Dyrk1A Phosphorylates α-Synuclein and Enhances Intracellular Inclusion Formation*

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. α-Synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with α-synuclein and subsequently affects intracellular α-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to α-synuclein in transformed and primary neuronal cells. α-Synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased α-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked α-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate α-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated α-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type α-synuclein. These findings suggest α-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. ␣-Synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with ␣-synuclein and subsequently affects intracellular ␣-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to ␣-synuclein in transformed and primary neuronal cells. ␣-Synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased ␣-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked ␣-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate ␣-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated ␣-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type ␣-synuclein. These findings suggest ␣-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.
␣-Synuclein is a major component of Lewy bodies (LBs) 2 found in Parkinson disease (PD), dementia with LB, Alzheimer disease (AD), and multiple system atropy (1). In these neurodegenerative disorders (collectively referred to as synucleinopathies), LBs are characterized by fibrillar, cytoplasmic ␣-synuclein aggregates within selective populations of neurons and glial cells (2). ␣-Synuclein inclusion formation is clearly involved in the pathogenic process of PD. ␣-Synuclein was first identified as a partial fragment in AD amyloid plaques (41), and subsequently three missense mutations in the ␣-synuclein gene were reported in early onset familial PD of some kindred (3,4).
Down syndrome (DS) is the most common genetic disorder, with a frequency of 1 in every 700 -800 live births, and is caused by an extra copy of all or part of chromosome 21 (5). In addition to characteristic physical features, DS individuals have congenital heart defects, gastrointestinal malformations, immune and endocrine system defects, a high incidence of leukemia, and early onset of Alzheimer-like dementia. DS individuals also exhibit mild to severe mental retardation (6 -8). Efforts to isolate the gene(s) responsible for DS mental retardation identified Dyrk1A as a candidate gene (9,10).
The Drosophila melanogaster minibrain (Mnb) gene encodes a serine/threonine protein kinase essential in cell proliferation and neuronal differentiation during postembryonic neurogenesis (10). Dual specificity tyrosine-regulated kinase-1A (Dyrk1A), the Mnb kinase human homolog, maps to the DS critical region on chromosome 21. Dyrk1A is thought to be responsible for the DS neurological defects. In DS fetal brains, Dyrk1A expression increases 1.5-fold, and transgenic mice overexpressing Dyrk1A exhibit neurodevelopmental delays, motor abnormalities, and cognitive deficits (11,12).
Interestingly, ␣-synuclein-positive LBs and neuritic processes frequently occur in DS brains with AD phenotypes (18). In addition, LB formation frequency in DS patient brains with AD is greater than in sporadic AD cases (19). To study the molecular mechanisms leading to LB formation in DS patients, we examined whether Dyrk1A interacts with ␣-synuclein and affects cytoplasmic inclusion formation in hippocampal neuroprogenitor cells. Our data show that Dyrk1A phosphorylates ␣-synuclein at the Ser-87 residue. Additionally, Dyrk1A-mediated ␣-synuclein phosphorylation facilitates its aggregation.
Cell Culture and DNA Transfection-Conditionally immortalized hippocampal (H19-7) cell lines were cultured as described previously (21). The neuroblastoma SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with penicillin and streptomycin. Rat fetal brain lysates and primary cortical neurons were prepared as described previously (22). The cells were transfected with Lipofectamine Plus reagent (Invitrogen), according to the supplier's instructions. To prepare cell lysates, cells were rinsed with ice-cold phosphate-buffered saline and solubilized in lysis buffer (10 mM Tris, pH 7.9, containing 1.0% Nonidet P-40, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10% glycerol, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, 1 g/ml aprotinin, 10 mM NaF, and 0.2 mM phenylmethylsulfonyl fluoride). Cells were scraped, and supernatants were collected after centrifugation for 10 min at 14,000 ϫ g at 4°C. Protein concentrations were determined using the detergent-compatible protein assay kit (Bio-Rad).
Cell Viability-Cell survival quantitation was performed using the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide extraction method, as described previously (23). Statistical analyses were completed with the aid of the StatView II program (Abacus Concepts, CA). All data were analyzed by one-way analysis of variance and preplanned comparisons with the control were performed by means of Dunnett's T-statistic.
