Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M102621200 on July 25, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38193-38200, October 12, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/41/38193    most recent
M102621200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kins, S.
Right arrow Articles by Götz, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kins, S.
Right arrow Articles by Götz, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Reduced Protein Phosphatase 2A Activity Induces Hyperphosphorylation and Altered Compartmentalization of Tau in Transgenic Mice*

Stefan KinsDagger , Arames CrameriDagger , David R. H. Evans§, Brian A. Hemmings§, Roger M. NitschDagger , and Jürgen GötzDagger ||

From the Dagger  Division of Psychiatry Research, University of Zürich, 8008 Zürich, Switzerland, and the § Friedrich-Miescher-Institute, 4002 Basel, Switzerland

Received for publication, March 23, 2001, and in revised form, July 3, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neurodegenerative diseases including Alzheimer's disease (AD)1 and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) are characterized by neurofibrillary lesions that are composed of hyperphosphorylated isoforms of tau, a microtubule-associated protein (1, 2). Because hyperphosphorylated tau binds poorly to microtubules and thereby alters the stability of microtubules, tau hyperphosphorylation could affect other cytoskeletal constituents, intracellular transport, cellular geometry, and neuronal viability (3-6).

A fundamental question for understanding the mechanism of neurofibrillary degeneration is why and how tau becomes abnormally hyperphosphorylated. To date, more than 20 phosphorylation sites have been identified in hyperphosphorylated tau that are either serine or threonine residues. Several protein kinases have been implicated in the phosphorylation of tau in AD (7-11), and there is accumulating evidence that reduced activities of phosphatases are also involved (12, 13). The serine/threonine-specific protein phosphatases PP2A, PP2B, and, to a lesser extent, PP1 were shown to efficiently dephosphorylate tau isolated from AD brain (14, 15). Pharmacological inhibition in vivo further suggested that PP2A is involved in tau dephosphorylation (16-18). A role for PP2A in tau dephosphorylation is also supported by the finding that PP2A is localized on microtubules and that it binds directly to tau (19). FTDP-17-associated mutations in tau induce a decrease in binding affinity for PP2A, suggesting that altered interactions between PP2A and tau may contribute to FTDP-17 pathogenesis (20). The prolyl isomerase Pin1, which co-purifies with tau filament preparations, catalyzes prolyl isomerization of specific Ser/Thr-Pro motifs in tau and thereby restores the function of tau and facilitates dephosphorylation by PP2A (21). Together, these data demonstrate the importance of PP2A for tau function in FTDP-17 and AD.

Attempts to investigate the role of PP2A in tau phosphorylation in vivo by a gene targeting approach in mice were complicated by the fact that the selective knockout of the PP2A catalytic subunit Calpha was embryonically lethal (22, 23).

Therefore, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P (24, 25), under the control of a neuron-specific promoter in transgenic mice. In brain homogenates of transgenic mice, PP2A activity was chronically reduced to 66% of the activities in wild-type mice. Endogenous tau was distinctly phosphorylated at the AT8 epitope Ser-202/Thr-205 and at the phosphorylated serine 422 (pS422) epitope Ser-422. It accumulated in aggregates in the somatodendritic compartment and was co-localized with ubiquitin, reflecting an early step in neurofibrillary lesion formation in AD.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constructs and Transgenic Mice-- The cDNA of the human PP2A Calpha mutant L199P was isolated from yeast plasmid YEpDE2.5.12 that carries a 978-base pair HindIII/BamHI fragment encoding the HA-PP2ACalpha -2512 mutant allele. The single hemagglutinin epitope present in this allele is located immediately downstream of the start codon. The HA-PP2ACalpha -2512 allele contains the T196C transition mutation encoding a L199P amino acid substitution (numbering is for the untagged PP2ACalpha ). It also encodes a C215T base change, which is a silent mutation (24). The 978-base pair fragment was subcloned into the neuron-specific murine Thy1.2 expression vector (26, 27). Vector sequences were removed, and DNA was purified by ultracentrifugation for 30 min at 40,000 rpm (TLA45 rotor) to remove residual agarose before microinjection. Transgenic mice were produced by pronuclear microinjection of B6D2F1 × B6D2F1 embryos, as described previously (28). Founders were identified by polymerase chain reaction analysis of lysates from tail biopsies using oligonculeotides O-144 (5'-GTTCACCAAGGAGCTGGACCAG-3') and O-145 (5'-ACAGGAAGTAGTCTGGGGTACG-3'), and an amplification product of the 915-base pair fragment was obtained. Founder animals were intercrossed with C57BL/6 mice to establish lines.

Antibodies-- The following tau antibodies directed against different phosphoepitopes (Table I) of tau were used: (a) AT8 (Innogenetics Inc., Temse, Belgium; 1:20 dilution) (29), (b) AT100 (Innogenetics Inc.; 1:100 dilution) (30), (c) AT180 (Innogenetics Inc.; 1:50 dilution) (30), (d) 12E8 (Dr. Peter Seubert, Elan Pharmaceuticals, South San Francisco, CA; 1:100 dilution) (31), (e) TG3 (Dr. Peter Davies, Bronx, NY; 1:20 dilution) (32), (f) PHF1 (Dr. Peter Davies; 1:50 dilution) (33), (g) AD2 (Dr. Chantal Mourton-Gilles, Lille, France; 1:500 dilution) (34), (h) MC1 (Dr. Peter Davies; 1:10 dilution) (35), (i) R145d (Dr. Khalid Iqbal, Staten Island, NY; 1:3000 dilution) (36), and (j) S199P (Dr. André Delacourte, Lille, France; 1:100 dilution) (37, 38). For immunohistochemistry, we used conditions that have been previously optimized in tau transgenic mouse models (39). To detect ubiquitin, a commercially available polyclonal anti-ubiquitin antibody (Calbiochem) was used at a dilution of 1:100. For immunoblotting, the following anti-tau antibodies were used: phosphorylation-independent monoclonal antibody TAU-5 (Neo Markers Inc., Fremont, CA; 1 µg/ml) and phosphorylation-dependent antibodies/antisera AT8 (1:150 dilution), AT100 (1:1000 dilution), AT180 (1:150 dilution), AD2 (1:10,000 dilution), pS422 (BIOSOURCE; 1:500 dilution) (40), and S199P (1:5000 dilution). For detection of the HA epitope by immunohistochemistry and immunoblotting, two monoclonal anti-HA antibodies (Roche Molecular Biochemicals (immunohistochemistry, 1:200 dilution; immunoblotting, 1:500 dilution) and Santa Cruz Biotechnology, Santa Cruz, CA (immunohistochemistry, 1:100 dilution; immunoblotting, 1:500 dilution)) and a polyclonal anti-HA antibody (Santa Cruz Biotechnology; immunohistochemistry, 1:500 dilution; immunoblotting, 1:1000 dilution) were used. The rabbit anti-PP2AC antiserum V598A (Promega, Madison, WI) directed against a carboxyl-terminal sequence shared between human and mouse PP2ACalpha and PP2ACbeta was used at a 1:100 dilution for immunoblot analysis. For immunofluorescence, secondary antibodies were obtained from Molecular Probes (ALEXA-FLUORTM series; Eugene, OR), and for immunoblotting, horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA) were used.

