critical region (DSCR) on chromosome 21 (4-megabase region containing 60–100 genes between the markers D21S17 and ETS2). Abnormalities in Dyrk1A have been suggested to play an important role in the production of mental retardation of DS patients (Smith et al., 1997; Antonarakis et al., 2004). Transgenic mice that overexpress Dyrk1A exhibit symptoms similar to DS, including neurodevelopmental delay, cognitive deficits, and significant impairment in hippocampal-dependent memory tasks (Altafaj et al., 2001; Ahn et al., 2006). These findings support a role of Dyrk1A in brain development and synaptic plasticity. In addition, we have shown that Dyrk1A is important in neuronal differentiation, via activation of the transcription factor CREB (Yang et al., 2001). Furthermore, Dyrk1A phosphorylates the microtubule-associated protein tau in vitro (Woods et al., 2001). Adults with DS develop Alzheimer's disease (AD) in a progressive age-dependent manner (Wisniewski et al., 1985; Mann et al., 1987; Evenhuis, 1990; Teller et al., 1996). AD patients present a characteristic neuropathology that includes the deposition of extracellular neuritic plaques and intracellular accumulation of neurofibrillary tangles (NFT) in neurons (Selkoe, 1994). Senile plaques are largely composed of β-amyloid (Aβ), whereas NFTs are composed of hyperphosphorylated tau organized into filamentous structures (Ihara et al., 1986; Grundke-Iqbal et al., 1986a,b). Progression of AD occurs through a series of neurochemical events. Proteolytic cleavage of an integral membrane protein, βamyloid precursor protein (APP), results in the generation of 40- or 42-residue Aβ peptides that accumulate in high amounts in the

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

271

brain regions important for memory and cognition. Specially, APP is cleaved by β-secretase to generate C-terminal fragment (CTF), which is subsequently cleaved by γ-secretase to generate Aβ. In the present study, we demonstrate that overexpression of Dyrk1A in hippocampal progenitor H19-7 cells caused abnormal modification of several AD-pathogenic proteins, such as tau and APP. In addition, Dyrk1A-overexpressing hippocampal cells showed a large increase in apoptotic cell death under serum deprivation as well as a remarkable reduction in neuronal differentiation. The characteristic processing of AD target proteins and the change in cell viability and differentiation indicate that Dyrk1A-overexpressing hippocampal cells could serve as a good model in which the detailed molecular mechanism of DS neuropathogenesis can be further clarified. Results Establishment of H19-7 cells stably transfected with Dyrk1A A conditionally immortalized hippocampal cell line (H19-7) was generated by transduction of a temperature-sensitive SV40 large T antigen into rat embryonic day 17 hippocampal cells (Eves et al., 1992). To make H19-7 cells that would stably overexpress Dyrk1A, cells were co-transfected with a plasmid encoding HAtagged Dyrk1A (pSVL-HA-Dyrk1A) plus the selection vector (pTK-hyg) and screened by hygromycin B. To select a clonal cell line (H19-7/Dyrk1A) showing the suitable overexpression level of Dyrk1A, several screened cells were lysed and immunoanalyzed with either anti-HA antibody or anti-Dyrk1A antibody. As shown in Figs. 1A and B, the established H19-7/Dyrk1A cells showed approximately 2.2-fold induction of Dyrk1A expression, compared to parental control cells (H19-7/pTK). Increased phosphorylation and inclusion formation of tau in stable Dyrk1A-overexpressing cells Using the constructed H19-7/Dyrk1A cells, we first examined the effect of Dyrk1A overexpression on the formation of microtubule-associated protein tau inclusion. Based on the report that DYRK1A and DYRK2 phosphorylate Thr212 residue of tau in vitro (Woods et al., 2001), the cells were immunostained with anti-phospho-Tau (Thr212) as well as anti-Tau antibody. As shown in Fig. 2A, immunocytochemical analysis revealed the remarkable formation of tau inclusion in H19-7/Dyrk1A cells. In addition, the presence of phosphorylated tau was detected within the intracellular tau inclusion (Fig. 2A). When the number of total cells having the formation of remarkable intracellular inclusions with tau and its hyperphosphorylated protein were counted and quantitatively analyzed, it was increased by approximately 6.3-fold in Dyrk1Aoverexpressing H19-7 cells, compared to parental control cells (Fig. 2B). In addition, an increase in phosphorylated tau levels within the stable Dyrk1A-overexpressing cells was confirmed by immunoblot analysis of cell lysates with anti-phospho-Tau antibody at Thr212 (Fig. 2C). Quantification of the band intensity demonstrated that the levels of phosphorylated tau proteins were increased by approximately 1.27-fold in Dyrk1A-overexpressing H19-7 cells, compared to parental control cells (Fig. 2C). To examine possible interactions between Dyrk1A and tau, HEK293 cells were co-transfected with plasmids encoding HisXpress-tagged wild type Dyrk1A and V5-His-tagged tau. Total cell lysates were immunoprecipitated with anti-V5 antibody, followed

Fig. 1. Establishment of stable Dyrk1A-overexpressing H19-7 cells. (A) H19-7 cells were co-transfected with pTK-Hyg and pSVL-HADyrk1A, and screened with 250 μg/ml of hygromycin B. To detect levels of Dyrk1A in each cell line, 75 μg of each cell lysate was analyzed by immunoblotting with anti-Dyrk1A, anti-HA, and anti-Hsp90 antibodies, as indicated. The results are representative of four independent experiments. (B) The intensity of Dyrk1A and Hsp90 bands was quantified using ImageJ, image analysis software from NIH (http://rsb.info.nih.gov/ij/), and the levels of Dyrk1A were normalized by Hsp90 expression. The graph represents the mean of four independent experiments ± S.D. in arbitrary units (A.U.). Statistical significance was determined by Student's t-test (⁎⁎ p b 0.01).