Immunoprecipitation and Western Blot Analysis-One microgram of suitable antibodies was incubated with 0.5-1 mg of cell extracts in cell lysis buffer overnight at 4°C. Fifty microliters 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. Bound proteins were dissociated by boiling in SDS-PAGE sample buffer, and samples were separated on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore). Membranes were blocked in TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween 20) plus 3% nonfat dry milk for 3 h and then incubated overnight at 4°C in TBST buffer with 3% nonfat dry milk and the appropriate antibodies. Membranes were washed several times in TBST and then incubated with secondary IgG-coupled horseradish peroxidase antibody (Zymed Laboratories Inc.). After 60 min, the blots were washed several times with TBST and visualized by ECL.
Immunocytochemistry-Cells were seeded overnight, at 70% confluence, onto coverslips in 6-well dishes and transfected with the appropriate plasmids the following day for 24 h. After washing with phosphate-buffered saline, the cells were fixed with neutral buffered 4% (w/v) paraformaldehyde and permeabilized with 1% bovine serum albumin containing 0.1% Triton X-100 for 1 h. Cells were incubated at 4°C for 24 h with the appropriate primary antibody and diluted in phosphate-buffered saline containing 1% bovine serum albumin. After washing with phosphate-buffered saline, either rhodamine-or fluorescein isothiocyanate-coupled secondary antibodies were added and incubated for 2 h at room temperature. Fixed cells were analyzed by confocal or fluorescence microscopy.
Hematoxylin and Eosin Staining-Hematoxylin and eosin staining were performed according to the manufacturer's instructions (Sigma). We counted cells with eosinophilic inclusions in six different fields, ϳ1000 cells/experimental condition.
In Vitro Kinase Assay-Confluent cells were harvested in lysis buffer. Soluble cell lysate fraction was incubated for 2 h at 4°C with suitable antibodies. After the addition of protein A-Sepharose beads, the reaction mixture was incubated for 2 h at 4°C and rinsed with lysis and kinase buffers. Immunocomplex kinase assays were performed by incubating the cell lysates for 2 h at 30°C with the substrate in the reaction buffer (0.2 mM sodium orthovanadate, 2 mM dithiothreitol, 10 mM MgCl 2 , 2 mCi of [␥-32 P]ATP, and 20 mM HEPES, pH 7.4). After reaction termination, the mixtures were analyzed by SDS-PAGE, and phosphorylated substrates were visualized by autoradiography.
Protein Aggregation Analysis-␣-Synuclein aggregation was monitored with both turbidity and thioflavin-T binding fluorescence, as described previously (23). Purified ␣-synuclein samples were concentrated to 7 mg/ml using Centricon-3 spin filters (Amicon). After concentration, samples were centrifuged for 10 min at 100,000 ϫ g to remove any aggregates that could have formed during the concentration step. The supernatants were adjusted to a final concentration of 7 mg/ml using Tris-buffered saline, 20 mM Tris, pH 7.5, and 0.2 M NaCl. Samples were dispensed into 1.5 ml of Beckman ultracentrifuge microtubes and incubated at 37°C. At various time points, samples were centrifuged at 100,000 ϫ g for 10 min, and supernatants were removed and diluted 10 times with Tris-buffered saline. These dilutions were analyzed by their absorbance at 280 nm. The remaining incubations were vortexed for 30 s to resuspend the pellets. For electron microscopy of ␣-synuclein aggregates, the samples were prepared as described previously (23). Samples were sectioned using an ultramicrotome, doublestained with uranyl acetate and lead citrate, and observed by transmission electron microscopy (Philips CM-10).

Construction of Dyrk1A siRNA Duplexes and Transfection-
The competitive silencing and noncompetitive control Dyrk1A siRNAs were designed as reported by Sitz et al. (24) and provided by Sigma-Proligo (Boulder, CO). Dyrk1A siRNA duplexes were transfected into cells using the Lipofectamine Plus reagent according to the manufacturer's instructions.