Immunohistochemistry-- Brains from 12-month-old transgenic mice and an equal number of age-matched control mice (C57BL/6 mice) were used for immunohistochemical analysis. Animals were perfused transcardially with 4% paraformaldehyde in saline/sodium phosphate buffer, pH 7.4. Immunohistological stainings were performed on 4-µm methanol-permeabilized, paraffin-embedded parasaggital sections from brain by using standard published procedures (22). In addition, 40-µm vibratome sections were permeabilized with 0.1% Nonidet P-40 (Calbiochem). Some of the sections were pretreated with either 5 µg/ml proteinase K or 0.1% Triton X-100 in Tris-buffered saline or phosphate-buffered saline at 37 °C for 2.5 min for signal enhancement. Sections were dehydrated in an ascending series of ethanol and flat embedded between glass slides and coverslips in Mowiol 4-88 (Roche Molecular Biochemicals) containing 2.5% (w/v) diazabicyclo[2.2.2]octane (Sigma).

Northern Blot and Immunoblots-- RNA was prepared according to the manufacturer's recommendations (RNeasy; Qiagen). 20 µg of total RNA were separated by formaldehyde gel electrophoresis and transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech). Prehybridization was done for 30 min in 1% (w/v) bovine serum albumin, 0.2 M sodium phosphate buffer, pH 7.2, 35% (v/v) deionized formamide, 1 mM EDTA, and 7% (w/v) SDS. For hybridization, a gamma -32P-labeled oligonucleotide hybridizing to exon 7 of the human PP2A Calpha gene (5'-GACATGTGGCTCGCCTCTAC-3') was used (22). For normalization, a random-labeled probe derived from a 2-kilobase human beta -actin cDNA fragment was used. After hybridization, membranes were washed several times under stringent conditions at 65 °C. Membranes were exposed to a Kodak x-ray film at -80 °C using an intensifying screen.

To analyze tau expression and phosphorylation, brains from transgenic and control mice were weighed and Dounce homogenized in 2.5% (v/v) perchloric acid in phosphate-buffered saline, allowed to stand on ice for 30 min, and centrifuged for 10 min at 10,000 × g. The supernatants were dialyzed against 50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride and used for immunoblot analysis as described previously, using equal amounts of protein extracts (41). Staining of the membranes with Ponceau S was included to confirm loading of comparable amounts of protein. To analyze levels of PP2A expression, protein extracts were prepared according to standard procedures (42) and normalized for protein contents using the DC Protein Assay from Bio-Rad (Hercules, CA). Extracts were separated by 10% SDS-polyacrylamide gel electrophoresis, followed by electrophoretic transfer onto a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech). The membrane was blocked and reacted with primary and horseradish peroxidase-conjugated secondary antibodies as described previously (42).

Protein Phosphatase Activity Measurements-- Total brain extracts from 5- and 12-month-old mice were prepared with a Teflon homogenizer (15 strokes at 100 rpm) in 1× Tris-buffered saline and 1% (v/v) Triton X-100 in the presence of protease inhibitors (CompleteTM with EDTA; Roche Molecular Biochemicals). Phosphatase assays were performed with these extracts using the phosphatase kit V2460 from Promega. First, endogenous free phosphate was removed from the homogenates, and then the homogenates were normalized for protein content. Release of phosphate from a chemically synthesized phosphopeptide (RRA(pT)VA; pT = phosphothreonine) (43) was measured over a period of 45 min. Homogenates were prepared in duplicate from six transgenic and control brains, and two assays were performed with each homogenate. Mean values and standard deviations were calculated. The amount of released phosphate was determined by measuring the absorbance of a molybdate-malachite green-phosphate complex at 595 nm. The interassay variance was <1%. Brain homogenates were also incubated with okadaic acid (okadaic acid, sodium salt; Calbiochem), a potent inhibitor of PP2A, at concentrations of 1 and 10 nM, respectively.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal Expression of the Dominant Negative Mutant Form of PP2A Calpha , L199P-- The neuron-specific elements of the mouse Thy1.2 promoter were used to express the cDNA of human PP2A Calpha mutant L199P (Fig. 1A). This expression vector mediates a postnatal neuronal expression of the inserted cDNA (44). The L199P mutant has previously been shown to act as a dominant negative mutant of PP2A in Saccharomyces cerevisiae (24). A HA epitope was fused amino-terminally (24) to facilitate later detection of the mutant protein. The construct was microinjected into oocytes as described previously (28), and six founders were obtained, four of which transmitted the transgene in a Mendelian fashion. These lines expressed PP2A Calpha L199P mRNA in brain, as determined with a human PP2A Calpha exon 7-specific oligonucleotide as probe (Fig. 1B, top panel) (22). To control for equal loading, a human beta -actin probe was used (Fig. 1B, bottom panel). An antibody directed against the HA epitope of the transgene detected on immunoblots a protein with the expected molecular mass in three transgenic lines. This protein was absent from the wild-type litter mate control (Fig. 1C, top panel). Immunoblotting of the same protein extracts with an antibody directed against a conserved epitope between human and murine PP2A C revealed that the total amount of PP2A C was not significantly changed upon expression of the mutant PP2A Calpha L199P protein (Fig. 1C, bottom panel). For comparison, in heterozygous PP2A Calpha knockout mice, PP2A C protein levels were identical to wild-type levels (22), suggesting that protein levels of PP2A C are tightly controlled (45, 46). PP2A Calpha L199P mice were overtly normal. Size and fertility were not reduced compared with nontransgenic litter mates.