by immunoblot analysis with the anti-Xpress antibody. As shown in Fig. 3A, Dyrk1A interacts with tau in an ectopic expression system. Then, we examined whether Dyrk1A could phosphorylate tau in vitro. H19-7 cells were transiently transfected with a plasmid encoding either HA-tagged wild type Dyrk1A or its kinasedeficient mutant. Total cell lysates were immunoprecipitated with the anti-Dyrk1A antibody. An in vitro kinase assay of the immunocomplexes was performed, using the bacterial recombinant tau protein as a substrate. As shown in upper panel of Fig. 3B, the antiDyrk1A immnunocomplexes from cells transfected with wild type Dyrk1A could phosphorylate tau in vitro. However, increased tau phosphorylation was not detected from cells transfected with the kinase-deficient Dyrk1A mutant (top panel of Fig. 3B). Additionally, when the immunoprecipitated Dyrk1A activity was assessed by immunoblotting with an anti-phosphotyrosine antibody, a significant activation of Dyrk1A was observed in cells transfected with wild type Dyrk1A, but not by its kinase-deficient mutant (lower panel of Fig. 3B). Furthermore, we tested whether Dyrk1A overexpression causes increased phosphorylation of endogenous tau protein in H19-7 cells (Fig. 3C). While transient transfection with either wild type

272

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

Fig. 2. Increased tau phosphorylation at Thr212 and the formation of intracellular tau inclusion in stable H19-7/Dyrk1A cells. (A) After washing with PBS buffer, H19-7/Dyrk1A and control H19-7/pTK cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.2% Triton-X-100/PBS, and blocked with 1% BSA in PBS. Then, the cells were stained with either anti-phospho-Tau (Thr212) antibody followed by TRITC-conjugated secondary antibody or anti-Tau or anti-αtubulin antibody followed by FITC-conjugated secondary antibody, as indicated. The immunostained cells were analyzed by confocal microscopy (LSM 510 META by Carl Zeiss). Every scale bar represents 10 μm. Where specified, arrowhead indicates phosphorylated tau inclusions. (B) The percentage of tau inclusion-positive cells was calculated by dividing the number of cells showing one or more intracellular tau inclusions by total tau-stained cell numbers. The graph represents the mean of randomly chosen fixed areas (n = 9) of three independent experiments ± S.D. (⁎⁎⁎ p b 0.0001). (C) Fifty microgram of each H19-7/ pTK and H19-7/Dyrk1A cell lysate was immunoblotted with anti-phospho-Tau (Thr212) and anti-Tau antibodies, as indicated. The intensity of phospho-tau (Thr212) and tau bands was quantified using ImageJ, and phosphorylated tau (Thr212) levels were normalized by the levels of intact tau protein. The gel pictures are representative of four independent experiments, and the values represent the mean of four independent experiments in arbitrary units (A.U.) (⁎⁎ p b 0.01).

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

Dyrk1A or its kinase-deficient mutant form (K188R) did not affect the endogenous level of tau protein, a significant increase in the level of phosphorylated tau protein at Thr212 residue was detected by approximately 1.24-fold in cells transfected with wild type Dyrk1A, but not in that transfected with kinase-inactive Dyrk1A mutant (Fig. 3C). These results show that Dyrk1A appears to phosphorylate tau directly, and that the up-regulation of Dyrk1A causes the formation of intracellular tau inclusion in the hippocampal progenitor cells. Dyrk1A phosphorylates the intracellular domain of APP and causes Aβ production To determine the levels of full-length of APP in H19-7 cells stably transfected with Dyrk1A, total cell lysates were immunoblotted using the anti-APP antibody. As shown in Fig. 4A, the endogenous level of APP was not significantly changed in H19-7/

273

Dyrk1A cells compared to control H19-7/pTK cells. In order to assess the changes in total Aβ peptide production, levels of Aβ in cell lysates and culture media collection were determined by immunoblot analysis with anti-Aβ antibody, which is specific to Aβ1–42, and ELISA assay kits, separately. As shown in Fig. 4A, a significant increase in intracellular Aβ production was observed in Dyrk1A-overexpressing H19-7 cells compared to parental control cells. In addition, an approximately 4.8-fold induction of Aβ secretion into the extracellular space was confirmed by ELISA assay (Fig. 4B). To examine possible interactions between Dyrk1A and APP, H19-7 cell lysates were immunoprecipitated with either antiDyrk1A antibody or pre-immune IgG. Analysis of the immunocomplex with anti-APP and anti-Dyrk1A antibody revealed an interaction between Dyrk1A and APP in H19-7 cells (Fig. 5A). As a control, there was no obvious APP or Dyrk1A band in the immunocomplex prepared by IgG (Fig. 5A), suggesting that endogenous Dyrk1A specifically interacts with APP in H19-7 cells. Analysis of the APP amino acid sequence indicated that there are three phosphorylation sites within the APP intracellular domain (AICD), also known as C-terminal fragment of APP (CTF: Thr654, Ser655, and Thr668). These sites have been identified to be an in vitro target for protein kinase C (PKC), calcium/calmodulindependent protein kinase II (CaMKII), and Cdc2 kinase, respectively (Gandy et al., 1988; Suzuki et al., 1992, 1994). Since Dyrk1A is a proline-directed serine/threonine kinase, we next tested whether Dyrk1A could phosphorylate the CTF of APP. Total cell lysates from H19-7/Dyrk1A cells and control H19-7/pTK cells were immunoprecipitated with the anti-Dyrk1A antibody. Following immunoprecipitation, in vitro kinase assays were performed,

