The FASEB Journal • Research Communication
Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome Fei Liu,* Zhihou Liang,* Jerzy Wegiel,† Yu-Wen Hwang,‡ Khalid Iqbal,* Inge Grundke-Iqbal,* Narayan Ramakrishna,‡ and Cheng-Xin Gong*,1 *Department of Neurochemistry, †Department of Developmental Neurobiology, and ‡Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA Adults with Down syndrome (DS) develop Alzheimer neurofibrillary degeneration in the brain, but the underlying molecular mechanism is unknown. Here, we report that the presence of an extra copy of the dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) gene due to trisomy 21 resulted in overexpression of Dyrk1A and elevated kinase activity in DS brain. Dyrk1A phosphorylated tau at several sites, and these sites were hyperphosphorylated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase-3 (GSK-3). Dyrk1A-induced tau phosphorylation inhibited tau’s biological activity and promoted its self-aggregation. In Ts65Dn mouse brain, an extra copy of the Dyrk1A gene caused increased expression and activity of Dyrk1A and resulted in increased tau phosphorylation. These findings strongly suggest a novel mechanism by which the overexpression of Dyrk1A in DS brain causes neurofibrillary degeneration via hyperphosphorylating tau. Liu, F., Liang, Z., Wegiel, J., Hwang, Y.-W., Iqbal, K., GrundkeIqbal, I., Ramakrishna, N., Gong, C.-X. Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome. FASEB J. 22, 3224 –3233 (2008)
ABSTRACT
Key Words: tau 䡠 hyperphosphorylation 䡠 GSK-3 䡠 trisomy 21
Down syndrome (DS) is caused by partial or complete trisomy of chromosome 21. All individuals with DS develop typical Alzheimer disease (AD) histopathology, that is, fibrillar amyloid plaques and neurofibrillary tangles (NFTs) in the brain, during the fourth decade of life (1). The early onset of amyloidosis in DS brain is believed to result from overexpression of -amyloid precursor protein (APP) encoded by a gene proximal to the DS critical region of chromosome 21 (2). Why individuals with DS always develop NFTs is not completely understood. NFTs are formed by aggregation of abnormally hyperphosphorylated tau protein into bundles of paired helical filaments and straight filaments in the affected neurons (3). The abnormal hyperphosphorylation of tau appears to lead to neurofibrillary degeneration that is characterized by the formation of NFTs. Tau hyperphosphorylation is pivotal in the neu3224
rofibrillary pathology (4) that may underlie the neurodegeneration and dementia of AD and other tauopathies (5, 6). Many adults with DS develop dementia in addition to mental retardation (7). It was postulated recently that the elevated expression of Dyrk1A and of DSCR1, two genes located in the DS critical region (8 –10), contributes to many phenotypes of DS (11, 12). Dyrk1A (dual-specificity tyrosinephosphorylated and regulated kinase 1A), the mammalian ortholog of Drosophila minibrain kinase (Mnb), encodes a proline/arginine-directed serine/threonine kinase. A recent study showed that this kinase phosphorylates tau at Thr212 in vitro (13), suggesting that its overexpression as the result of trisomy 21 might contribute to the development of neurofibrillary pathology in DS. In this study, we demonstrate that Dyrk1A phosphorylated tau at several sites that are hyperphosphorylated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase-3 (GSK-3), inhibited tau’s biological activity, and promoted its self-aggregation. In Ts65Dn mouse brain, an extra copy of Dyrk1A gene caused increased expression and activity of Dyrk1A and resulted in increased tau phosphorylation. These findings provide a novel mechanism leading to neurofibrillary degeneration in DS by abnormal hyperphosphorylation of tau as a result of the overexpression of Dyrk1A.
MATERIALS AND METHODS Human brain tissue and transgenic mice Tissue from the temporal cortices of 6 DS and 6 normal control brains (Table 1) was obtained from the Brain Bank for Developmental Disabilities and Aging of our institute. Diagnosis of all DS cases was genetically confirmed, and Alzheimer lesions in the DS brains were histopathologically confirmed. The brain tissue samples were stored at ⫺70°C 1 Correspondence: Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, New York 10314, USA. E-mail:
[email protected] doi: 10.1096/fj.07-104539
0892-6638/08/0022-3224 © FASEB
TABLE 1. Human brain tissue used in this study Group
DS
Mean ⫾ sd Control
Mean ⫾ sd
Case no.