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 1.   Transgenic mice expressing a dominant negative mutant form of PP2A Calpha , L199P. A, the neuron-specific elements of the mouse Thy1.2 promoter were used to express the cDNA of human PP2A Calpha mutant L199P in transgenic mice. To discriminate transgenic mutant from endogenous wild-type PP2A Calpha , a HA epitope was fused to the amino terminus of PP2A Calpha L199P. B, Northern blot analyses revealed expression of PP2A Calpha L199P mRNA in every second transgenic line. C, whereas mRNA levels were different for lines 19 and 21, protein levels appeared to be similar as determined by Western blot analysis using an antibody directed against the HA epitope (C, top panel). Total PP2A C levels were not significantly different in transgenic brain homogenates as compared with wild-type brain homogenates, as determined with antiserum V598A (C, bottom panel). D, reduced protein phosphatase activities in brain extracts of PP2A Calpha L199P transgenic mice. Brain homogenates were obtained from transgenic (tg) and wild-type (wt) mice, and phosphatase activities were measured in the presence of 2 mM EDTA (n = 6). Phosphatase activity was reduced to 66 ± 9% (n = 6) in brain extracts of transgenic mice compared with wild-type mice. Significant differences (**), as compared with wild-type mice, are at the p = 0.01 level (U test).

The expression pattern of PP2A Calpha L199P was determined by immunohistochemistry with an antibody directed against the HA epitope (Fig. 2). This staining revealed a neuronal, cytoplasmic expression in most brain areas including the Purkinje cells of the cerebellum (Fig. 2A), layers I-V of the cortex (Fig. 2B), and the hippocampal formation (Fig. 2C), whereas no staining was detected in the same brain areas of wild-type mice (Fig. 2, D-F). Together, these data show that the PP2A Calpha mutant L199P is also expressed in such brain regions as the hippocampus and the cortex, which are affected by tau pathology and neurodegeneration in AD.


View larger version (95K):
[in this window]
[in a new window]
  		Fig. 2.   Neuronal expression of mutant PP2A Calpha L199P protein in transgenic mice. Parasaggital vibratome sections of transgenic mice (A-C) and age-matched wild-type controls (D and E) were stained with an antibody directed against the HA tag fused to the Calpha L199P protein. Immunoreactivity of mutant PP2A Calpha L199P was detected throughout the brain of transgenic mice. Cerebellar Purkinje cells (A), cortical pyramidal neurons (B), and pyramidal neurons of the CA1 region of the hippocampus (C) revealed a cytoplasmic and partly granular perinuclear staining. This staining was absent in the corresponding brain regions of wild-type mice (D-F). Scale bar, 100 µm.

Reduction of PP2A Activity in Transgenic Mice-- To determine whether expression of the mutant PP2A Calpha L199P in brain caused a long-term, chronic inhibition of PP2A activity, brain homogenates from 5- and 12-month-old transgenic and age-matched wild-type mice were analyzed for phosphatase activity by a colorimetric assay. Homogenates were obtained from six brains each, and activities were determined in duplicate samples by using as substrate a phosphopeptide that can be dephosphorylated by PP2A, PP2C, and, with substantially lower efficiencies, PP2B (47). PP1, in contrast, does not dephosphorylate peptide substrates at all (47). Assays were done with total extracts in a buffer optimized for PP2A to inhibit cation-dependent PP2B and PP2C activities. After 10 min of incubation, the assay revealed a reduction in activity of 34 ± 9% (n = 6; S.E.= ±3%) in PP2A Calpha L199P as compared with control homogenates (Fig. 1D). A similar decrease of PP2A activity was found in 5- and 12-month-old mice (data not shown). To further confirm that the measured decrease in phosphatase activity was due to a reduced activity of PP2A, brain homogenates were incubated with okadaic acid, a potent inhibitor of PP2A. Concentrations of 10 nM okadaic acid induced a half-maximal reduction of phosphatase activity in both wild-type and transgenic brain homogenates, normalized for the respective activities in wild-type and transgenic brain homogenates in the absence of okadaic acid. Because the ratios remained the same, our data indicate that PP2A and not another serine/threonine-directed phosphatase is inhibited in brains of PP2A Calpha L199P transgenic mice (data not shown). Additionally, assays were performed in the presence of 100 mM EGTA, which, as compared with standard assay conditions, had no influence on the ratio of inhibition, suggesting that PP2B is not a significant contaminant. Together, these data demonstrate that a neuronal, postmitotic expression of PP2A Calpha L199P was sufficient to induce a chronic, 34% reduction of PP2A activity in brains of transgenic mice.

Distinct Phosphorylation, Aggregation, and Ubiquitination of Endogenous Tau Protein-- To determine the functional consequences of reduced PP2A activity in mouse brains, we analyzed the phosphorylation and localization of the microtubule-associated protein tau by immunoblotting and immuno histochemistry.

Immunoblot analysis of tau protein in brain from transgenic mice as compared with controls revealed neither changes in tau expression levels nor changes in the relative distribution of the three murine tau isoforms (Fig. 3). To determine whether tau phosphorylation was altered in transgenic brain, we used a panel of different phosphorylation-dependent antibodies (Table I; Fig. 3). Tau phosphorylation was not detectable at the S199P and AT180 epitopes, and the AT100 and AD2 epitopes were not phosphorylated differently in brains from transgenic mice as compared with control brains. Phosphorylation of the AT8 epitope was only barely detectable, if detectable at all. In contrast, this epitope was readily detectable in brain extracts obtained from both human wild-type and FTDP-17 mutant tau transgenic mice (Fig. 3) (27, 28, 39). The pS422 epitope was phosphorylated in PP2A L199P transgenic brains, but not in controls (Fig. 3).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.   Immunoblot analysis of tau protein in brain from transgenic mice. Tau protein extracted from the brains of transgenic and control mice was treated with alkaline phosphatase and analyzed by immunoblotting using phosphorylation-independent anti-tau antibody Tau-5 (lanes 1 and 2). No differences in isoform composition were revealed. Tau proteins from a human wild-type tau transgenic brain (27) before and after alkaline phosphatase treatment (lanes 3 and 4) were analyzed with phosphorylation-dependent anti-tau antibody AT8. The molecular mass × 10-3 is shown to the left. Tau proteins from the brain of transgenic (lanes 5, 7, 9, 11, 13, and 15) and control (lanes 6, 8, 10, 12, 14, and 16) mice were analyzed by immunoblotting using the phosphorylation-dependent anti-tau antibodies AT8, AT100, AT180, AD2, pS422, and S199P.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Specificity of anti-tau antibodies employed in this study
Several phosphorylation- and conformation-dependent antibodies were used in this study. The respective phosphoepitopes are listed.