Fig. 3. Dyrk1A phosphorylates tau at Thr212. (A) Where specified, HEK293 cells were transfected with plasmids encoding 6xHis-Xpress-tagged wild type Dyrk1A and/or V5-6xHis-tagged wild type tau. Five hundred micrograms of cell lysates were immunoprecipitated with the anti-V5 antibody. The immunocomplexes were immunoblotted with anti-Xpress or anti-V5 antibodies, as indicated. The proper expression of transientlytransfected proteins in cell lysates was identified with immunoblot analysis using anti-Xpress or anti-V5 antibodies. The asterisks indicated intact V5tagged tau and the upper two bands were thought to be post-translational modification forms of V5-tagged tau. (B) H19-7 cells were transiently transfected with plasmids encoding HA-tagged wild type Dyrk1A (WT) or its kinase-deficient mutant (K188R). Total cell lysates were immunoprecipitated with the anti-Dyrk1A antibody. Where specified, in vitro kinase assays were performed using Dyrk1A-bound immunoprecipitates and bacterially expressed GST fusion protein containing wild type Tau protein as an exogenous substrate. Kinase reaction products were resolved by 10% SDS-PAGE, and phosphorylated tau levels were determined by autoradiography. Additionally, the activity of immunoprecipitated Dyrk1A was checked by immunoblotting with an anti-phosphotyrosine antibody, as indicated (lower panel). (C) H19-7 cells were transiently transfected with plasmids encoding 6xHis-Xpress-tagged wild type Dyrk1A or its kinasedeficient mutant (K188R). Thirty micrograms of each cell lysates were immunoblotted with anti-phospho-Tau (Thr212), anti-Tau, anti-Xpress, and anti-Hsp90 antibodies, as indicated. The proper expression of transfected Dyrk1A was analyzed by immunoblotting with anti-Xpress antibody, and equal loading of sample was examined by immunoblotting with anti-Hsp90 antibody. Each immunoblotting with anti-Tau, anti-Xpress, and anti-Hsp90 antibodies was performed by repetitive re-probing of the same membrane and stripping with 100 mM citric acid. The intensity of phospho-tau (Thr212) and tau bands was quantified using ImageJ, and phosphorylated tau (Thr212) levels were normalized by tau levels. The gel pictures are representative of three independent experiments.

274

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

(Fig. 5C). Taken together, these results show that Dyrk1A phosphorylates the intracellular domain of β-amyloid precursor protein, which then causes the Aβ production in hippocampal progenitor cells. Enhancement of serum deprivation-induced apoptotic cell death in stable Dyrk1A-overexpressing cells While adults with DS develop Alzheimer's disease, development of the DS brain is also associated with decreased neuronal number, neuronal degeneration, and abnormal neuronal differentiation. The current findings show that Dyrk1A-overexpressing hippocampal progenitor cells (H19-7/Dyrk1A) showed typical AD-like pathology, including increased phosphorylated tau inclusion formation and the significant induction of Aβ production. Next, we determined the susceptibility of Dyrk1A-overexpressing cells to apoptotic cell death. To assess this possibility, the pattern of cell death induced by serum deprivation was measured in control and H19-7/Dyrk1A cells. When compared with parental control cells, stable H19-7/Dyrk1A cells showed lower viability when incubated with serum-free N2 media (Fig. 6A). Cell viability decreased by approximately two- (at 24 h) to seven-fold (at 72 h) in H19-7/Dyrk1A cells compared to parental control cells (Fig. 6A). In order to define the mode of cell death, we counted the number of cells in sub-G0/G1 phase using FACS analysis. Cell

Fig. 4. Enhancement of Aβ production in stable H19-7/Dyrk1A cells. (A) H19-7/Dyrk1A and control H19-7/pTK cell lysates were immunoblotted with either anti-APP or anti-Aβ antibodies, as indicated. After the levels of APP were analyzed by immunoblotting using anti-APP antibody (top panel), the membrane was re-probed with anti-Hsp90 antibody as a control for equal sample loading (lower panel). (B) The culture media were collected from H19-7/Dyrk1A and control H19-7/pTK cells, and concentrated by ultrafiltration (Amicon). The level of extracellular Aβ was determined by using an ELISA kit (Sigma-Aldrich) as described in manufacturer's protocol. The graph represents the mean of three independent experiments ± S.D. (⁎⁎ p b 0.001).

using wild type CTF-APP as a substrate. As shown in Fig. 5B, Dyrk1A could phosphorylate the C-terminal fragment of APP in vitro. APP phosphorylation at its cytoplasmic Thr668 residue was reported to stimulate intracellular Aβ levels (Lee et al., 2003). When the substitution of threonine 668 to alanine (T668A) was made within CTF-APP, it caused no phosphorylation of CTF-APP by Dyrk1A (Fig. 5B). This finding suggests that at least Dyrk1A specifically phosphorylates Thr668 residue of β-amyloid precursor protein. To confirm the involvement of Dyrk1A in Thr668 phosphorylation of the APP intracellular domain, HEK293 cells were co-transfected with wild type Dyrk1A together with the plasmid encoding either wild type or T668A mutant of APP Cterminal fragment fused with GFP (Fig. 5C). The transfected cell lysates were then immunoprecipitated with an anti-GFP antibody. The immunocomplexes were analyzed by immunoblotting with anti-phosphothreonine IgGs. As shown in Fig. 5C, phosphorylation of the wild type APP intracellular domain was increased by Dyrk1A overexpression. However, this remarkable induction was not observed in cells transfected with APP-T668A mutant

Fig. 5. Dyrk1A phosphorylates the intracellular domain of APP. (A) H19-7 cell lysates were immunoprecipitated with either anti-Dyrk1A antibody or normal mouse IgG, followed by immunoblotting with anti-APP and antiDyrk1A antibodies, as indicated. (B) After cell lysates were immunoprecipitated with anti-Dyrk1A antibody, in vitro kinase assays were performed by using a bacterially expressed GST protein fused with either wild type Cterminal fragment of APP or its T668A mutant as a substrate, as described in Experimental methods. (C) Where specified, HEK293 cells were transiently transfected with plasmids encoding 6xHis-Xpress-tagged wild type Dyrk1A, GFP-tagged wild type CTF-APP, and/or T668A mutant CTF-APP. The cell lysates were then immunoprecipitated with anti-GFP antibody, followed by immunoblotting with either anti-phosphothreonine or anti-GFP antibodies. Proper expression of transiently expressed Dyrk1A was confirmed by immunoblotting with the anti-Xpress antibody.