Gender
Age at death
PMI (h)
69 1139 1162 1238 1283 1342
M F F M F M
241 244 248 252 255 256
F M F F F M
65 58 55 55 59 61 58.8 ⫾ 3.8 67 86 61 68 67 59 68.0 ⫾ 9.5
4.5 5 5 6 6 3 4.9 ⫾ 1.1 2.5 1.5 7 3 4 6 4.0 ⫾ 2.1
PMI, postmortem interval.
until used. Ts65Dn transgenic mice and nomosomic control mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Frozen human brain tissue and vertebrate animals were used in accordance with the U.S. National Institutes of Health guidelines, and the protocols were approved by the institutional review committees of the Institute for Basic Research in Developmental Disabilities. Proteins, peptides, enzymes, and antibodies Mammalian expression vector pcDNA3 containing rat Dyrk1A and recombinant Dyrk1A were prepared as described previously (14). The longest isoform of human brain tau (tau441) and cyclin-dependent kinase 5 (cdk5) and its activator p25 were prepared as described previously (15, 16). The catalytic subunit of cAMP-dependent protein kinase (PKA) and GSK-3 were purchased from Sigma (St. Louis, MO, USA) and Calbiochem (San Diego, CA, USA), respectively. Tubulin was purchased from Cytoskeleton (Denver, CO, USA). Dynatide 3 was synthesized by Genemed Synthesis, Inc. (South San Francisco, CA, USA). Monoclonal antibody 8D9 was raised against a histidine-tagged protein containing the first 160 residues of rat Dyrk1A (17). It recognizes Dyrk1A from rat, mouse, and human brains, as well as recombinant rat Dyrk1A expressed in cultured cells and has no significant cross reaction to other brain proteins or to proteins from 3T3 cells (data not shown). The monoclonal antibody 43D and polyclonal antibody R134d against tau in a phosphorylationindependent manner were raised at our institute (18). Phosphorylation-dependent and site-specific tau antibodies pT181, pS199, pS202, pT205, pT212, pS214, pT217, pT231, pS262, pS396, pS400, pS404, pS409, and pS422 were purchased from BioSource International (Camarillo, CA, USA). Anti--actin was bought from Sigma. Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); enhanced chemiluminescence kit was from Amersham Pharmacia (Costa Mesa, CA, USA); and [␥-32P]ATP was from MP Biomedicals (Irvine, CA, USA). Western blots, immuno-dot-blots, and Dyrk1A transfection The level of specific proteins in tissue samples and the site-specific phosphorylation of tau were determined by Western blots developed with the appropriate antibodies. In some experiments, the phosphorylation of tau at each specific site DYRK1A AND TAU PATHOLOGY IN DOWN SYNDROME
was also measured by using an immuno-dot-blot assay, as described (19). Transient transfection of COS7 cells with pCI/tau441 and pcDNA3/Dyrk1A was carried out by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The cells were harvested and lysed in SDS-PAGE sample buffer 48 h after transfection, and the lysates were analyzed by Western blotting. The levels of tau441 and Dyrk1A expression in the COS7 cells at this time point were approximately 5⫻ higher than those in brain neurons, as estimated by Western blot analysis. Tau phosphorylation, enzyme kinetics, and Dyrk1A kinase assays For in vitro tau phosphorylation by Dyrk1A, tau441 (0.2 mg/ml) was incubated with various concentrations of Dyrk1A in a phosphorylation buffer consisting of 50 mM Tris-HCl (pH 7.4), 10 mM -mercaptoethanol, 0.1 mM EGTA, 10 mM MgCl2, and 0.2 mM [␥-32P]ATP (500 cpm/pmol). After incubation at 30°C for 30 min, the reaction was stopped, the 32 P-labeled tau was separated from [␥-32P]ATP by ascending paper chromatography, and the radioactivity of tau was determined by Cerenkov counting. For kinetic experiments, Dyrk1A activity was assayed by incubating recombinant tau441 (2–130 M) with 5.0 g/ml Dyrk1A at 30°C for 10 min. The phosphate incorporation into tau was plotted against substrate (tau) concentration. The same data were also plotted by the Lineweaver-Burk double-reciprocal method, from which the apparent Km value and Vmax value were calculated. To determine the apparent Km values of tau phosphorylation at individual phosphorylation sites, [␥-32P]ATP was replaced by nonradioactive ATP, and the site-specific phosphorylation of tau was measured by using an immuno-dot-blot assay developed with phosphorylation-dependent and site-specific