To map the regional phosphorylation of tau in brain, parasaggital sections from transgenic mice expressing PP2A Calpha L199P and age-matched controls were stained with a panel of phosphorylation- and conformation-dependent anti-tau antibodies (Table I), using conditions previously optimized in tau transgenic mouse models (27, 39). Stainings with antibody AT8 revealed that tau was phosphorylated at the epitope Ser-202/Thr-205 in several brain areas of L199P transgenic mice. AT8-immunoreactive tau was present in axonal (Fig. 4A) and somatodendritic compartments of cerebellar Purkinje cells (Fig. 4, C-F). In cortical neurons, expression levels were highest in layer IV (Fig. 5, A and C-G). No AT8 immunoreactivity was detected in age-matched nontransgenic controls (Figs. 4B and 5B) These data show that inhibition of PP2A by expression of a mutant form of PP2A Calpha caused hyperphosphorylation of the tau epitope Ser-202/Thr-205 and aggregation of tau in the somatodendritic compartment.


View larger version (115K):
[in this window]
[in a new window]
  		Fig. 4.   Tau phosphorylation in the cerebellum of PP2A Calpha L199P mutant mice. Parasaggital sections of paraffin-embedded brains from transgenic mice expressing PP2A Calpha L199P (A, C, E, and F) and age-matched wild-type mice (B and D) were stained with anti-tau antibody AT8 (red; B and C). AT8 immunoreactivity was detected in axons (A) and cell bodies of cerebellar Purkinje cells (C), demonstrating phosphorylation of tau at epitope Ser-202/Thr-205. No AT8 immunoreactivity was found in age-matched wild-type controls (B and D). In PP2A Calpha L199P mutant mice, AT8-positive tau accumulated in cell bodies of Purkinje cells and formed small aggregates. A single Purkinje cell is shown (E and F, differential interference contrast image). Scale bar: 100 µm (A and B), 20 µm (C and D), and 5 µm (E and F).


View larger version (143K):
[in this window]
[in a new window]
  		Fig. 5.   Tau phosphorylation in the cortex of PP2A Calpha L199P mutant mice. Parasaggital sections of paraffin-embedded brains from transgenic mice expressing PP2A Calpha L199P (A and C (at higher magnification)) and age-matched wild-type mice (B) stained with anti-tau antibody AT8. In PP2A Calpha L199P mutant mice, as for Purkinje cells, AT8-positive tau accumulated in the cell bodies of cortical neurons and formed small aggregates (two representative neurons: E and D, differential interference contrast image; and G and F, DIC image). Scale bar: 100 µm (A and B), 30 µm (C), and 5 µm (E-G).

Because additional phosphoepitopes of tau have been shown to be dephosphorylated by PP2A in vitro (15, 36, 48-52), we included the phosphorylation-dependent anti-tau antibodies AT100, AT180, 12E8, PHF1, AD2, R145d, S199P, and TG3 in our analysis (Table I). Immunohistochemical analysis revealed no significant differences between transgenic and control brains for any of these epitopes. Even antiserum R145d that was reactive in immunoblots did not reveal phosphorylation of epitope Ser-422 by immunohistochemistry. This suggests that this epitope, in contrast to the AT8 epitope, is phosphorylated in many neurons of our L199P mice at only very low levels. Strong staining of hyperphosphorylated tau by this antiserum was detected in brain sections from pharmacologically treated P301L mutant tau transgenic mice under identical experimental conditions,2 which excludes the possibility that antiserum R145d was not reactive under the conditions employed. Somatodendritic localization of tau was not accompanied or preceded by an altered conformation of tau as determined by conformation-dependent anti-tau antibodies TG3 and MC1 (Table I).

Interestingly, AT8 immunoreactivity in the cell bodies always appeared granular, which could be due to aggregation of abnormally localized and phosphorylated tau. Because aggregated tau is highly ubiquitinated in AD brains, we analyzed transgenic and control brains by confocal microscopy for AT8 and ubiquitin staining (Fig. 6). We found that AT8 and ubiquitin co-localized in the majority of AT8-positive tau aggregates (Fig. 6C). However, some of the tau aggregates were ubiquitin-negative, and this did not correlate with the size of the AT8-positive granular material or the number of AT8-postive aggregates/cell.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 6.   Aggregated tau co-localizes with ubiquitin. Parasaggital sections of transgenic mice were stained with the AT8 antibody (A, shown in red) and an anti-ubiquitin antibody (B, shown in green) and analyzed by confocal microscopy. Serial sections of confocal images were recorded sequentially. In the Purkinje cells of the cerebellum, hyperphosphorylated tau accumulated in large, perinuclear aggregates that were co-stained with ubiquitin (C, shown in yellow). Not all AT8-positive aggregates are also ubiquitin-positive (A, shown in red). m, molecular layer; g, granular layer; scale bar, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our data show that expression of a dominant negative mutant form of the catalytic subunit Calpha of PP2A in transgenic mice, in up to 1-year-old mice, was sufficient to permanently reduce PP2A activity by 34% as compared with wild-type controls. The reduced activity was associated with a distinct neuronal hyperphosphorylation of the microtubule-associated protein tau at the AT8 epitope Ser-202/Thr-205 and at the pS422 epitope Ser-422 and the accumulation of hyperphosphorylated tau in both the axonal and somatodendritic compartments. Furthermore, AT8-positive tau aggregates were partly co-localized with ubiquitin, suggesting that tau was targeted for degradation.