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

275

in stable H19-7/Dyrk1A cells under the same conditions (Fig. 7A). A ratio measure of normal neuronal differentiation was calculated by dividing the number of differentiated cells by the number of total surviving cells (Fig. 7B). Differentiated cells were defined as cells with refractile cell bodies extending at least two neurites that were longer than the diameter of the cell body. As shown in Fig. 7B, the normal neuronal differentiation in H19-7/Dyrk1A cells was exceedingly reduced after 24 h and 48 h compared to control H19-7/pTK cells. Based on our previous report showing that Dyrk1A-mediated CREB phosphorylation plays an important role in the differentiation of H19-7 cells (Yang et al., 2001), the profile of CREB phosphorylation was examined in control and H19-7/Dyrk1A cells under neuronal differentiation conditions. As shown in Fig. 7C, the immunoblot analysis of cell lysates with anti-phospho-CREB antibody at Ser133 showed that the addition of bFGF induces rapid and significant induction of CREB phosphorylation within 2 h in control H19-7/pTK cells. This was consistent with our previous finding (Yang et al., 2001). However, the pattern of CREB phosphorylation in stable H19-7/Dyrk1A cells was different from the control cells. As shown in Figs. 7C and D, the considerable CREB phosphorylation was not detected in stable H19-7/Dyrk1A cells. These results suggest that stable overexpression of Dyrk1A may also cause the defect of neuronal differentiation in hippocampal progenitor cells. Discussion

Fig. 6. Enhanced vulnerability to serum deprivation-induced apoptotic cell death of stable H19-7/Dyrk1A cells. (A) H19-7/Dyrk1A and control H19-7/ pTK cells were incubated with N2 media for 0 to 72 h, as indicated. The surviving cells were washed with PBS, trypsinized, and counted by hemocytometer. The cell viability relative to 0 h is illustrated. The graph represents the mean of six independent experiments ± S.E.M. in arbitrary units (A.U.) (⁎⁎ p b 0.001). (B) The cells were treated with N2 media for 0 to 16 h and trypsinized. The suspended cells were washed with PBS and fixed with 70% ethanol for overnight at 4 °C. After repeated washing with PBS, the fixed cells were treated with 20 μg of RNase A for 30 min at 37 °C and stained with 100 μg of propidium iodide. Then, the stained cells were counted with a BD FACScalibur. The graph represents the mean of three independent experiments ± S.D. (⁎⁎ p b 0.001).

were stained with propidium iodide (PI) and counted. The number of H19-7/Dyrk1A cells in sub-G0/G1 phase was increased by approximately 1.5-fold during a short time of N2 treatment compared to control H19-7/pTK cells (Fig. 6B). Taken together, these cells show that Dyrk1A overexpression in H19-7 cells caused an enhancement of cellular susceptibility to serum deprivationinduced apoptotic cell death. Defective neuronal differentiation in stable H19-7/Dyrk1A cells Next, we determined the effects of Dyrk1A overexpression on neuronal differentiation in hippocampal neuroprogenitor H19-7 cells. Treatment of H19-7 cells with bFGF for 24 to 48 h caused the cells to differentiate at 39 °C, as judged by morphology and neurite extension. However, no significant differentiation occurred

The present study demonstrates that Dyrk1A mapped to the DSCR is closely involved in the formation of hyperphosphorylated tau aggregates, APP processing, and Aβ production. We have also shown that two pathogenic proteins associated with Alzheimer's disease, tau and β-amyloid precursor protein could be substrates of Dyrk1A. The substrates of the proline-directed serine/threonine kinase Dyrk1A have consensus amino acid sequences in their phosphorylation site, as [RPX(S/T)P] is similar to that of ERK2 [PX (S/T)P] (Himpel et al., 2000). Through the multiple alignment analyses, tau and APP were found to have similar amino acid sequences of Dyrk1A consensus targeting sequence (Fig. 8). As shown in Fig. 8, their potentially targeting serine or threonine sites are near a proline residue with an adjacent arginine residue: 209RsrTP213 of tau and 668TPeeR672 of APP. Although the sequences of two proteins are not identical, the Dyrk1A-targeting sites are very similar, sufficiently enough to be the consensus sequence. Kimura and his colleagues reported that Dyrk1A may bridge tau to its intracellular NFT formation (Kimura et al., 2007). In fact, a correlation between the DYRK family and tau had already been reported, since DYRK1A and DYRK2 phosphorylate Thr212 of tau in vitro (Woods et al., 2001). The present study also showed an interaction between Dyrk1A and tau, and provided evidence of Dyrk1A-mediated phosphorylation of tau. Additionally, Dyrk1A overexpression in H19-7 cells caused an increase in tau phosphorylation at Thr212 residue; phosphorylated tau at Thr212 has been observed in the brain of AD patients (Gong et al., 2005). Immunocytochemical analysis revealed the formation of tau inclusions in stable H19-7/Dyrk1A cells. Moreover, the autophosphorylation of Dyrk1A was observed in stable H19-7/Dyrk1A cells. This auto-phosphorylation leads to Dyrk1A activation (Yang et al., 2001). Active Dyrk1A could also directly phosphorylate tau. Neurofibrillary tangles (NFTs) are mainly composed of hyperphosphorylated tau and are present mainly in the hippocampus and