tau antibodies, as described previously (19). To study the effect of Dyrk1A-induced phosphorylation on the subsequent phosphorylation of tau by other kinases, tau441 (1.0 mg/ml) was first phosphorylated by Dyrk1A (50 g/ml) in the above buffer. After 30 min of incubation at 30°C, the reaction was terminated by precipitating the proteins with 8% trichloroacetic acid. The precipitated tau was then washed with ethanol and dried, followed by reconstitution in 5 mM 2-(N-morpholino)ethane sulfonic acid (MES; pH 6.8) and 0.1 mM EGTA. The control tau (con-tau) was treated the same way in parallel, except that Dyrk1A was replaced with the same amount of BSA. The prephosphorylated tau (P-tau) and the con-tau (both 0.2 mg/ml) were further incubated with GSK-3 or cdk5/p25 in a buffer containing 50 mM HEPES (pH 7.4), 10 mM -mercaptoethanol, 10 mM MgCl2, and 0.2 mM [␥-32P]ATP (500 cpm/pmol), or with PKA in a buffer containing 50 mM HEPES (pH 6.8), 10 mM -mercaptoethanol, 1 mM EGTA, 10 mM MgCl2, and 0.2 mM [␥-32P]ATP (500 cpm/pmol). After further incubation at 30°C for the indicated periods of time, aliquots of the reaction mixture were removed to determine the 32P-incorporation of tau, as described above. To determine Dyrk1A activity in brain tissue, the Triton X-100 extracts of the tissue homogenates were incubated in a mixture (20 l) containing 50 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 4.0 M cyclosporine A, 0.2 M okadaic acid, 1.0 mM Na3VO4, 10 mM MgCl2, 0.2 mM [␥-32P]ATP (1500 cpm/ pmol), and 40 g/ml Dynatide 3. After incubation at 30°C for 20 min, 20 l of 150 mM phosphoric acid was added to terminate the reaction, and then aliquots of a 10-l mixture containing Dynatide 3 were spotted onto P81 phosphocellulose membrane. After extensive washing with 75 mM phosphoric acid 5 times, the 32P-incorporation into Dynatide 3 was 3225
Figure 1. Level and activity of Dyrk1A are both elevated in DS brain. A) Western blot analysis of homogenates (10 g/lane) of brain tissue from six cases each of adult DS and controls, developed with anti-Dyrk1A 8D9 and anti--actin (loading control), respectively. B) Dyrk1A level in the brain tissue. The blots in A were quantitated densitometrically and normalized by those of -actin. Data are presented as means ⫾ sd. C) Dyrk1A activity of various concentrations of crude mouse brain extracts, assayed by using Dynatide 3 as a substrate in the presence (F) or absence (f) of 10 M EGCG. Assays without Dynatide 3 (E, 䡺) were also included as background controls. D) Kinase activity of purified Dyrk1A, assayed under the same conditions in the presence (E) or absence (䡺) of 10 M EGCG. E) Dyrk1A activity of the brain extracts. *P ⬍ 0.05 vs. control group. counted in a scintillation counter. This assay is specific to the kinase activity of Dyrk1A from crude mixture (Fig. 1C, D). Microtubule assembly assays and tau aggregation assays Recombinant tau441 (1.0 mg/ml) was first phosphorylated by Dyrk1A, precipitated, and reconstituted, as described above. The microtubule assembly assay was carried out by adding (0.07 mg/ml) the P-tau and the con-tau (treated in parallel except Dyrk1A was added after the reaction mixtures were mixed with trichloroacetic acid) into microtubule assembly mixture (100 mM MES, pH 6.8; 1.0 mM EGTA; 1.0 mM MgCl2; 1.0 mM GTP; and 2.0 mg/ml tubulin) to initiate assembly. The microtubule assembly was carried out in quartz microcuvettes at 35°C in a thermostatically controlled Cary 1 recording spectrophotometer (Varian, Inc., Palo Alto, CA, USA). Solution turbidity was continuously monitored at 350 nm. Steady-state values were determined by measuring the total change in optical density at 350 nm (OD350) after a turbidity plateau was reached. For aggregation assays, tau441 (0.5 mg/ml) was first phosphorylated by incubating it with 50 g/ml Dyrk1A or 2.2 g/ml GSK-3 or both at 30°C for 3 h and then incubated in 50 mM Tris (pH 7.4) at 37°C for 12 h for aggregation. At the end of incubation, one part of the mixture was centrifuged at 4°C at 30,000 g for 20 min, and tau in the resulting supernatants was quantitated by both Western blot analysis and immuno-dot-blots using 43D as the primary antibody. The other part of the mixture was examined by negative stain electron microscopy (20).