Impaired PP2A Function in Mice Expressing Mutant L199P-- Deregulation of phosphorylation-dephosphorylation mechanisms has been postulated in AD (2, 41, 53-57). PP2A, PP2B and, to a lesser extent, PP1 were found to be involved in the dephosphorylation of tau in normal brain (18, 58, 59). In our studies, we showed that expression of the human PP2A Calpha mutant L199P in brains of transgenic mice reduced PP2A activity by 34% by using as substrate a short phosphopeptide that is preferentially dephosphorylated by PP2A (47).

Mutant L199P was initially isolated in a mutagenesis study aimed at predicting the role of specific PP2A C residues in yeast (24). These studies showed that L199P bound the endogenous yeast PR65/A structural subunit, suggesting that, although catalytically impaired, it folded sufficiently to bind PP2A-interacting proteins, thereby contributing to its interfering effect on PP2A C function. Because the core dimer composed of the catalytic C and the structural PR65/A subunit serves as an essential scaffold for the recruitment of B-type regulatory subunits (60-64), PP2ACalpha mutant L199P may effectively titrate other subunits, interfering with targeting and/or substrate specificity of the wild-type enzyme (24, 25). Because expression of the human PP2A Calpha mutant interfered with murine PP2A function in our transgenic mice, it is likely that the mechanism of PP2A inhibition is identical to that in yeast. This suggests a functional conservation between murine, human, and yeast PP2A that reflects conservation at the sequence level (65).

In a related study, a mutant form of the structural subunit A of PP2A with impaired binding of regulatory B subunits was expressed under control of the beta -actin promoter in transgenic mice. This expression caused a 30% decrease in the activity of the holoenzyme in heart homogenates (66, 67). In our L199P mutant mice, a reduction of PP2A activity similar to that found in the PP2A subunit A mutant mice was seen. Despite differences in the construct design and expression pattern, the mechanisms responsible for the reduction in PP2A activity are likely similar. They involve a titration of either the A or the C subunit and thereby prevent the formation of active holoenzyme complexes. This approach suggests the expression of mutant forms of PP2A under the control of additional promoters to assess the role of PP2A during development and in terminally differentiated tissues.

Distinct Phosphorylation and Aggregation of Tau-- Phosphorylation in general and that of tau in particular are determined by the relative activities of kinases and phosphatases. PP2A can exert its effects on substrates either directly or indirectly via dephosphorylation of substrate-specific kinases (13, 46). Here, we show that reduction of PP2A activity in transgenic mice was sufficient to induce a distinct hyperphosphorylation of the AT8 epitope Ser-202/Thr-205 of tau (Table I). This finding is consistent with several studies that suggested a role of PP2A in dephosphorylating this epitope (15, 51, 55, 68, 69). Short treatments with the PP2A inhibitor okadaic acid on rat brain slices increased both tau levels and the phosphorylation of the Tau-1 epitope (Ser-199/Ser-202) (18), which overlaps, in parts, with the AT8 epitope (Ser-202/Thr-205) of tau. In our mice, reduction of PP2A activity induced the AT8 epitope, but not epitope S199P, suggesting that Ser-202 is a substrate of PP2A. Obviously, other phosphatases fail to take over the function of PP2A in dephosphorylating the AT8 epitope, whereas they may compensate for the reduced PP2A activity and thus dephosphorylate tau at the S199 epitope. Strong reactivity of the AT8 epitope by immunoblotting was not observed because few cells in the brain significantly phosphorylated this epitope. These findings are consistent with those obtained in transgenic mice that express human tau under the control of the human Thy1 promoter. In these mice, the AT8 epitope also was phosphorylated in few neurons and therefore failed to reveal phosphorylation by immunoblotting (28).

Treatment of brain slices with okadaic acid induced additional phosphoepitopes of tau including 12E8 and R145d. By immunoblotting, we found that epitope Ser-422, a substrate of antisera R145d and pS422, was phosphorylated in transgenic brain homogenates. There are two possible reasons for the failure to detect hyperphosphorylation of this epitope by immunohistochemistry: (a) either low levels of tau may be phosphorylated at epitope Ser-422 in many neurons in transgenic mice, or (b) the antiserum may not be suitable for immunohistochemistry. The latter is unlikely because both antisera detect tau hyperphosphorylated at Ser-422 in pharmacologically treated P301L mutant tau transgenic mice.2 Remarkably, transgenic mice expressing human wild-type tau under the control of the murine Thy1.2 promoter also reveal weak phosphorylation of the Ser-422 epitope by immunoblotting but fail to do so by immunohistochemistry (27). In vitro studies suggested that in addition to AT8 and R145d/pS422, tau phosphoepitopes AD2, 12E8, PHF1, and AT180 (15, 36, 48-52) are also dephosphorylated by PP2A. However, the 34% reduction of PP2A activity in our transgenic mice was not associated with changes in the phosphorylation- and/or conformation-dependent tau epitopes PHF1 and AD2, 12E8, AT100, AT180, S199P, MC1, and TG3 (Table I). The phosphorylation-dependent epitopes are conserved between mice and men, whereas murine tau may not adopt a conformation that is recognized by the conformation-dependent antibodies MC1 and TG3.

Previous studies showed that in brain homogenates of AD patients compared with those of control subjects, tau phosphatase activity was decreased by ~30% (12), and PP2A mRNA expression was quantitatively decreased in AD (70). Because the reduction of PP2A activity in PP2A Calpha L199P transgenic mice resembled that in AD brain (12), and because the AT8 epitope is an early epitope in AD pathogenesis (71), our data support a critical role of PP2A in the early stages of AD.

Interestingly, in our mice, AT8 staining was most pronounced in the cerebellum and cortex, whereas staining was weaker in the hippocampus. This correlates with the expression level of the mutant L199P, which was much lower in the hippocampus as compared with cerebellum and cortex. Differences in AT8 staining may also be due to differences in the activities of kinases and phosphatases in different brain areas. This view is supported by the finding that in brains of transgenic mice lacking the catalytic subunit Aalpha of PP2B, hyperphosphorylation of tau was restricted to the axonal mossy fiber projection of the hippocampus (72).