276

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

Fig. 7. Defect in neuronal differentiation in stable H19-7/Dyrk1A cells. (A) H19-7/Dyrk1A and control H19-7/pTK cells were stimulated with 10 ng/ml bFGF under differentiation condition for 48 h. The cells were then fixed with 4% formaldehyde in PBS. Cells were photographed under an optical microscope. (B) Normally differentiated cells were counted. The ratio of differentiation was calculated by dividing the number of differentiated cells by the number of total surviving cells. The graph represents the mean of two independent experiments ± S.D. Statistical significance was determined by Student's t-test (⁎⁎ p b 0.001, ⁎ p b 0.01). (C) Where specified, H19-7/Dyrk1A and control H19-7/pTK cells were stimulated with bFGF for the indicated times under conditions of neuronal differentiation. Thirty micrograms of each cell lysates were immunoblotted with anti-phospho-CREB (Ser133) antibody. After stripping with 100 mM citric acid followed by repetitive washing with TBST, the membrane was analyzed by re-probing with anti-CREB antibody. The equal loading of each cell lysates was analyzed by immunoblotting with anti-Hsp90 antibody. The pictures of immunoblot analysis are representative of two independent experiments. (D) The intensity of phospho-CREB (Ser133) and CREB bands was quantified using ImageJ, and phosphorylated CREB (Ser133) levels were normalized by the levels of intact CREB protein. The graph represents the mean of two independent experiments ± S.D. in arbitrary units (A.U.). Statistical significance was determined by Student's t-test (⁎⁎ p b 0.01, ⁎ p b 0.05).

amygdala (Mandelkow et al., 1995). As shown in Fig. 2A, the expression of phosphorylated tau at Thr212 residue were exclusively detected within the intracellular inclusion-like tau aggregates in Dyrk1A-overexpressing cells, suggesting that the hyperphosphorylation of tau caused by Dyrk1A overexpression may result in the formation of its insoluble inclusion. Comparing with Figs. 2A and C, the increase in the levels of phospho-tau protein detected by immunoblot analysis was much less than that by immunocytochemical analysis. This inconsistency may come from that hyperphosphorylated tau by Dyrk1A overexpression may precipitate to form the insoluble inclusions and the remaining soluble protein lysates were used for immunoblot analysis. Tau is multifunctional microtubule-associated protein that plays major roles in the assembly of microtubules and the stabilization of microtubules against dynamic instability (Maccioni and Cambiazo, 1995). The most relevant protein kinases involved in tau modifications in neurofibrillary degeneration are GSK-3β (Imahori and Uchida, 1997) and Cdk5 (Michel et al., 1998). Hyperphosphorylated tau-mediated cytotoxicity is well associated with induction

of apoptotic cell death. Tau expression specifically sensitizes cells to other apoptotic stimuli such as staurosporine (Fath et al., 2002). Consistent with this finding, Dyrk1A-mediated tau modification is thought to contribute to the increased sensitivity of stable H19-7/ Dyrk1A cells to apoptotic stimuli such as serum deprivation. We demonstrated that Dyrk1A could phosphorylate Thr668 of the APP intracellular domain and induce Aβ production. Since APP phosphorylation at its cytoplasmic Thr668 residue was

Fig. 8. Potential Dyrk1A-phosphorylation sites in Tau and APP protein are similar to Dyrk1A target consensus sequence, R(x)xx(S/T)(P/V) or (S/T) (P/V)xx(x)R. Diagram of potential Dyrk1A-phosphorylation sequences showing homologous sites between mouse, rat, and human sequences.

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

reported to stimulate intracellular Aβ levels (Lee et al., 2003), Dyrk1A-mediated APP phosphorylation may also play a role in the pathologic Aβ deposition of DS patients. Indeed, an increase in Aβ caused up-regulation of Dyrk1A mRNA, and Dyrk1A transcripts are significantly elevated in the hippocampus of AD patients (Kimura et al., 2007). Key features of the DS brain include reduced neuronal number and defects in neuronal differentiation, although the mechanisms are not well understood (Mann, 1988). Loss of neurons in the DS brain may occur via apoptotic cell death. Since the increase of Aβ caused up-regulation of Dyrk1A mRNA (Kimura et al., 2007), it is important to determine whether Dyrk1A overexpression is involved in apoptotic cell death. The current study showed that up-regulation of Dyrk1A exacerbates apoptotic cell death under the conditions of serum deprivation. In addition, increased production of Aβ and the formation of inclusions composed of phosphorylated tau may be involved in the induction of apoptotic cell death in stable H19-7/Dyrk1A cells. In fact, intracellular Aβ production induces neuronal apoptosis (Kienlen-Campard et al., 2002). Furthermore, Dyrk1A-overexpressing cells showed severe defects in neuronal differentiation. Several molecular and clinical similarities between AD and DS have been shown. The most remarkable feature is abnormal accumulation of β-amyloid in the brains of individuals affected with AD and in aging DS patients (Iwatsubo et al., 1995). AD and DS could be related genetically because AD families exhibit a higher rate of DS cases. This was further supported by the finding that transgenic mice overexpressing Dyrk1A exhibited clear phenotypes such as significant defects in learning and memory and neurodevelopmental abnormalities (Altafaj et al., 2001; Ahn et al., 2006). The current findings that show characteristic processing of AD target proteins, increased apoptotic neuronal death, and differentiation defects in stable H19-7/Dyrk1A cell lines suggest that this could be a good model to further clarify the detailed molecular mechanisms of neuropathogenesis in DS and sporadic AD. Further characterization of the molecular mechanisms leading to the Dyrk1A-mediated abnormal processing of APP and tau will help to clarify the biological function(s) of Dyrk1A. Such knowledge will be very relevant to understanding the pathogenesis of AD in DS patients as well as sporadic AD. In summary, the present finding demonstrates that Dyrk1A overexpression causes abnormal processing of several AD-related pathogenic proteins. These findings also strongly implicate Dyrk1A in production of AD phenotype in DS patients.