RESULTS Dyrk1A is increased in DS brain To test our hypothesis that overexpression of Dyrk1A contributes to neurofibrillary degeneration in DS, we first studied whether trisomy 21 indeed causes increased expression and activity of this kinase in adult DS brains. Western blot analysis of Dyrk1A indicated that its level in DS brain was increased to ⬃1.5-fold of the controls (Fig. 1A, B), which is consistent with the presence of an extra copy of the gene in DS. For determining Dyrk1A activity in the crude brain 3226
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extracts, we first developed a Dyrk1A kinase assay by using a specific substrate Dynatide 3, which is a 14-mer peptide derived from the Dyrk1A-phosphorylating site of dynamin 1 (21). To verify the specificity of the assay, we added 10 M epigallocatechin-3-gallate (EGCG), a selective Dyrk1A inhibitor (22) into the assay mixture and found that EGCG inhibited ⬎80% of the kinase activity in the brain extracts (Fig. 1C). This inhibition was close to a ⬃90% inhibition of purified Dyrk1A by EGCG under the same conditions (Fig. 1D). These studies indicate that this assay can determine Dyrk1A activity in crude extracts with a reasonably high specificity. In another set of experiments, overexpression of dominant negative Dyrk1A in two different cell lines (PC12 and HN2–5) almost diminished the kinase activity of the crude cell lysates, as determined by using the same assay (data not shown). These independent experiments further confirmed the specificity of the assay. By using this assay, we have found that the Dyrk1A activity is increased by ⬃1.5-fold in DS brain (Fig. 1E). Dyrk1A phosphorylates tau at sites seen in DS brain We then explored whether Dyrk1A phosphorylated tau to a biologically significant level and mapped the phosphorylation sites of tau by the kinase. We found that in vitro phosphorylation of recombinant tau by recombinant Dyrk1A reached a stoichiometry of 2.0 mol Pi/mol tau. Kinetic studies revealed an apparent Km of 21.4 M and an apparent Vmax of 0.4 mol Pi incorporation per minute per milligram (Fig. 2A). This Km toward tau is similar to those of GSK-3 (12 M) (23), cdk5 (27–33 M) (24) and PKA (31 M) (unpublished observations), which are the most likely tau kinases and play a key role in abnormal hyperphosphorylation of tau and in Alzheimer pathogenesis (4). This Km is also close to the intraneuronal tau concentration (5–10 M) in human brain (25). These studies suggest that Dyrk1A is able to phosphorylate tau in vivo and that the Dyrk1A-mediated tau phosphorylation might have biological relevance.
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Figure 2. Dyrk1A phosphorylates tau at several phosphorylation sites. A) Kinetics of tau441 phosphorylation catalyzed by recombinant Dyrk1A. Tau phosphorylation assayed under various concentrations is plotted against substrate (tau) concentration or by the Lineweaver-Burk double-reciprocal method (inset). The apparent Km and Vmax values calculated from the Lineweaver-Burk plot are 21.4 M and 0.4 mol/min/mg, respectively. B) Phosphorylation of tau at multiple sites by Dyrk1A in vitro. Tau441 was phosphorylated by indicated concentrations of Dyrk1A in vitro and then analyzed by Western blotting developed with site-specific and phosphorylation-dependent tau antibodies, as indicated under each blot. Hyperphosphorylated tau isolated from AD brain (ⴱ) was included as a positive control of the blots. C) Kinetics of Thr212 phosphorylation of tau catalyzed by Dyrk1A. Thr212 phosphorylation with various tau concentrations, determined by using immuno-dot-blots, is plotted against tau concentration or by the Lineweaver-Burk method (inset). D) Western blot analysis of tau phosphorylation in COS7 cells transiently transfected with tau441 and either empty vector (⫺) or Dyrk1A (⫹). Data are representative of 3 independent experiments with similar results. E) Relative phosphorylation levels at individual phosphorylation sites of tau in the homogenates of temporal cortices from six adult DS and six control brains, analyzed by quantitative Western blot and normalized by total tau level. No phosphorylation of tau is detectable at Thr212, Thr217, Ser-262, Ser-396, or Ser-422 in the control group, so the fold increase in DS at these sites becomes infinity (⬁). *P ⬍ 0.05 vs. control group.
Phosphorylation sites of tau by Dyrk1A were mapped by Western blot analysis developed with antibodies that recognize tau only when it is phosphorylated at the specific individual phosphorylation site, which is a well-established and commonly used method for mapping tau phosphorylation sites. We found that tau was phosphorylated by Dyrk1A at Thr181, Ser-199, Ser-202, Thr205, Thr212, Thr217, Thr231, Ser-396, Ser-400, Ser-404, and Ser-422, but not at Ser-214, Ser-262, or Ser-409 in vitro (Fig. 2B). Among these phosphorylation sites, Thr212 was the predominant site because its phosphorylation level reached its maximum at the lowest Dyrk1A concentration used. This finding is consistent with a previous report that identified, by mass spectrometry, Dyrk1A-catalyzed tau phosphorylation only at Thr212 (13) and with our recent report showing that Dyrk1A mainly phosphorylates the proline-rich region of tau (26). Kinetic analysis revealed that the apparent Km of Thr212 phosphorylation of tau by Dyrk1A was 5.0 M (Fig. 2C), which is lower than the Km of 21.4 M for total Pi incorporation to tau (Fig. 2A). The other tau phosphorylation sites are not the known Dyrk1A consensus sites (those preceded by arginine at the -2 or -3 DYRK1A AND TAU PATHOLOGY IN DOWN SYNDROME
position) (12, 13, 27). Those phosphorylation sites add additional examples of the recently reported exceptions of the known Dyrk1A consensus sites (28 –30). When COS7 cells were transiently transfected with tau and Dyrk1A, increased phosphorylation of tau at Thr181, Ser-199, Ser-202, Thr212, and Thr217 was also observed, as compared to transfection with tau alone (Fig. 2D). These observations are consistent with our in vitro data (Fig. 2B). In contrast to in vitro phosphorylation, however, expression of Dyrk1A did not increase tau phosphorylation at Ser-396 or Ser-404 and even decreased tau phosphorylation at Thr205, Ser-214, and Ser-262 (Fig. 2D). The failure to observe phosphorylation of Ser-396 and Ser-404 by Dyrk1A might be due to the already high level of tau phosphorylation at these sites in COS7 cells, as a recent study showed phosphorylation of tau at Ser-404 (Ser-396 was not examined in that study) by transfected Dyrk1A in the tau-transfected HEK293T cells (30). The reason why the phosphorylation level of Thr205, Ser-214, and Ser-262 of tau was decreased on DyrK1A expression in COS7 cells is not presently understood. Possible explanations include tau conformational changes due to 3227
Dyrk1A-induced tau phosphorylation and/or changes in the activities of other tau kinases and/or phosphatases caused by Dyrk1A transfection. To determine whether tau is hyperphosphorylated at Dyrk1A-catalyzed phosphorylation sites in adult DS brain, we measured phosphorylation levels of tau at individual phosphorylation sites in the temporal cortex of individuals with DS. We found that the majority of the Dyrk1Amediated phosphorylation sites of tau are dramatically hyperphosphorylated in DS brain (Fig. 2E), suggesting that tau hyperphosphorylation in DS brain might be caused by overexpression of Dyrk1A. The pattern of site-specific tau hyperphosphorylation in DS brain is different from that in sporadic AD brains (unpublished observations), further supporting the causative role of Dyrk1A in tau hyperphosphorylation in DS brain. Dyrk1A promotes tau phosphorylation with GSK-3 Because Dyrk1A phosphorylates tau up to a stoichiometry of only 2.0 mol Pi/mol tau in vitro, the phosphor-
ylation at most of the sites except Thr212 observed above in vitro and in cultured cells is likely to be far below the stoichiometric level. Thus, the marked increase in tau phosphorylation at most of the sites observed in DS brain can hardly be explained by the ⬃1.5-fold increase in DyrK1A activity. It has been shown that phosphorylation of tau by PKA dramatically promotes the subsequent tau phosphorylation by GSK-3 both in vitro (31) and in vivo (32). A previous study also reported that Dyrk2-mediated tau phosphorylation at Thr212 could prime tau for phosphorylation at Ser-208 with GSK-3 (13). Therefore, we investigated whether Dyrk1A-mediated tau phosphorylation also primes tau for phosphorylation with GSK-3. Our in vitro studies demonstrated that prephosphorylation by Dyrk1A promoted further phosphorylation of tau by GSK-3, from ⬃2 mol Pi/mol tau to 4 mol Pi/mol tau (Fig. 3A). This priming effect appears to be specific for GSK-3-catalyzed tau phosphorylation, because cdk5and PKA-catalyzed tau phosphorylation was slightly
Figure 3. Phosphorylation of tau by Dyrk1A primes its subsequent phosphorylation by GSK-3. A) In vitro phosphorylation of tau441 by GSK-3, cdk5, or PKA before (E) and after (F) being prephosphorylated by Dyrk1A. B) Western blots of tau441 phosphorylated by Dyrk1A and then GSK3, cdk5, or PKA. C) Time kinetic analysis of tau phosphorylation catalyzed by GSK3 with (F) and without (E) prephosphorylation by Dyrk1A, as measured by immuno-dot-blots using phosphorylation-dependent and site-specific tau antibodies. The phosphorylation levels presented in the graphs were quantitated after normalization by the total level of tau. Data are representative of 3 separate experiments with the same results. 3228
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inhibited rather than promoted by prephosphorylation with Dyrk1A (Fig. 3A). We further studied the effects of Dyrk1A-mediated phosphorylation on the subsequent tau phosphorylation at each individual phosphorylation site by GSK-3, cdk5, and PKA. Consistent with the data in Fig. 3A, prephosphorylation of tau by Dyrk1A promoted its subsequent phosphorylation by GSK-3 at Ser-181, Ser199, Ser-202, Thr205, and Ser-208 (Fig. 3B, C; compare lanes 8 and 1), but not by cdk5 (Fig. 3B, compare lanes 7 and 2) or PKA (Fig. 3B, compare lanes 6 and 3) at these sites. In contrast to GSK-3-catalyzed tau phosphorylation, prephosphorylation of tau by Dyrk1A inhibited cdk5-catalyzed phosphorylation at Thr217, and PKA-catalyzed phosphorylation at Ser-214 of tau, which is consistent with the data shown in Fig. 3A. These results suggest that overexpression of Dyrk1A in DS brain not only directly phosphorylates tau (e.g., Thr212) but probably also induces hyperphosphorylation by promoting GSK-3-mediated phosphorylation of tau. Dyrk1A-mediated tau phosphorylation inhibits its biological activity and promotes its self-aggregation The abnormal hyperphosphorylation of tau in AD brain inhibits its ability to stimulate microtubule assembly (33, 34) and promotes its self-aggregation into NFTs (35, 36). We therefore studied whether Dyrk1A-mediated tau phosphorylation affects its activity and promotes its self-aggregation. In vitro microtubule-assembly assays indicated that tau’s biological activity is reduced on phosphorylation by Dyrk1A (Fig. 4A). Dyrk1A-in-
duced phosphorylation also promoted tau’s self-aggregation, as determined by both Western blot analysis (Fig. 4B) and immuno-dot-blots (Fig. 4C, D) after incubation at 37°C for 12 h and then centrifugation at 30,000 g. GSK-3-induced phosphorylation promoted tau aggregation to a greater extent (60%) than Dyrk1Ainduced phosphorylation (30%) (Fig. 4B–D). Phosphorylation of tau by Dyrk1A and then by GSK-3 had a stronger effect in promoting its aggregation than phosphorylation by the single kinase had, leading to ⬃75% of tau aggregated in the pellets (Fig. 4C, D). When the incubated tau samples were examined under electron microscope, we found numerous protein aggregates (100 to 500 nm in sizes) in tau samples after phosphorylation by Dyrk1A, but not in control tau samples (Fig. 4E–G). Occasionally, filament-like structures were seen in Dyrk1A-phosphorylated tau samples (Fig. 4G). In GSK-3-phosphorylated tau samples, more protein aggregates and thin filaments were observed (Fig. 4H). Some larger filaments were seen in tau samples after phosphorylation by Dyrk1A and then by GSK-3 (Fig. 4I). Tau is hyperphosphorylated in Dyrk1A-overexpressed mouse brains To confirm the role of Dyrk1A overexpression in neurofibrillary pathology in DS brain, we studied the most commonly used transgenic mouse model of DS, Ts65Dn (37). These mice carry an additional copy of segmental murine chromosome 16, which contains the equivalent of the DS critical region of human chromo-
Figure 4. Dyrk1A-mediated tau phosphorylation impairs its biological activity and promotes its self-aggregation. A) Microtubule assembly activity (OD350) in control-treated tau441 (con-tau) and Dyrk1A-phosphorylated tau441 (P-tau). B, C) Tau441 preparations without (con) and with phosphorylation by Dyrk1A or GSK-3 or both were incubated at 37°C for 12 h and centrifuged at 30,000 g for 20 min. Tau in the samples before centrifugation (T) and in the resulting supernatants (S) was analyzed by Western blots (B) and immuno-dot-blots (C) developed with monoclonal antibody 43D. For the latter method, all samples were analyzed in four dilutions and in triplicate. D) Percentages of tau in the resulting supernatants as quantitated by immuno-dot-blots. *P ⬍ 0.05 vs. control. E–I) Electron microscopy of negatively stained tau441 aggregates after incubation without (E) or with Dyrk1A (F, G), GSK-3 (H), or both (I). Scale bars ⫽ 500 nm. DYRK1A AND TAU PATHOLOGY IN DOWN SYNDROME
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of triplicated human chromosome 21 gene orthologs including Dyrk1A (38), and of the transgenic mice that overexpress human Dyrk1A (30).
DISCUSSION
Figure 5. Overexpression of Dyrk1A leads to hyperphosphorylation of tau in the brains of Ts65Dn transgenic mice. A) Western blots of brain homogenates (10 g/lane) from 5 Ts65Dn mice (15 months old) and 5 littermates developed with anti-Dyrk1A (8D9) and anti--actin antibodies, respectively. B) Dyrk1A level in the brain tissue. The blots in A were quantitated densitometrically and normalized by those of -actin. Data are presented as means ⫾ sd. C) Dyrk1A activity of the brain extracts. D) Relative phosphorylation levels at individual phosphorylation sites of tau in the mouse brain homogenates, analyzed by quantitative Western blots and quantitated after normalization by the total tau level. *P ⬍ 0.05 vs. control groups.
some 21 and includes the Dyrk1A gene. Western blot analysis indicated ⬃1.5-fold greater expression of Dyrk1A in the brains of Ts65Dn mice than in the littermate controls (Fig. 5A, B), which is similar to the ratio of Dyrk1A elevation in DS brain (Fig. 1A, B). The Dyrk1A activity was also ⬃1.5-fold higher in Ts65Dn mouse brain as compared to the controls (Fig. 5C). The increased Dyrk1A activity in Ts65Dn mouse brain was accompanied by increased phosphorylation of tau at most of the phosphorylation sites relevant to Dyrk1A and GSK-3 (Fig. 5D). The increase in tau phosphorylation in Ts65Dn brains was not as pronounced as in DS brain, probably because mouse brain is more resistant to abnormal tau hyperphosphorylation and neurofibrillary pathology than human brain. Normal aged mice never develop tau pathology, whereas tau pathology is common in aged humans. Nevertheless, these results clearly demonstrate that overexpression of Dyrk1A in the transgenic mouse brain results in hyperphosphorylation of tau, as is similarly observed in DS brain. Hyperphosphorylation of tau was also found in brains of another DS mouse model, Ts1Cje, which has a subset 3230
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Increased Dyrk1A transcription in DS brain has been reported previously (39). We now demonstrate that both the protein level and the activity of Dyrk1A are increased to ⬃1.5-fold in adult DS brains as compared to controls. Our observations are consistent with a recent study that, by using a different method and different brain samples, reported an increase of Dyrk1A protein level in various brain areas of DS as compared to control brains (40). Although Dyrk1A is expressed predominantly in the nucleus of certain cells (41), it is present in both the nucleus and cytoplasm of neurons in adult human brain (17, 42, 43) and in mouse brain (44). Nuclear and cytoplasmic Dyrk1A may contribute to DS via different mechanisms. Two independent studies have demonstrated that nuclear Dyrk1A phosphorylates NFAT, a nuclear transcription factor essential for vertebrate development and function, and facilitates its exit into the cytoplasm, leading to the inactivation of its transcriptional activity (11, 12). Overexpression of Dyrk1A may impair brain and heart development and contributes to the DS phenotype by reducing NFAT activity, because either overexpression of Dyrk1A alone (45) or NFAT knockout (11) produces many phenotypes similar to DS. Although Dyrk1A can phosphorylate and regulate dynamin 1 (14) and amphiphysin 1 (46), proteins that are involved in endocytic synaptic vesicle recycling, the biological and pathological role of cytoplasmic Dyrk1A is hardly understood. The present findings suggest its participation in regulating tau phosphorylation and provide a novel mechanism leading to neurofibrillary pathology in DS. On the basis of our findings, we propose that overexpression of Dyrk1A due to trisomy 21 phosphorylates tau at Thr212 and several other sites which, in turn, promotes its further phosphorylation by GSK-3 into an abnormally hyperphosphorylated form. The abnormal hyperphosphorylation, on one hand, causes the loss of tau’s biological function of stimulating microtubule assembly and the gain of the toxic activity of sequestering normal tau and other microtubule-associated proteins (MAPs), leading to neurodegeneration (4). On the other hand, it promotes selfaggregation of tau into NFTs. This novel mechanistic hypothesis of neurofibrillary degeneration in DS (Fig. 6) is also supported by immunohistochemical studies showing a high level of cytoplasmic Dyrk1A in many pyramidal neurons of the hippocampus but minimally in granular neurons of the dentate gyrus of DS and AD brains (17, 42). These findings coincide with the neurofibrillary degeneration seen in many pyramidal neurons but not in the granular neurons in DS and AD brains. Our hypothesis is also consistent with a recent report showing that Dyrk1A interacts with and directly
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Figure 6. Proposed mechanism by which neurofibrillary degeneration occurs in adults with DS. P in pink bullet on the primed tau represents phosphate catalyzed by increased Dyrk1A. P in red bullet on the hyperphosphorylated tau represents phosphates catalyzed by GSK-3, which was promoted by Dyrk1A-induced phosphorylation. P in yellow bullet represents phosphates catalyzed by GSK-3, not affected by Dyrk1A-induced phosphorylation.
phosphorylates tau in immortalized hippocampal progenitor H19 –7 cells (47). Overexpression of Dyrk1A in these cells induces a marked increase in apoptotic cell death under conditions of serum deprivation and causes defects in neuronal differentiation. In the brains of transgenic mice that overexpress the human Dyrk1A, a significant increase in tau phosphorylation at Ser-202, Thr212, and Ser-404 was observed (30). Although only three phosphorylation sites of tau were examined in the above study, their results are consistent with our conclusions and suggest that the extra copy of the Dyrk1A gene contributes to the early onset of tau pathology. Other factors could also contribute to neurofibrillary degeneration in DS. It has been hypothesized that APP overexpression and/or A overproduction can accelerate neurofibrillary pathology in AD (48). Because neurofibrillary pathology could not be induced by APP overexpression in many APP transgenic mouse models that develop lots of amyloid plaques in the brain, it is unlikely that neurofibrillary pathology in DS results from APP overexpression alone. However, it is possible that Dyrk1A and APP overexpression induces neurofibrillary degeneration synergistically in DS brain. In addition to promoting neurofibrillary degeneration, overexpression of Dyrk1A might also contribute to amyloid pathology in DS brain. Ryoo et al. (49) have recently shown that APP is phosphorylated at Thr668 by Dyrk1A in vitro and in mammalian cells and that the amounts of phospho-APP and A are increased in the brains of transgenic mice that overexpress the human Dyrk1A. Furthermore, the levels of APP and phosphoAPP are also elevated in human DS brain. These results DYRK1A AND TAU PATHOLOGY IN DOWN SYNDROME
suggest that overexpression of Dyrk1A may contribute to both tau and amyloid pathologies of AD in DS brain. There is recent evidence showing that Dyrk1A might also play a role in AD. A genetic association study of late-onset AD using 374 Japanese patients and 375 population-based controls resulted in 17 genetic risk markers, of which Dyrk1A gene showed the highest significance in logistic regression (50). The same study also showed an increased mRNA level of Dyrk1A in the hippocampus of patients with AD as compared with pathological controls. Many protein kinases can phosphorylate tau in vitro. Among these kinases, GSK-3 is the most important kinase in regulation of tau phosphorylation and in hyperphosphorylation of tau in AD (51, 52). Although there is no conclusive evidence showing an up-regulated GSK-3 in AD brain, tau can be hyperphosphorylated by normal level of GSK-3 if it is primed by other kinases (32). Therefore, we investigated Dyrk1Aprimed tau phosphorylation by GSK-3. Our observations that Dyrk1A promoted tau phosphorylation with GSK-3 further suggest a significant role of Dyrk1A overexpression in neurofibrillary degeneration in DS brain. Recent studies demonstrated that casein kinase 1␦ also phosphorylates numerous sites of tau in vitro, as GSK-3 does (53) and probably in vivo as well (54), suggesting that it may also play a role in tau pathology. By using double-labeled fluorescence immunohistochemistry, casein kinase 1 isoforms were found to associate differentially with neurofibrillary and granulovacuolar degeneration lesions in AD brain (55) and with tau-bearing lesions of inclusion body myositis (56). Therefore, future studies should examine whether Dyrk1A also promotes tau phosphorylation with casein kinase 1. DSCR1, another important gene located in the DS critical region (10), encodes calcipressin that is an endogenous inhibitor of calcineurin (also called protein phosphatase 2B) (57). Calcineurin can dephosphorylate tau at multiple sites in vitro (58). Thus, it is theoretically possible that hyperphosphorylation of tau could result from calcineurin inhibition due to overexpression of calcipressin in DS brain. However, it is well established that protein phosphatase 2A is the major tau phosphatase in vivo, and calcineurin only contributes to ⬃7% of the total tau phosphatase activity in the human brain (59). In contrast to inhibition of protein phosphatase 2A, inhibition of calcineurin in metabolically active rat brain slices does not result in hyperphosphorylation of tau (60). More importantly, there is no difference in calcineurin activity between DS brain and the age-matched control human brains or between TS65Dn and control mouse brains (unpublished observation). Thus, neurofibrillary pathology in DS brain is unlikely to be caused by calcineurin inhibition. Ermak et al. (61) recently reported that DSCR1 stimulates expression of GSK-3, providing another possibility that DSCR1 overexpression might contribute to neurofibrillary degeneration via up-regulating GSK-3 in DS. To test this possibility, we determined GSK-3 levels by 3231
quantitative Western blots and found no difference between DS and age-matched control brains or between TS65Dn and control mouse brains (data not shown). Taking together, it is unlikely that DSCR1 overexpression could contribute to abnormal hyperphosphorylation of tau and neurofibrillary degeneration via inhibiting calcineurin activity or stimulating GSK-3 expression in DS. In summary, our present findings suggest a molecular mechanism explaining the neurofibrillary degeneration in DS. Further study of this newly identified mechanism will help in development of novel therapeutic strategies to prevent or inhibit neurofibrillary degeneration in DS and, thus, treat the dementia in this syndrome.
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This work was supported, in part, by the New York State Office of Mental Retardation and Developmental Disabilities and by grants from the U.S. National Institutes of Health (AG027429 to C.-X.G., HD043960 to J.W., HD038295 to Y.-W.H., AG019158 to K.I.), the U.S. Alzheimer’s Association (IIRG-05–13095 to C.-X.G.) and the Li Foundation, Inc. (to C.-X.G.). We thank Dr. A. del C. Alonso for help with electron microscopy, Dr. D. Miller for support and advice, Dr. E. El-Akkad for preparation of recombinant tau441, Dr. J. Currie for critical reading of the manuscript and suggestions, Ms. M. Marlow for editorial suggestions, and Ms. J. Murphy for secretarial assistance.
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