In AD, the axonal protein tau is hyperphosphorylated and accumulates in the somatodendritic compartment, where it eventually aggregates, forming filaments. With maturation, this tau becomes ubiquitinated (73-75). In our mice, some of these steps were recapitulated. A fraction of AT8-positive tau was present not only in axonal but also in somatodendritic compartments, where it accumulated as perinuclear aggregates that partly co-localized with ubiquitin, suggesting that tau is targeted for degradation. Interestingly, after deafferentation of the hippocampus, similar AT8-positive granules form that resemble those present in argyrophilic grain disease, another AD-related tauopathy (76, 77).

Taken together, our results support the hypothesis that PP2A is involved in the phosphorylation of tau in vivo. Our PP2A Calpha L199P mice are characterized by a chronic reduction of PP2A activity in brain that is sufficient to induce both hyperphosphorylation and somatodendritic accumulation of tau. This suggests that a defect in PP2A activity contributes to the pathogenesis in AD (5, 19, 78). Mating of PP2A dominant negative mutant mice with AD transgenic models, including tau filament-forming mice (39, 79), will allow the role of PP2A in AD to be further addressed.

    ACKNOWLEDGEMENTS

We thank Eva Moritz and Daniel Schuppli for excellent technical assistance and James Opoku for animal care. We thank Dr. Khalid Iqbal for antiserum R145d, Dr. Peter Davies for antibodies TG3, PHF1, and MC1, Dr. Chantal Mourton-Gilles for antiserum AD2, Dr. André Delacourte for antiserum S199P, and Dr. Peter Seubert for antibody 12E8.

    FOOTNOTES

* This work was supported in part by grants from the Bayer Alzheimer Research Network (to J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a grant from the Human Frontiers Science Program. Present address: Program in Molecular Pharmacology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.

|| To whom correspondence should be addressed: Division of Psychiatry Research, University of Zürich, August Forel Str. 1, 8008 Zürich, Switzerland. Tel.: 41-1-634-8873; Fax: 41-1-634-8874; E-mail: goetz@ bli.unizh.ch.