277

phosphotyrosine antibodies and mouse immunoglobulins (IgGs) were purchased from Santa Cruz Biotechnology. Anti-Dyrk1A, anti-HA, anti-Tau, anti-phospho-Tau (Thr212), and anti-APP (6E10) antibodies were purchased from BD Biosciences, BAbCo, Upstate Biotechnology, Biosource, and Signet Laboratories, respectively. Anti-Aβ antibody specific to 1–42 was purchased from GeneTex. Plasmids encoding HA-tagged wild type Dyrk1A (pSVL-HA-Dyrk1A WT) and its K188R mutant (pSVL-HADyrk1A K188R) were kindly provided by W. Becker (Institute of Pharmacology and Toxicology, RWTH, Aachen, Germany). In order to make constructs encoding 6xHis-Xpress-tagged Dyrk1A, wild type and K188R mutant Dyrk1A were amplified by PCR with primers; 5′-GAATTCTCATGCATACAGGAGGAGAGA-3′ and 5′-GCGGCCGCTCACGAGCTAGCTACAGG-3′, and sub-cloned into pcDNA4/HisMax-A vector (Invitrogen). Plasmids encoding V5-tagged wild-type Tau (pcDNA3.1/V5-6xHis-Tau) were kindly provided by L. Petrucelli (Mayo Clinic, Jacksonville). Plasmids encoding enhanced green fluorescent protein (EGFP)tagged wild type and T668A mutant C-terminus of APP (pEGFPN1-CTF-APP WT and pEGFP-N1-CTF-APP T668A) were provided by Y.H. Suh (Seoul National University, College of Medicine). Constructs encoding GST-tagged wild type and T668A mutant C-terminus of APP (pGEX-5X-1-CTF-APP WT and pGEX5X-1-CTF-APP T668A) were provided by K.F. Lau (Institute of Psychiatry, King's College). Cell culture and preparation of cell lysates Immortalized hippocampal H19-7 cells and HEK293 cells were grown on poly-L-lysine-coated and non-coated cell culture dishes, respectively, in DMEM containing 10% FBS and 100 unit/ml penicillin–streptomycin. To prepare cell lysates, cells were rinsed twice with ice-cold phosphate-buffered saline and solubilized in lysis buffer as described previously (Yang et al., 2001). Establishment of stably Dyrk1A-overexpressing cells In order to construct H19-7 cells that stably overexpress Dyrk1A, plasmids encoding wild type Dyrk1A (pSVL-HADyrk1A WT) were transfected into the cells together with pTKHyg as a selectable marker. Cells were selected with 250 μg/ml of hygromycin B (Invitrogen). Then, single clonal cells that showed the proper expression of Dyrk1A were chosen as a stably Dyrk1Aoverexpressing cell line, whereas the cells transfected only with pTK-Hyg vector were used as a control.

Experimental methods Immunoprecipitation and Western blot analysis Materials Secondary goat anti-IgG horseradish peroxidase-, FITC-, and TRITC-conjugated antibodies, Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), LipofectAMINE PLUS reagents, anti-Xpress and anti-V5 antibodies were purchased from Invitrogen. Glutathione-Sepharose 4B and Protein A-Sepharose were obtained from Amersham Biosciences. Enhanced chemiluminescence (ECL) reagents and [γ-32P] ATP were purchased from PerkinElmer Life and Analytical Sciences. Anti-phosphothreonine antibody and all other used chemicals were commercial products of analytical grade purchased from Sigma. Anti-phospho-CREB (Ser133) and anti-CREB antibodies were purchased from Cell Signaling Technology. Anti-GFP, anti-α-tubulin, anti-Hsp90, anti-

One microgram of suitable antibodies was incubated with 0.5 to 1 mg of cell extract in cell lysis buffer overnight at 4 °C. Fifty microliter of a 1:1 Protein A-Sepharose bead suspension was added and incubated for 2 h at 4 °C with gentle rotation. Beads were pelleted and washed extensively with cell lysis buffer. The immunocomplexes were dissociated by boiling in SDS-PAGE sample buffer, separated onto SDS-PAGE gel, and transferred to a nitrocellulose membrane (Amersham Biosciences). Membranes were then blocked in TBST buffer (25 mM Tris, pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20) plus 5% nonfat dry milk for 1 h at room temperature, and incubated overnight at 4 °C in TBST buffer with 3% nonfat dry milk and the appropriate primary antibodies. Membranes were washed three times in TBST, and then incubated with appropriate secondary

278

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279

IgG-coupled horseradish peroxidase antibodies for 2 h at room temperature. The membranes were washed three times with TBSTand visualized with ECL reagent. Immunocytochemistry analysis Dyrk1A-overexpressing H19-7 cells and parental control cells were seeded into poly-L-lysine-coated coverslips with approximately 50–70% confluency at 33 °C. Twenty-four hours later, cells were washed twice with PBS (pH 7.4) and immediately fixed with 4% formaldehyde during overnight at 4 °C. After fixation, cells were permeabilized with 0.2% Triton-X-100 for 30 min, and blocked with 1% bovine serum albumin for 30 min at room temperature followed by immunostaining with anti-phospho-Tau (Thr212), anti-Tau, or anti-α-tubulin antibodies. Then, either FITCconjugated or TRITC-conjugated secondary antibody was used to detect the primary antibodies. The stained samples were stained with DAPI, mounted, and analyzed by LSM 510 META confocal microscopy (Carl Zeiss). The data were processed by Zeiss LSM Image Browser (Carl Zeiss). In vitro kinase assay After cell lysis in lysis buffer, 600 μg of protein was incubated with anti-Dyrk1A antibody for overnight at 4 °C. The immunocomplexes were mixed with protein A-Sepharose beads, and kinase reactions were carried out at 30 °C for 60 min in 20 μl of kinase buffer containing 20 mM HEPES (pH 7.2), 5 mM MnCl2, 200 μM sodium orthovanadate, 10 μg of acid-treated enolase, 10 μM ATP, 5 μCi of [γ-32P] ATP, and 5 μg of GST-tau and GST– CTF–APP as substrate. The reaction was stopped by adding SDSsample buffer, and analyzed by SDS-PAGE followed by autoradiography. Analysis of cell viability and neuronal differentiation H19-7 cells were grown on poly-L-lysine-coated 24-well dishes. To assess cell viability, N2 medium was added to the cells before reaching 70–90% confluence. The cells were then cultured at 39 °C for 0 to 72 h. Then, the surviving cells were washed with PBS and counted by hemocytometer. To examine apoptotic cell death, we also performed FACS analysis. Cells were cultured in N2 media for 0 to 16 h at 39 °C. After trypsinization, the cells were washed with ice-cold PBS (pH 7.4) and fixed in 70% ethanol overnight at 4 °C. The fixed cells were washed with ice-cold PBS, treated with 20 μg of RNase A for 30 min at 37 °C and incubated with 100 μg of propidium iodide. Then, the stained cells were analyzed by BD FACScalibur. For the analysis of neuronal differentiation, cells were grown on poly-L-lysine-coated 6-well dishes and switched to N2 media for 48 h at 39 °C, treated with 10 ng/ml bFGF for 48 h, and analyzed for morphological changes. Differentiated cells were defined as cells with refractile cell bodies extending at least two neurites that were longer than the diameter of the cell body. Acknowledgments We are deeply grateful to W. Becker, L. Petrucelli, Y.H. Suh, and K.F. Lau for generously providing cDNA constructs. This study was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science

and Technology (M103KV010011-06K2201-01110 to K.C.C.), by Basic Research Grant from the Korea Science and Engineering Foundation (KOSEF; R01-2004-000-10673-0 to K.C.C.), and by grants from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A050181 and A060440 to K.C.C.). This work was also supported by KOSEF through the National Research Lab. Program fund (R04-2007-00020014-0 to K.C.C.), partly supported by the Korea Research Foundation Grant (KRF-2004005-E0017), the Brain Korea 21 Projects from Korea Research Foundation (to J.P.), and supported in part by Seoul Science Fellowship Program from Seoul Metropolitan Government (to J.P.). References Ahn, K.J., Jeong, H.K., Choi, H.S., Ryoo, S.R., Kim, Y.J., Goo, J.S., Choi, S.Y., Han, J.S., Ha, I., Song, W.J., 2006. DYRK1A BAC transgenic mice show altered synaptic plasticity with learning and memory defects. Neurobiol. Dis. 22, 463–472. Altafaj, X., Dierssen, M., Baamonde, C., Marti, E., Visa, J., Guimera, J., Oset, M., Gonzalez, J.R., Florez, J., Fillat, C., Estivill, X., 2001. Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down's syndrome. Hum. Mol. Genet. 10, 1915–1923. Antonarakis, S.E., Lyle, R., Dermitzakis, E.T., Reymond, A., Deutsch, S., 2004. Chromosome 21 and Down syndrome: from genomics to pathology. Nat. Rev., Genet. 5, 725–738. Becker, W., Joost, H.G., 1999. Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity. Prog. Nucleic Acid Res. Mol. Biol. 62, 1–17. Evenhuis, H.M., 1990. The natural history of dementia in Down's syndrome. Arch. Neurol. 47, 263–267. Eves, E.M., Tucker, M.S., Roback, J.D., Downen, M., Rosner, M.R., Wainer, B.H., 1992. Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression. Proc. Natl. Acad. Sci. U. S. A. 89, 4373–4377. Fath, T., Eidenmuller, J., Brandt, R., 2002. Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer's disease. J. Neurosci. 22, 9733–9741. Gandy, S., Czernik, A.J., Greengard, P., 1988. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/ calmodulin-dependent protein kinase II. Proc. Natl. Acad. Sci. U. S. A. 85, 6218–6221. Gong, C.X., Liu, F., Grundke-Iqbal, I., Iqbal, K., 2005. Post-translational modifications of tau protein in Alzheimer's disease. J. Neural Transm. 112, 813–838. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.C., Zaidi, M.S., Wisniewski, H.M., 1986a. Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J. Biol. Chem. 261, 6084–6089. Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M., Binder, L.I., 1986b. Abnormal phosphorylation of the microtubuleassociated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. U. S. A. 83, 4913–4917. Guimera, J., Casas, C., Pucharcos, C., Solans, A., Domenech, A., Planas, A. M., Ashley, J., Lovett, M., Estivill, X., Pritchard, M.A., 1996. A human homologue of Drosophila minibrain (MNB) is expressed in the neuronal regions affected in Down syndrome and maps to the critical region. Hum. Mol. Genet. 5, 1305–1310. Himpel, S., Tegge, W., Frank, R., Leder, S., Joost, H.G., Becker, W., 2000. Specificity determinants of substrate recognition by the protein kinase DYRK1A. J. Biol. Chem. 275, 2431–2438. Ihara, Y., Nukina, N., Miura, R., Ogawara, M., 1986. Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease. J. Biochem. 99, 1807–1810. Imahori, K., Uchida, T., 1997. Physiology and pathology of tau protein kinases in relation to Alzheimer's disease. J. Biochem. 121, 179–188.

J. Park et al. / Mol. Cell. Neurosci. 36 (2007) 270–279 Iwatsubo, T., Mann, D.M., Odaka, A., Suzuki, N., Ihara, Y., 1995. Amyloid beta protein (A beta) deposition: A beta 42(43) precedes A beta 40 in Down syndrome. Ann. Neurol. 37, 294–299. Kienlen-Campard, P., Miolet, S., Tasiaux, B., Octave, J.N., 2002. Intracellular amyloid-beta 1–42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis. J. Biol. Chem. 277, 15666–15670. Kimura, R., Kamino, K., Yamamoto, M., Nuripa, A., Kida, T., Kazui, H., Hashimoto, R., Tanaka, T., Kudo, T., Yamagata, H., Tabara, Y., Miki, T., Akatsu, H., Kosaka, K., Funakoshi, E., Nishitomi, K., Sakaguchi, G., Kato, A., Hattori, H., Uema, T., Takeda, M., 2007. The DYRK1A gene, encoded in chromosome 21 Down syndrome critical region, bridges between beta-amyloid production and tau phosphorylation in Alzheimer disease. Hum. Mol. Genet. 16, 15–23. Lee, M.S., Kao, S.C., Lemere, C.A., Xia, W., Tseng, H.C., Zhou, Y., Neve, R., Ahlijanian, M.K., Tsai, L.H., 2003. APP processing is regulated by cytoplasmic phosphorylation. J. Cell Biol. 163, 83–95. Maccioni, R.B., Cambiazo, V., 1995. Role of microtubule-associated proteins in the control of microtubule assembly. Physiol. Rev. 75, 835–864. Mandelkow, E.M., Biernat, J., Drewes, G., Gustke, N., Trinczek, B., Mandelkow, E., 1995. Tau domains, phosphorylation, and interactions with microtubules. Neurobiol. Aging 16, 355–362. Mann, D.M., 1988. Alzheimer's disease and Down's syndrome. Histopathology 13, 125–137. Mann, D.M., Yates, P.O., Marcyniuk, B., Ravindra, C.R., 1987. Loss of neurons from cortical and subcortical areas in Down's syndrome patients at middle age. Quantitative comparisons with younger Down's patients and patients with Alzheimer's disease. J. Neurol. Sci. 80, 79–89. Michel, G., Mercken, M., Murayama, M., Noguchi, K., Ishiguro, K., Imahori, K., Takashima, A., 1998. Characterization of tau phosphorylation in glycogen synthase kinase-3beta and cyclin dependent kinase-5 activator (p23) transfected cell. Biochem. Biophys. Acta. 1380, 177–182. Raich, W.B., Moorman, C., Lacefield, C.O., Lehrer, J., Bartsch, D., Plasterk, R.H., Kandel, E.R., Hobert, O., 2003. Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A. Genetics 163, 571–580. Selkoe, D.J., 1994. Normal and abnormal biology of the beta-amyloid precursor protein. Annu. Rev. Neurosci. 17, 489–517. Shindoh, N., Kudoh, J., Maeda, H., Yamaki, A., Minoshima, S., Shimizu, Y., Shimizu, N., 1996. Cloning of a human homolog of the Drosophila

279

minibrain/rat Dyrk gene from “the Down syndrome critical region” of chromosome 21. Biochem. Biophys. Res. Commun. 225, 92–99. Smith, D.J., Stevens, M.E., Sudanagunta, S.P., Bronson, R.T., Makhinson, M., Watabe, A.M., O'Dell, T.J., Fung, J., Weier, H.U., Cheng, J.F, Rubin, E.M., 1997. Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome. Nat. Genet. 16, 28–36. Song, W.J., Sternberg, L.R., Kasten-Sportes, C., Keuren, M.L., Chung, S.H., Slack, A.C., Miller, D.E., Glover, T.W., Chiang, P.W., Lou, L., Kurnit, D.M., 1996. Isolation of human and murine homologues of the Drosophila minibrain gene: Human homologue maps to 21q22.2 in the Down syndrome “Critical Region”. Genomics 38, 331–339. Suzuki, T., Nairn, A.C., Gandy, S.E., Greengard, P., 1992. Phosphorylation of Alzheimer amyloid precursor protein by protein kinase C. Neuroscience 48, 755–761. Suzuki, T., Oishi, M., Marshak, D.R., Czernik, A.J., Nairn, A.C., Greengard, P., 1994. Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. EMBO J. 13, 1114–1122. Teller, J.K., Russo, C., DeBusk, L.M., Angelini, G., Zaccheo, D., DagnaBricarelli, F., Scartezzini, P., Bertolini, S., Mann, D.M., Tabaton, M., Gambetti, P., 1996. Presence of soluble amyloid beta-peptide precedes amyloid plaque formation in Down's syndrome. Nat. Med. 2, 93–95. Wisniewski, K.E., Laure-Kamionowska, M., Wisniewski, H.M., 1984. Evidence of arrest of neurogenesis and synaptogenesis in brains of patients with Down's syndrome. New Engl. J. Med. 311, 1187–1188. Wisniewski, K.E., Wisniewski, H.M., Wen, G.Y., 1985. Occurrence of neuropathological changes and dementia of Alzheimer's disease in Down's syndrome. Ann. Neurol. 17, 278–282. Woods, Y.L., Cohen, P., Becker, W., Jakes, R., Goedert, M., Wang, X., Proud, C.G., 2001. The kinase DYRK phosphorylates proteinsynthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase. Biochem. J. 355, 609–615. Yang, E.J., Ahn, Y.S., Chung, K.C., 2001. Protein kinase Dyrk1 activates cAMP response element-binding protein during neuronal differentiation in hippocampal progenitor cells. J. Biol. Chem. 276, 39819–39824.