RESULTS
Dyrk1A Binds to and Phosphorylates ␣-Synuclein in H19-7 Cells-We examined whether the ␣-synuclein and Dyrk1A interaction occurs in mammalian neuronal cells, such as in immortalized hippocampal H19-7 cells. We previously reported that the endogenous ␣-synuclein protein levels in H19-7 cells were undetectable (25,26). Therefore, we transiently transfected H19-7 cells with ␣-synuclein cDNA, immunoprecipitated ␣-synuclein, and immunoblotted for Dyrk1A. Exogenously expressed ␣-synuclein binds to endogenous Dyrk1A (Fig. 1A). We confirmed this interaction by reverse co-immunoprecipitation and detecting the HA-tagged Dyrk1A-␣-synuclein interaction in the dopaminergic neuroblastoma SH-SY5Y cell line (Fig. 1, A and B). These data demonstrate that ␣-synuclein interacts with Dyrk1A in H19-7 and SH-SY5Y cells.  In vivo interaction between Dyrk1A and ␣-synuclein in rat brain and primary cortical neurons. Whole brain tissue (A) or primary cortical neurons (B) were prepared from rat embryonic 17-day fetus. Whole cell lysates from rat brain (A) or primary cortical neurons (B) were immunoprecipitated (IP) with anti-␣-synuclein or anti-Dyrk1A antibodies, followed by immunoblot analysis with anti-Dyrk1A or anti-␣-synuclein antibodies, respectively. As a control, the cell extracts were immunoprecipitated with preimmune IgG (IgG) or empty protein A-beads (Bead). As an input control, ␣-synuclein and Dyrk1A expression was monitored by Western analysis. C, rat E17 cortical neurons were fixed, permeabilized, labeled subsequently with either anti-␣-synuclein or anti-Dyrk1A antibodies, with fluorescein isothiocyanate-or rhodamine-attached secondary antibodies, and with 4Ј,6-diamidino-2-phenylindole. Immunostained preparations were examined using confocal microscopy. These results are representative of three independent experiments.
Next, we asked if endogenous Dyrk1A interacts with ␣-synuclein in the mammalian central nervous systems. ␣-Synuclein and Dyrk1A are highly expressed in rat brain lysates and primary cortical neurons (Fig. 2, A and B). As shown in Fig. 2, ␣-synuclein selectively associates with Dyrk1A, and we did not observe nonspecific interaction with preimmune IgG or protein A beads (Fig. 2, A and B). Moreover, immunostaining of primary cortical neurons showed that Dyrk1A and ␣-synuclein co-localize in the cytoplasm (Fig. 2C). These data suggest that the Dyrk1A and ␣-synuclein interaction is not an artifact observed in transformed cell lines but occurs in the mammalian central nervous system.
Dyrk1A Phosphorylates ␣-Synuclein upon the Stimulation with bFGF in H19-7 Cells-Previously, we showed that basic fibroblast growth factor (bFGF) addition to H19-7 cells causes Dyrk1A activation, which plays an important role during neuronal differentiation of H19-7 cells (21). Based on these findings, we tested whether active Dyrk1A directly phosphorylates ␣-synuclein. Transient ␣-synuclein expression in H19-7 cells, followed by bFGF stimulation under the neuronal differentiation conditions, enhanced Ser/Thr phosphorylation within 6 h, reaching a maximum at 1 h (Fig. 3A). Since Dyrk1A acts as a dual specificity protein kinase, which catalyzes the tyrosinedirected autophosphorylation as well as serine/threonine residue phosphorylation in exogenous substrates (27), ␣-synuclein could also be phosphorylated at tyrosine residues. We performed a similar experiment to determine whether ␣-synuclein was phosphorylated on tyrosine residue(s). As shown in Fig. 3B, A, H19-7 cells were transiently transfected with ␣-synuclein for 24 h and stimulated with bFGF for the indicated times in differentiation conditions. Immunoprecipitation was performed with anti-␣-synuclein (Syn) antibody, and the immunocomplexes were analyzed by Western blotting (WB) with either anti-␣-synuclein or phosphoserine/phosphothreonine (p-Ser/Thr) antibodies. B, as performed in A, except using phosphotyrosine (p-Tyr) antibodies. C, H19-7 cells were transiently transfected with ␣-synuclein plasmid alone (No T or Con) or together with a kinase-deficient Dyrk1A (mDyrk). Where indicated, ␣-synuclein was co-transfected with either silencing Dyrk1A siRNA (siRNA) or noncompetitive control siRNA (nsRNA). After 24 h, cells were left untreated (No T) or stimulated with bFGF for 1 h. Immunoprecipitation was performed with anti-␣-synuclein IgG. Immunocomplexes were examined by Western analysis with anti-phosphoserine/phosphothreonine antibodies (left). The blocking of Dyrk1A expression by siRNA was determined by Western blotting with anti-Dyrk1A antibodies (right). ␤-Tubulin expression showed equal loading. D, H19-7 cells were either untreated or stimulated with bFGF for 1 h under differentiation conditions, and cell lysates were immunoprecipitated with anti-Dyrk1A antibodies. By using anti-Dyrk1A immunocomplexes, in vitro kinase was performed with either GST or GST fused with ␣-synuclein (GST-Syn) protein as a substrate. Phosphorylated GST-␣-synuclein levels were visualized by autoradiography (Ag) (top). The purity of each GST or GST-␣synuclein was assayed by Western blot with anti-GST IgG (bottom). E, H19-7 cells were mock-transfected (Con) or transfected with kinase-defective Dyrk1A, competitive (siRNA), or noncompetitive siRNA for 24 h, and stimulated with bFGF for 1 h. After, Dyrk1A was immunoprecipitated for an in vitro kinase assay using ␣-synuclein as a substrate. These results are representative of two or three independent experiments. A, four recombinant ␣-synuclein proteins, full length (residues 1-140), amphipathic N-terminal region (residues 1-60), NAC region (residues 61-95), or C-terminal acidic tail region (residues 96 -140) were tested for phosphorylation by immunoprecipitated Dyrk1A (top). Dyrk1A was immunoprecipitated from differentiating H19-7 cells. Only two ␣-synuclein recombinant proteins were phosphorylated (filled arrowhead). The bottom panel shows input recombinant ␣-synuclein proteins via Western blot analysis. B, H19-7 cells were mock-transfected or transfected for 24 h with ␣-synuclein wild type or point mutants, S4A, S42A, S87A, and S129A. After 1 h of bFGF stimulation in differentiation conditions, cell lysates were prepared and immunoprecipitated (IP) with anti-synuclein (Syn) IgG, followed by immunoblotting with either anti-␣-synuclein or anti-phosphoserine/phosphothreonine (p-S/T) antibodies. These results are representative of two independent experiments. C, the recombinant ␣-synuclein has been incubated with in vitro phosphorylation system in the absence or presence of casein kinase 1 (CK1) for 3 h. The occurrence of ␣-synuclein phosphorylation at Ser-129 has been examined by immunoblotting with anti-PSer-129 antibody. D, cells were transfected with Dyrk1A for 24 h, and cell lysates were immunoprecipitated with anti-Dyrk1A IgG. In vitro phosphorylation by anti-Dyrk1A immunocomplexes was performed with either GST-CREB or recombinant ␣-synuclein as a substrate. The phosphorylation of CREB at Ser-133 or ␣-synuclein at Ser-129 was determined by immunoblotting with their specific antibodies, as indicated.
we did not detect tyrosine-phosphorylated ␣-synuclein within 6 h of bFGF treatment, in H19-7 cells.
To clarify whether ␣-synuclein phosphorylation is due to active Dyrk1A, we tested whether a kinase-deficient Dyrk1A mutant or Dyrk1A siRNA duplex affects ␣-synuclein phosphorylation. As shown in Fig. 3C, ␣-synuclein phosphorylation was significantly diminished by expressing a kinase-dead Dyrk1A mutant and Dyrk1A siRNA, as compared with control cells. As a negative control, the transient expression of nonsilencing siRNAs did not affect ␣-synuclein phosphorylation (Fig. 3C). As expected, the Dyrk1A siRNA duplex, but not nonspecific siRNAs, blocked the endogenous Dyrk1A expression in a dosedependent manner (Fig. 3C).
The Kinetics and Neurotoxicity Induced by Intact ␣-Synuclein Inclusions Are Different from the Phosphorylated Forms by Dyrk1A-We monitored protein aggregation of recombinant or phosphorylated wild type ␣-synuclein samples by measuring FIGURE 5. Dyrk1A-induced ␣-synuclein phosphorylation promotes intracellular inclusion formation and increases cytotoxicity. Aggregation kinetics of intact ␣-synuclein or phosphorylated proteins by active Dyrk1A immunocomplexes was evaluated with turbidity (A) and thioflavin-T binding fluorescence (B). ␣-Synuclein protein aggregations were performed using ␣-synuclein (50 g) with or without active Dyrk1A immunoprecipitates (ϳ0.4 g). Turbidity (A) and amyloid formation (B) in the presence (open circles) and absence (closed circles) of anti-Dyrk1A immunocomplexes were measured by absorbance at 405 nm and thioflavin-T binding fluorescence. The proteins were immunoblotted with anti-␣-synuclein antibody to analyze for ␣-synuclein phosphorylation by a gel shift assay in the inset (A). Intact ␣-synuclein (Syn) and its phosphorylated bands (P-Syn) are indicated with arrows. C, cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide extract assay, after treatment with either intact ␣-synuclein aggregate (Syn) or the ␣-synuclein aggregate incubated with active Dyrk1A immunocomplexes (Syn ϩ Dyrk1A) (**, p Ͻ 0.01 versus control). D, ␣-Synuclein aggregate morphological differences were examined. ␣-Synuclein aggregates prepared in the absence (Syn) or the presence of bFGF-induced active Dyrk1A (Syn ϩ Dyrk1A) were examined by electron microscopy (magnification, ϫ78,000). Scale bars, 0.5 m. These results are representative of two or three independent experiments. the turbidity (Fig. 5A). As monitored by UV absorption at 280 nm, incubating purified ␣-synuclein at 37°C led to insoluble aggregate formation. Phosphorylated ␣-synuclein exhibited more aggressive aggregate formation as compared with unphosphorylated, wild type ␣-synuclein (Fig. 5A). Measurement of thioflavin-T binding fluorescence confirmed that phosphorylated ␣-synuclein exhibited enhanced aggregation as compared with unphosphorylated ␣-synuclein, although the difference was not as large as observed in the turbidity measurements (Fig. 5B).
We next asked whether the enhanced aggregation of phosphorylated ␣-synuclein proteins exhibited greater cytotoxicity. Phosphorylated ␣-synuclein protein aggregates were more cytotoxic than unphosphorylated ␣-synuclein aggregates (Fig.  5C). Transmission electron microscopy analysis of the aggregated granular structure formation during the ␣-synuclein aggregation in the presence of protein phosphorylation was microscopically distinct from those aggregates from unphosphorylated ␣-synuclein (Fig. 5D). Intact ␣-synuclein aggregates contained only the fibrillar forms, whereas the inclusions obtained with the Dyrk1A-induced phospho-␣-synuclein showed that spherical granular forms are also present, in addition to the fibrillar aggregates (Fig. 5D).
The ␣-Synuclein Phosphorylation via Active Dyrk1A Promotes Intracellular ␣-Synuclein Inclusion Formation-To validate the consequences of ␣-synuclein Ser-87 phosphorylation, we tested whether Dyrk1A could influence the insoluble ␣-synuclein aggregate formation, in H19-7 cells. Previously, we showed that transient ␣-synuclein expression in H19-7 cells leads to neuronal cell death. This effect is closely associated with the formation of intracytoplasmic ␣-synuclein-positive inclusions, which have similar composition to LBs found in PD patients (26). After H19-7 cells were transfected with ␣-synuclein, the expression pattern of ␣-synuclein within the cells was compared in the absence or presence of bFGF. Consistent with our previous finding, the distribution of ␣-synuclein was uneven and took the form of granular aggregates (Fig. 6A). Stimulation with bFGF enhanced intracytoplasmic ␣-synuclein inclusion formation (Fig. 6A). Additionally, ␣-synuclein co-expression with the dominant negative Dyrk1A diminished ␣-synuclein aggregates, as compared with ␣-synuclein alone (Fig. 6A). Cells expressing the ␣-synuclein S87A mutant also exhibited reduced aggregate formation (Fig. 6B). Quantification of intracellular eosinophilic protein aggregates found a 2-fold increase when cells were transfected with ␣-synuclein plus GFP and stimulated with bFGF (Table 1). However, we did not observe this increase in cells transfected with either ␣-synuclein S87A or kinase-deficient Dyrk1A in the absence of bFGF stimuli (Table 1).
Following transient ␣-synuclein expression, we measured cell viability after 24 h. As shown in Table 1, ␣-synuclein overexpression significantly decreased the cell viability by ϳ42% in H19-7 cells. Consistent with a previous report that H19-7 cells

TABLE 1 ␣-Synuclein phosphorylation effects on cell viability and eosinophilic inclusion formation in hippocampal H19-7 cells
also undergo apoptosis upon differentiation (28), bFGF addition resulted in a 25% loss of the total cell population, within 24 h (Table 1). However, co-transfection with wild type ␣-synuclein plus dominant-negative Dyrk1A diminished the neuronal cell death (Table 1). Furthermore, when cells were transfected with ␣-synuclein S87A, cytotoxicity was significantly reduced in response to bFGF. As a control, transfection of either kinase-dead Dyrk1A or ␣-synuclein S87A mutant in the absence of fibroblast growth factor stimulation reduced the toxic effect (Table 1). These data indicate that active Dyrk1A increases intracellular ␣-synuclein aggregates and potentiates its cytotoxicity in H19-7 cells.
␣-Synuclein can be phosphorylated in vitro at several residues, including serines 87 and 129 and three C-terminal tyrosine residues (tyrosines 125, 133, and 136). Several protein kinases were reported to phosphorylate ␣-synuclein in vitro and/or in vivo. For example, G protein-coupled receptor kinase-2 phosphorylates Ser-129 in vivo and enhances ␣-synuclein toxicity (16). Casein kinase 1 and 2 can also phosphorylate Ser-129 of ␣-synuclein in cultured cells (13). In addition, the ␣-synuclein Tyr-125 residue is phosphorylated by c-Src and Fyn (34,35). Although ␣-synuclein is constitutively phosphorylated at Ser-87 as well as at Ser-129 residues (13), the kinase targeting serine 87 residue and its physiological role have not been described. We examined whether ␣-synuclein is a Dyrk1A phosphorylation target. The current study shows that Dyrk1A can phosphorylate ␣-synuclein at Ser-87, and this enhances cytoplasmic aggregate formation. In addition to Ser-129, the Ser-87 residue plays an important role modulating cytoplasmic ␣-synuclein inclusion formation. Dyrk1A catalyzes the tyrosine-directed autophosphorylation and serine/ threonine phosphorylation in exogenous substrates (27). Therefore, we could not exclude the possibility that Dyrk1A can phosphorylate ␣-synuclein at tyrosine residues. Western blot analysis of anti-␣-synuclein immunocomplexes with antiphosphotyrosine IgGs revealed that the tyrosine residues within the ␣-synuclein are not probably phosphorylated in response to bFGF-induced active Dyrk1A, in H19-7 cells.
Previously, we reported that active Dyrk1A phosphorylates CREB, which subsequently leads to the stimulation of cAMPresponse element-mediated gene transcription, during neuronal differentiation (21). These data strongly suggest that Dyrk1A may play an important role during the neurogenic factor-induced differentiation of central nervous system neuronal cells. In addition, our recent findings show that huntingtininteracting protein-1 phosphorylation by Dyrk1A has an important role in neuronal differentiation and cell death (36). The present study demonstrates that ␣-synuclein is a Dyrk1A phosphorylation target, and this modification results in enhanced aggregate formation, which potentiates the proapoptotic effects of ␣-synuclein. Supporting our current findings are other reports that show that Dyrk1A phosphorylates human microtubule-associated tau protein at Thr-212, a residue that is hyperphosphorylated in AD and tauopathies (37). Abnormally increased Dyrk1A immunoreactivity is found in AD and DS (38), suggesting a possible involvement of Dyrk1A with neurofibrillary tangle pathology.
Through the aggregation assay in vitro, we observed the generation of previously unrecognized positive regulation of ␣-synuclein aggregation through protein phosphorylation. The aggregation of ␣-synuclein in the unstimulated quiescent condition can be positively regulated by active Dyrk1A. In addition, electron microscopy shows the aggregates prepared from phosphorylated ␣-synuclein protein have a globular protofibrillar structure quite distinct from the fibril forming from intact ␣-synuclein. Caughey and Lansbury (39) and Lansbury et al. (40) demonstrated that in vitro fibril formation by ␣-synuclein from its soluble monomeric form does not follow a simple onestep transition but is a rather complex process involving one or more discrete intermediates, termed protofibrils. During the process, a protofibril intermediate rather than the fibril itself may be more pathogenic. Additionally, studies have characterized several ␣-synuclein oligomers, which are much smaller than fibrils and appear early in the fibrillization process as granular forms (39,40). Consistent with this view, the morphological difference between the two protein aggregations is reflected by their distinct aggregation patterns, kinetics, and neurotoxicity. Further studies to characterize the molecular mechanisms leading to intracellular ␣-synuclein aggregate formation as well as the important signal transduction pathway(s) will give us insight into the mechanism of LB formation and the pathogenesis of PD and DS.