Published, JBC Papers in Press, July 25, 2001, DOI 10.1074/jbc.M102621200

2 Gotz, J., Chen, F., van Dorpe, J., and Nitsch, R. M. (2001) Science, 293, 1491-1495.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; PP, protein phosphatase; FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome 17; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., Zaidi, M. S., and Wisniewski, H. M. (1986) J. Biol. Chem. 261, 6084-6089
2. Grundke-Iqbal, I., Iqbal, K., Tung, Y. C., Quinlan, M., Wisniewski, H. M., and Binder, L. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4913-4917
3. Alonso, A. C., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5562-5566
4. Alonso, A. C., Grundke-Iqbal, I., and Iqbal, K. (1996) Nat. Med. 2, 783-787
5. Sontag, E., Nunbhakdi-Craig, V., Lee, G., Bloom, G. S., and Mumby, M. C. (1996) Neuron 17, 1201-1207
6. Price, D. L., Tanzi, R. E., Borchelt, D. R., and Sisodia, S. S. (1998) Annu. Rev. Genet. 32, 461-493
7. Ishiguro, K., Shiratsuchi, A., Sato, S., Omori, A., Arioka, M., Kobayashi, S., Uchida, T., and Imahori, K. (1993) FEBS Lett. 325, 167-172
8. Matsushita, M., Matsui, H., Itano, T., Tomizawa, K., Tokuda, M., Suwaki, H., Wang, J. H., and Hatase, O. (1995) Neuroreport 6, 1267-1270
9. Sironi, J. J., Yen, S. H., Gondal, J. A., Wu, Q., Grundke-Iqbal, I., and Iqbal, K. (1998) FEBS Lett. 436, 471-475
10. Schneider, A., Biernat, J., von Bergen, M., Mandelkow, E., and Mandelkow, E. M. (1999) Biochemistry 38, 3549-3558
11. Reynolds, C. H., Betts, J. C., Blackstock, W. P., Nebreda, A. R., and Anderton, B. H. (2000) J. Neurochem. 74, 1587-1595
12. Gong, C. X., Shaikh, S., Wang, J. Z., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1995) J. Neurochem. 65, 732-738
13. Sontag, E. (2001) Cell. Signal. 13, 7-16
14. Gong, C. X., Grundke-Iqbal, I., Damuni, Z., and Iqbal, K. (1994) FEBS Lett. 341, 94-98
15. Wang, J. Z., Gong, C. X., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1995) J. Biol. Chem. 270, 4854-4860
16. Garver, T. D., Oyler, G. A., Harris, K. A., Polavarapu, R., Damuni, Z., Lehman, R. A., and Billingsley, M. L. (1995) Mol. Pharmacol. 47, 745-756
17. Arendt, T., Holzer, M., Fruth, R., Bruckner, M. K., and Gartner, U. (1995) Neuroscience 69, 691-698
18. Gong, C. X., Lidsky, T., Wegiel, J., Zuck, L., Grundke-Iqbal, I., and Iqbal, K. (2000) J. Biol. Chem. 275, 5535-5544
19. Sontag, E., Nunbhakdi-Craig, V., Lee, G., Brandt, R., Kamibayashi, C., Kuret, J., White, C. L., III, Mumby, M. C., and Bloom, G. S. (1999) J. Biol. Chem. 274, 25490-25498
20. Goedert, M., Satumtira, S., Jakes, R., Smith, M. J., Kamibayashi, C., White, C. L., III, and Sontag, E. (2000) J. Neurochem. 75, 2155-2162
21. Zhou, Z. X., Kops, O., Werner, A., Lu, J. P., Shen, M., Stoller, G., Kullertz, G., Stark, M., Fischer, G., and Lu, P. K. (2000) Mol. Cell 6, 873-883
22. Gotz, J., Probst, A., Ehler, E., Hemmings, B., and Kues, W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12370-12375
23. Gotz, J., Probst, A., Mistl, C., Nitsch, R. M., and Ehler, E. (2000) Mech. Dev. 93, 83-93
24. Evans, D. R., Myles, T., Hofsteenge, J., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 24038-24046
25. Evans, D. R., and Hemmings, B. A. (2000) Genetics 156, 21-29
26. Luthi, A., Putten, H., Botteri, F. M., Mansuy, I. M., Meins, M., Frey, U., Sansig, G., Portet, C., Schmutz, M., Schroder, M., Nitsch, C., Laurent, J. P., and Monard, D. (1997) J. Neurosci. 17, 4688-4699
27. Probst, A., Gotz, J., Wiederhold, K. H., Tolnay, M., Mistl, C., Jaton, A. L., Hong, M., Ishihara, T., Lee, V. M., Trojanowski, J. Q., Jakes, R., Crowther, R. A., Spillantini, M. G., Burki, K., and Goedert, M. (2000) Acta Neuropathol. 99, 469-481
28. Gotz, J., Probst, A., Spillantini, M. G., Schafer, T., Jakes, R., Burki, K., and Goedert, M. (1995) EMBO J. 14, 1304-1313
29. Goedert, M., Jakes, R., and Vanmechelen, E. (1995) Neurosci. Lett. 189, 167-169
30. Goedert, M., Jakes, R., Crowther, R. A., Cohen, P., Vanmechelen, E., Vandermeeren, M., and Cras, P. (1994) Biochem. J. 301, 871-877
31. Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M.-Y. (1995) J. Biol. Chem. 270, 18917-18922
32. Dickson, D. W., Crystal, H. A., Bevona, C., Honer, W., Vincent, I., and Davies, P. (1995) Neurobiol. Aging 16, 285-298, discussion 298-304
33. Greenberg, S. G., Davies, P., Schein, J. D., and Binder, L. I. (1992) J. Biol. Chem. 267, 564-569
34. Buee-Scherrer, V., Condamines, O., Mourton-Gilles, C., Jakes, R., Goedert, M., Pau, B., and Delacourte, A. (1996) Brain Res. Mol. Brain Res. 39, 79-88
35. Jicha, G. A., Bowser, R., Kazam, I. G., and Davies, P. (1997) J. Neurosci. Res. 48, 128-132
36. Tanaka, T., Zhong, J., Iqbal, K., Trenkner, E., and Grundke-Iqbal, I. (1998) FEBS Lett. 426, 248-254
37. Delacourte, A., Sergeant, N., Wattez, A., Gauvreau, D., and Robitaille, Y. (1998) Ann. Neurol. 43, 193-204
38. Sergeant, N., Wattez, A., and Delacourte, A. (1999) J. Neurochem. 72, 1243-1249
39. Gotz, J., Chen, F., Barmettler, R., and Nitsch, R. M. (2001) J. Biol. Chem. 276, 529-534
40. Bussiere, T., Hof, P. R., Mailliot, C., Brown, C. D., Caillet-Boudin, M. L., Perl, D. P., Buee, L., and Delacourte, A. (1999) Acta Neuropathol. 97, 221-230
41. Goedert, M., Spillantini, M. G., Cairns, N. J., and Crowther, R. A. (1992) Neuron 8, 159-168
42. Kins, S., Betz, H., and Kirsch, J. (2000) Nat. Neurosci. 3, 22-29
43. Donella Deana, A., Mac Gowan, C. H., Cohen, P., Marchiori, F., Meyer, H. E., and Pinna, L. A. (1990) Biochim. Biophys. Acta 1051, 199-202
44. Kollias, G., Spanopoulou, E., Grosveld, F., Ritter, M., Beech, J., and Morris, R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1492-1496
45. Wera, S., Fernandez, A., Lamb, N. J., Turowski, P., Hemmings-Mieszczak, M., Mayer-Jaekel, R. E., and Hemmings, B. A. (1995) J. Biol. Chem. 270, 21374-21381
46. Janssens, V., and Goris, J. (2001) Biochem. J. 353, 417-439
47. Donella-Deana, A., Krinks, M. H., Ruzzene, M., Klee, C., and Pinna, L. A. (1994) Eur. J. Biochem. 219, 109-117
48. Saito, T., Ishiguro, K., Uchida, T., Miyamoto, E., Kishimoto, T., and Hisanaga, S. (1995) FEBS Lett. 376, 238-242
49. Xie, H., Litersky, J. M., Hartigan, J. A., Jope, R. S., and Johnson, G. V. (1998) Brain Res. 798, 173-183
50. Gartner, U., Janke, C., Holzer, M., Vanmechelen, E., and Arendt, T. (1998) Neurobiol. Aging 19, 535-543
51. Kim, D., Su, J., and Cotman, C. W. (1999) Brain Res. 839, 253-262
52. Lee, J., Hong, H., Im, J., Byun, H., and Kim, D. (2000) Neurosci. Lett. 282, 49-52
53. Vincent, I. J., and Davies, P. (1990) Brain Res. 531, 127-135
54. Drewes, G., Mandelkow, E. M., Baumann, K., Goris, J., Merlevede, W., and Mandelkow, E. (1993) FEBS Lett. 336, 425-432
55. Mawal-Dewan, M., Henley, J., Van de Voorde, A., Trojanowski, J. Q., and Lee, V. M. (1994) J. Biol. Chem. 269, 30981-30987
56. Goedert, M., Spillantini, M. G., Jakes, R., Crowther, R. A., Vanmechelen, E., Probst, A., Gotz, J., Burki, K., and Cohen, P. (1995) Neurobiol. Aging 16, 325-334
57. Trojanowski, J. Q., and Lee, V. M. (1995) FASEB J. 9, 1570-1576
58. Bennecib, M., Gong, C. X., Grundke-Iqbal, I., and Iqbal, K. (2000) FEBS Lett. 485, 87-93
59. Bennecib, M., Gong, C. X., Grundke-Iqbal, I., and Iqbal, K. (2001) FEBS Lett. 490, 15-22
60. Mayer, R. E., Hendrix, P., Cron, P., Matthies, R., Stone, S. R., Goris, J., Merlevede, W., Hofsteenge, J., and Hemmings, B. A. (1991) Biochemistry 30, 3589-3597
61. Hendrix, P., Mayer-Jackel, R. E., Cron, P., Goris, J., Hofsteenge, J., Merlevede, W., and Hemmings, B. A. (1993) J. Biol. Chem. 268, 15267-15276
62. Hendrix, P., Turowski, P., Mayer-Jaekel, R. E., Goris, J., Hofsteenge, J., Merlevede, W., and Hemmings, B. A. (1993) J. Biol. Chem. 268, 7330-7337
63. McCright, B., and Virshup, D. M. (1995) J. Biol. Chem. 270, 26123-26128
64. Csortos, C., Zolnierowicz, S., Bako, E., Durbin, S. D., and DePaoli-Roach, A. A. (1996) J. Biol. Chem. 271, 2578-2588
65. Gotz, J., and Kues, W. (1999) Biol. Chem. 380, 1117-1120
66. Ruediger, R., Brewis, N., Ohst, K., and Walter, G. (1997) Virology 238, 432-443
67. Brewis, N., Ohst, K., Fields, K., Rapacciuolo, A., Chou, D., Bloor, C., Dillmann, W., Rockman, H., and Walter, G. (2000) Am. J. Physiol. 279, H1307-H1318
68. Baum, L., Seger, R., Woodgett, J. R., Kawabata, S., Maruyama, K., Koyama, M., Silver, J., and Saitoh, T. (1995) Brain Res. Mol. Brain Res. 34, 1-17
69. Merrick, S. E., Demoise, D. C., and Lee, V. M. (1996) J. Biol. Chem. 271, 5589-5594
70. Vogelsberg-Ragaglia, V., Schuck, T., Trojanowski, J. Q., and Lee, V. M. (2001) Exp. Neurol. 168, 402-412
71. Braak, E., Braak, H., and Mandelkow, E. M. (1994) Acta Neuropathol. 87, 554-567
72. Kayyali, U. S., Zhang, W., Yee, A. G., Seidman, J. G., and Potter, H. (1997) J. Neurochem. 68, 1668-1678
73. Wisniewski, H. M., and Wrzolek, M. (1988) Ciba Found. Symp. 135, 224-238
74. Lowe, J., Mayer, R. J., and Landon, M. (1993) Brain Pathol. 3, 55-65
75. Iqbal, K., Alonso, A. C., Gong, C. X., Khatoon, S., Pei, J. J., Wang, J. Z., and Grundke-Iqbal, I. (1998) J. Neural Transm. 53 (suppl.), 169-180
76. Mudher, A. K., Yee, B., Smith, A. D., and Perry, V. H. (2001) Neurosci. Lett. 301, 5-8
77. Tolnay, M., Spillantini, M. G., Goedert, M., Ulrich, J., Langui, D., and Probst, A. (1997) Acta Neuropathol. 93, 477-484
78. Gong, C. X., Singh, T. J., Grundke-Iqbal, I., and Iqbal, K. (1993) J. Neurochem. 61, 921-927
79. Gotz, J. (2001) Brain Res. Rev. 35, 266-286

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Yotsumoto, T. Saito, A. Asada, T. Oikawa, T. Kimura, C. Uchida, K. Ishiguro, T. Uchida, M. Hasegawa, and S.-i. Hisanaga
Effect of Pin1 or Microtubule Binding on Dephosphorylation of FTDP-17 Mutant Tau
J. Biol. Chem., June 19, 2009; 284(25): 16840 - 16847.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
A. Deshpande, K. M. Win, and J. Busciglio
Tau isoform expression and regulation in human cortical neurons
FASEB J, July 1, 2008; 22(7): 2357 - 2367.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
U. Narayanan, V. Nalavadi, M. Nakamoto, D. C. Pallas, S. Ceman, G. J. Bassell, and S. T. Warren
FMRP Phosphorylation Reveals an Immediate-Early Signaling Pathway Triggered by Group I mGluR and Mediated by PP2A
J. Neurosci., December 26, 2007; 27(52): 14349 - 14357.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
H.-W. Klafki, M. Staufenbiel, J. Kornhuber, and J. Wiltfang
Therapeutic approaches to Alzheimer's disease
Brain, November 1, 2006; 129(11): 2840 - 2855.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Liu, K. Iqbal, I. Grundke-Iqbal, S. Rossie, and C.-X. Gong
Dephosphorylation of Tau by Protein Phosphatase 5: IMPAIRMENT IN ALZHEIMER'S DISEASE
J. Biol. Chem., January 21, 2005; 280(3): 1790 - 1796.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Schneider, G. W. Araujo, K. Trajkovic, M. M. Herrmann, D. Merkler, E.-M. Mandelkow, R. Weissert, and M. Simons
Hyperphosphorylation and Aggregation of Tau in Experimental Autoimmune Encephalomyelitis
J. Biol. Chem., December 31, 2004; 279(53): 55833 - 55839.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
U. Gergs, P. Boknik, I. Buchwalow, L. Fabritz, M. Matus, I. Justus, G. Hanske, W. Schmitz, and J. Neumann
Overexpression of the Catalytic Subunit of Protein Phosphatase 2A Impairs Cardiac Function
J. Biol. Chem., September 24, 2004; 279(39): 40827 - 40834.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Chen, M. A. Wollmer, F. Hoerndli, G. Munch, B. Kuhla, E. I. Rogaev, M. Tsolaki, A. Papassotiropoulos, and J. Gotz
Role for glyoxalase I in Alzheimer's disease
PNAS, May 18, 2004; 101(20): 7687 - 7692.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
E. Planel, T. Miyasaka, T. Launey, D.-H. Chui, K. Tanemura, S. Sato, O. Murayama, K. Ishiguro, Y. Tatebayashi, and A. Takashima
Alterations in Glucose Metabolism Induce Hypothermia Leading to Tau Hyperphosphorylation through Differential Inhibition of Kinase and Phosphatase Activities: Implications for Alzheimer's Disease
J. Neurosci., March 10, 2004; 24(10): 2401 - 2411.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
J. M. Shulman and M. B. Feany
Genetic Modifiers of Tauopathy in Drosophila
Genetics, November 1, 2003; 165(3): 1233 - 1242.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
C. R. Scherzer, R. V. Jensen, S. R. Gullans, and M. B. Feany
Gene expression changes presage neurodegeneration in a Drosophila model of Parkinson's disease
Hum. Mol. Genet., October 1, 2003; 12(19): 2457 - 2466.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. Kins, P. Kurosinski, R. M. Nitsch, and J. Gotz
Activation of the ERK and JNK Signaling Pathways Caused by Neuron-Specific Inhibition of PP2A in Transgenic Mice
Am. J. Pathol., September 1, 2003; 163(3): 833 - 843.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/41/38193    most recent
M102621200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kins, S.
Right arrow Articles by Götz, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kins, S.
Right arrow Articles by Götz, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement