©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Detection of Phosphorylated Ser in Fetal Tau, Adult Tau, and Paired Helical Filament Tau (*)

(Received for publication, March 2, 1995; and in revised form, May 12, 1995)

Peter Seubert (1) Madhumalti Mawal-Dewan (2) Robin Barbour (1) Ross Jakes (3) Michel Goedert (3) Gail V. W. Johnson (4) Joel M. Litersky (4) Dale Schenk (1) Ivan Lieberburg (1) John Q. Trojanowski (2) Virginia M.-Y. Lee (2)(§)

From the (1)Athena Neurosciences, Incorporated, South San Francisco, California 94080, the (2)Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283, (3)Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom, and the (4)Department of Psychiatry, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0017

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Paired helical filaments (PHFs) are the major structural elements of Alzheimer's disease neurofibrillary lesions, and these filaments are formed from hyperphosphorylated brain tau known as PHF-tau. Recent studies showed that many previously identified phosphorylated residues in PHF-tau also are phosphate acceptor sites in fetal and rapidly processed adult brain tau. However, Ser has been suggested to be uniquely phosphorylated in PHF-tau and a key regulator of the binding of tau to microtubules. For these reasons, we generated a monoclonal antibody (12E8) specific for phosphorylated Ser and showed that 12E8 binds to PHF-tau, rat and human fetal brain tau, as well as to rapidly processed adult rat and biopsy-derived human brain tau. Further, phosphorylation at Ser was developmentally regulated, and endogenous brain phosphatases rapidly dephosphorylated Ser in biopsy-derived brain tau isolates. Finally, the phosphorylation of Ser did not eliminate the binding of tau to microtubules. Thus, we speculate that the binding of tau to microtubules is regulated by phosphorylation at multiple sites and that the generation of PHF-tau in Alzheimer's disease results from the reduced efficiency of phosphatases leading to the incremental accumulation of hyperphosphorylated tau.


INTRODUCTION

Tau protein is the major component of paired helical filaments (PHFs) (^1)in Alzheimer's disease (AD) neurofibrillary tangles and related neurofibrillary lesions(1, 2, 3, 4, 5, 6) . It is expressed predominantly in axons where its major function is to bind to and stabilize microtubules (MTs)(7, 8, 9) . The MT binding domains of tau are 31- or 32-amino acid motifs repeated three or four times in the carboxyl-terminal half of the molecule. The presence or absence of 29- or 58-residue amino-terminal inserts in conjunction with three or four MT binding repeats gives rise to six tau isoforms in adult human brain (10, 11) , but only the shortest (``fetal'') isoform (three MT binding repeats, no amino-terminal inserts) is present in developing human brain(11) .

PHFs are comprised of all six tau isoforms in a hyperphosphorylated state (PHF-tau) relative to normal tau in adult brain(12) , and PHF-tau has a greatly reduced ability to bind to MTs(13, 14) . This is due to hyperphosphorylation, since dephosphorylated PHF-tau binds MTs like normal tau. Thus, hyperphosphorylation may be a key event in the transformation of normal brain tau into PHF-tau(5, 6) , and PHFs may result from the self-association of hyperphosphorylated tau through its MT-binding repeats(15, 16) . Mass spectrometry and immunological studies have identified a number of phosphorylation sites in PHF-tau, some of which are not phosphorylated in tau isolated from postmortem adult human brains(13, 17, 18, 19, 20, 21) . Many of these sites are Ser/Thr-Pro motifs and, except for Ser, are located outside the MT-binding region. Ser is located in the first MT-binding repeat of tau(18) . Except for Ser, many of the phosphorylation sites in PHF-tau also are phosphorylated in a significant fraction of fetal brain tau(13, 17, 18, 19) . Further, recent studies on adult rat brain tau (21, 22) and adult human brain tau isolated from biopsy samples (23) have demonstrated that some phosphorylation sites previously identified in PHF-tau and fetal tau also are phosphorylated in a fraction of adult tau. However, Ser has been reported to be phosphorylated only in PHF-tau(18, 19) .

Tau protein can be phosphorylated in vitro at many of the above sites by proline-directed protein kinases such as mitogen-activated protein kinase(24, 25, 26) , glycogen synthase kinase 3 (27, 28, 29) , and cyclin-dependent kinase 5(30, 31, 32) . In addition, cyclic AMP-dependent protein kinase(33) , Ca/calmodulin-dependent protein kinase II (34) , and protein kinase C (35) phosphorylate tau at specific sites in vitro, but among these sites, only Ser and Ser are phosphorylated in PHF-tau. Although none of the above protein kinases has been reported to phosphorylate Ser, Ser has been reported to be phosphorylated by a novel 35/41-kDa kinase partially purified from brain(36) .

Since the latter study emphasized the critical importance of Ser in regulating the binding of tau to MTs and the pivotal role that Ser may play in the pathogenesis of PHF-tau in AD(36) , we explored this issue further using a novel monoclonal antibody (MAb) raised to a synthetic phosphopeptide spanning amino acid residues 257-270 in human tau with a phosphoserine at position 262. Using this novel MAb, we show here that Ser is phosphorylated in PHF-tau as well as in normal brain tau and that phosphorylation at Ser alone cannot account for the inability of PHF-tau to bind to MTs.


EXPERIMENTAL PROCEDURES

Materials

A/J mice were purchased from Jackson Labs, and Sprague-Dawley rats were purchased from Charles River. Synthetic phosphorylated and non-phosphorylated peptides were made by BioServ Labs, and non-phosphorylated peptides were synthesized at Athena Neurosciences, Inc. Maleimidohexanoyl-N-hydroxysuccinimide was obtained from Boehringer. SDS-molecular weight standards, GTP, dithiothreitol, MES, and 2-nitro-5-thiocyanobenzoic acid (NTCB) were from Sigma; the mouse myeloma cell line SP2/O-Ag14 was from the American Type Culture Center; sheep anti-mouse IgG was from Jackson Immunochemicals; I-labeled goat-anti-mouse IgG was from ICN, and I-labeled protein A-agarose was purchased from DuPont NEN.

Preparation of Monoclonal Antibody 12E8

The immunogen was a synthetic phosphopeptide corresponding to amino acid residues 257-270 in tau (according to the numbering of the longest human tau isoform)(10) , with a phosphoserine at position Ser, as well as with two additional Gly residues and a Cys residue added at the carboxyl terminus for ease of coupling. This peptide (KSKIGS(PO(4))TENLKHQPGGC) was coupled to sheep anti-mouse IgG using maleimidohexanoyl-N-hydroxysuccinimide, and 100 µg of the immunogen was emulsified in Freund's complete adjuvant and injected subcutaneously into A/J mice. Additional boosting was done three times at biweekly intervals using 100 µg of immunogen emulsified in Freund's incomplete adjuvant. Mice were tittered against the phosphorylated peptide by radioimmunoassay (RIA, see below), and the mouse with the highest antibody titer was injected intravenously and intraperitoneally 1 month after the last boost with 50 µg of immunogen in phosphate-buffered saline. 3 days later, the mouse was sacrificed, the spleen was removed, and spleen cells were fused with the myeloma cell line SP2/O-Ag14 by a modification of the method of Kohler and Milstein(37) . Hybridoma-containing wells were screened by RIA for their ability to capture an I-labeled Ser-phosphorylated synthetic peptide. This peptide was similar to the immunogen except that a Tyr was substituted for Gly-Gly-Cys. The hybridoma with the highest titer was subcloned, and a stable hybridoma line was obtained that secreted a MAb designated 12E8. This MAb was produced and purified from ascites for use here except as noted.

Assessment of the Specificity of MAb 12E8 by RIA

The specificity of MAb 12E8 was determined by competitive RIA. Briefly, supernatants from late log phase 12E8 hybridoma cultures were harvested, and 50 µl of supernatant was incubated for 2 h at 37 °C with unlabeled peptides (at dilutions of 25 nM to 18 µM) with or without selective phosphorylation at Ser and with a Tyr residue at the carboxyl terminus. 50 µl of I-labeled Ser-phosphorylated synthetic peptide was added to each well and incubated for an additional 2 h at 37 °C. The antigen-antibody complexes were immunoprecipitated using rabbit anti-mouse IgG bound to protein A-Sepharose. The antigen-antibody Sepharose complexes were washed four times and counted using a Wallac Microbeta following the manufacturer's instructions.

Production and Phosphorylation of Purified Wild Type and Mutant Recombinant Tau

Tau constructs containing 4 MT binding repeats (4R tau) were used to produce the Ala, Ala, and Ala/Ala tau proteins by mutagenizing codons 262 and/or 356 from TCC to GCG as described(11) . cDNA clones encoding Ser/Ser 4R tau, Ala/Ser 4R tau, Ser/Ala 4R tau, and Ala/Ala 4R tau were subcloned into plasmid pRK172 as NdeI-EcoRI fragments and expressed in the Escherichia coli strain BL21 (DE3) following induction with isopropyl beta-D-thiogalactoside(11) . Wild type and mutant tau proteins were purified as described(11) . Wild type and mutant tau proteins were phosphorylated with a rat brain extract as described (21) . Control samples were processed identically, except that the brain extract was omitted.

Isolation of Tau from Adult Human Brain Biopsy Samples, Human Adult and Fetal Autopsy Brain and Rat Brain, as well as the Isolation of PHF-tau from Autopsy AD Brains

The isolation of enriched biopsy-derived brain tau was carried out as described(23) . Autopsy-derived normal human tau (from normal brains at 6-11 h postmortem) was extracted as described(4, 13) . Purified PHF-tau from postmortem AD brains (at postmortem intervals of 6-14 h) and enriched autopsy-derived fetal brain tau (at postmortem intervals of 1-14 h) were prepared exactly as reported(13) . Rat brain tau was obtained from Sprague-Dawley rats of different ages as described for biopsy-derived human tau. In these studies, biopsy-derived brain samples were processed for the isolation of tau within 10-15 min of surgical removal, and fresh rat brains were processed similarly within less than 2 min of sacrifice. We also isolated MT binding competent (BC) and binding incompetent (BI) tau from biopsy brain samples using previously published protocols(22) . Briefly, the high speed tissue extracts were supplemented with GTP and glycerol and incubated at 37 °C to induce MT assembly. The BC-tau was recovered in the pellet bound to MTs, leaving the BI-tau in the supernatant.

Immunoblot Analysis

SDS-PAGE and Western blot analyses of tau were performed as previously reported(11, 22, 23, 38) . In addition to the 12E8 MAb, several other antibodies against tau and PHF-tau were used. These included four antibodies (T14, T46, T49, 134) specific for phosphorylation-independent epitopes in tau (11, 22, 39, 40) and two antibodies (PHF1, AT8) specific for phosphorylation-dependent epitopes, i.e. Ser/Ser and Ser/Thr, respectively(41, 42, 43) . The concentration of antibodies used for Western blots were T14/T46 ascites, 1:1000; PHF1 supernatant, 1:250; 12E8 IgG, 1 µg/ml; AT8 IgG, 0.2 µg/ml; and 134 antiserum, 1:1000. The amount of bound primary antibody was quantitated using I-labeled secondary antibodies as described(23, 44) . The protein concentrations of the samples were determined using bichinchoninic acid as a dye reagent with bovine serum albumin as the standard(45) .

NTCB Cleavage

The NTCB cleavage of PHF-tau from AD brains, biopsy-derived adult human tau, and autopsy-derived fetal human tau was carried out essentially as described(46) . Briefly, trichloroacetic acid-precipitated enriched tau preparations or lyophilized samples of tau containing 10-25 µg of protein were resuspended in 25 µl of 40 mM EDTA, 20 mM dithiothreitol and incubated in a boiling water bath for 2 min. Guanidine HCl and Tris-HCl at pH 8.0 were added to each tube to give a final concentration of 6.4 and 0.1 M, respectively, in a final volume of 250 µl. Samples were incubated for 12 h at 37 °C, at which time 50 µl of 60 mM NTCB in 6.4 M guanidine HCl and 0.1 M Tris-HCl, pH 8.0, was added to each tube, and the incubation continued for another 48 h. The protein was precipitated by the addition of trichloroacetic acid to a final concentration of 6% followed by centrifugation. The protein pellet was resuspended in 0.1 M Tris-HCl, pH 8.0, and 4 volumes of methanol:chloroform:water (4:1:3) were added. The samples were then spun, and the top layer was removed. The protein at the interface was precipitated by adding cold methanol, incubated on ice for 1 h, and centrifuged. The final pellet was air-dried, resuspended in sample buffer, and electrophoresed as described above.

Preparation of Brain Samples for Immunohistochemistry

Small pieces of biopsy (control) or autopsy (AD cases)-derived brain were immersion-fixed in isotonic 70% ethanol (pH 7.4) at 4 °C for 18-24 h. The biopsy samples were normal residual fragments removed with epileptic foci from patients with intractable seizures(23) . Fixed tissues were infiltrated and embedded in paraffin, and 6-µm-thick sections were cut from paraffin blocks of the brain samples(23, 40) . Immunohistochemistry was performed using the peroxidase-anti-peroxidase method as described(23, 40) . The study of these tissues was approved by the Committee on Studies of Human Subjects at the University of Pennsylvania School of Medicine.


RESULTS

MAb 12E8 Recognizes Phosphorylated Ser and Ser in Tau

The specificity of the 12E8 MAb was determined initially by RIA, which showed that this MAb did not bind to the non-phosphorylated synthetic peptide, since binding of I-labeled peptide phosphorylated at Ser was not appreciably competed out by the non-phosphorylated peptide (Fig.1). Further, 12E8 also was able to bind strongly to the non-radiolabeled Ser-phosphorylated peptide, since the I-labeled phosphorylated synthetic peptide binding was effectively competed for by the unlabeled Ser-phosphorylated peptide (Fig.1). These results suggest that 12E8 recognized phosphorylated but not non-phosphorylated Ser. Since the sequence containing Ser (KIGSTEN) within the first MT binding repeat is similar to that in the fourth MT binding repeat (KIGSLDN), experiments were conducted to determine whether 12E8 also recognizes phosphorylated Ser in this motif or any other residues in tau phosphorylated by rat brain extracts. Purified wild type Ser/Ser 4R tau, as well as several tau mutants (i.e. Ala 4R tau, Ala 4R tau, and Ala/Ala 4R tau) were expressed in E. coli and then probed with antibodies 134 and 12E8 (Fig.2). As expected, none of the non-phosphorylated, recombinant wild type or mutant 4R tau proteins were labeled by 12E8 (Fig.2B, lanes1, 3, 5, and 7). By contrast, wild type 4R tau, Ala 4R tau, and Ala 4R tau, but not Ala/Ala 4R tau, were recognized by 12E8 following in vitro phosphorylation (Fig.2B, lanes2, 4, 6, and 8). This suggests that 12E8 binds only to tau proteins that are phosphorylated at Ser and/or Ser. Of the two sites, 12E8 recognized Ser better than Ser. Further, the phosphorylation of other residues in tau was not required for the recognition of tau by 12E8 since this MAb did not recognize the Ala/Ala double 4R tau mutant after phosphorylation. The latter treatment also revealed that the in vitro phosphorylation of Ser and/or Ser did not alter the electrophoretic mobility of these tau proteins, since wild type 4R tau and the 4R tau mutants migrated similarly before and after in vitro phosphorylation (Fig.2A).


Figure 1: RIA studies of the specificity of 12E8. The phosphorylated (filledcircles) or non-phosphorylated (opencircles) synthetic peptides spanning the tau sequence including Ser were compared by competitive RIA. The I-labeled ligand was prepared using chloramine-T and is present in the reaction mixtures at approximately 4 nM. Determinations were made in triplicate, and they varied less than 10% from each other.




Figure 2: Epitope mapping of 12E8. Wild type (WT) recombinant tau expressed from cDNA clone htau24 and mutated at Ser (Ala/Ser), Ser (Ser/Ala) or Ser and Ser (Ala/Ala) were immunoblotted with antiserum 134 (panelA) or MAb 12E8 (panelB) following incubation without (-) or with (+) a rat brain extract, resulting in the phosphorylation of tau. Antiserum 134 is not phosphorylation-dependent.



12E8 Binds to Autopsy-derived PHF-tau, Biopsy-derived Adult Human Brain Tau, and Autopsy-derived Fetal Human Brain Tau but not Autopsy-derived Adult Human Brain Tau

Since previous studies demonstrated that Ser is partially phosphorylated in PHF-tau, we probed PHF-tau from postmortem AD brains as well as tau from autopsy-derived adult and fetal brains using Western blots and a number of different anti-tau antibodies in addition to 12E8 (Fig.3). Fig. 3A demonstrates the profile of tau proteins present in these preparations as monitored with phosphate-independent antibodies. Fig.3C shows that 12E8 binds to PHF-tau like other phosphorylation-dependent anti-tau antibodies such as PHF1 (Fig.3B) and AT8 (Fig. 3D). Next, we probed Western blots of tau-rich isolates obtained from human biopsies processed immediately after surgical removal and from autopsy-derived adult and fetal human brains (Fig.3C), and we showed that 12E8 recognized both biopsy-derived normal human adult tau and autopsy-derived normal fetal brain tau but not to autopsy-derived adult tau. This suggests that Ser and/or Ser are normal in vivo sites of phosphorylation in tau. 12E8 also binds to all three major isoforms of biopsy-derived tau much like the phosphorylation-independent antibodies T14/T46 and the phosphorylation-dependent MAb PHF1 (compare Fig.3C with 3A and 3B). Quantitative assessment (Fig. 3, E-H) of the extent of phosphorylation of biopsy-derived brain tau, autopsy-derived adult and fetal brain tau, as well as of PHF-tau suggests that compared to PHF-tau only a fraction of the biopsy-derived adult tau (about 15-20%) and autopsy-derived fetal tau (about 28%) is phosphorylated at Ser and/or Ser.


Figure 3: Studies of PHF-tau and normal fetal and adult human tau with 12E8. Western blots comparing the site and extent of phosphorylation in the different tau preparations are shown in panelsA-D. Biopsy-derived human tau (B), autopsy-derived fetal tau (F), autopsy-derived adult human tau (A), and PHF-tau (PHF) were loaded onto 10% SDS-PAGE gels for immunoblot analysis. B(1)-B(3) denote tau from three different biopsy samples. The antibodies used here are shown on each blot. To obtain comparable levels of T14/T46 immunoreactive tau (i.e. as shown in E) in different tau preparations, the following amounts of total protein from each preparation were loaded: F, 2.5 µg; A, 5 µg; PHF, 2 µg; and B, 10 µg. The same amount of PHF was loaded for all antibodies. However, twice the amount of F and A and three times the amount of B were loaded for PHF1, 12E8, and AT8. PanelsE-H show quantitative data from PhosphorImager analysis of the immunoblots shown in panelsA-D using I-labeled goat anti-mouse IgG. The extent of phosphorylation of F, A, and B relative to PHF was calculated by taking into account the amount of protein loaded in each lane. The extent of phosphorylation in F, A, and B for each lane was normalized to PHF (for the antibodies PHF1, 12E8, and AT8).



Autopsy-derived Fetal Human Brain Tau, Biopsy-derived Adult Human Brain Tau, and Autopsy-derived PHF-tau Are Phosphorylated at Ser but not at Ser

Previous mass spectrometry studies suggested that Ser was variably phosphorylated in PHF-tau but not in fetal or adult tau (18, 19, 20) . To determine whether Ser alone, Ser alone, or Ser and Ser together are in vivo sites of phosphorylation, autopsy-derived fetal tau, biopsy-derived adult tau, and autopsy-derived PHF-tau were cleaved with NTCB. Since NTCB cleaves selectively at Cys residues(46) , this provides a strategy for distinguishing between the binding of 12E8 to Serversus Ser. Fetal tau contains a single Cys within the third MT binding repeat or repeat 3, whereas adult tau proteins contain 2 Cys residues in MT binding repeats 2 and 3, respectively(12) . Thus, cleavage of tau preparations with NTCB will result in amino-terminal fragments containing Ser (located within MT binding repeat 1) and carboxyl-terminal fragments containing Ser (located within MT binding repeat 4). We used T14 (an MAb that binds to a phosphorylation-independent epitope located within amino acid residues 141-178 of tau) to identify amino-terminal fragments of tau and T46 (a MAb that binds to a phosphorylation-independent epitope located between amino acid residues 404-441) to identify carboxyl-terminal fragments of tau. MAb 12E8 only detected tau fragments that were derived from the amino but not from the carboxyl terminus of tau (Fig.4). Thus, despite some heterogeneity in the tau cleavage products generated by NTCB, we showed that Ser but not Ser is an endogenous phosphorylation site in PHF-tau, fetal tau, and biopsy-derived adult tau.


Figure 4: Phosphorylation of Ser in PHF-tau as well as in fetal and adult human tau. Fetal and biopsy-derived normal human tau and PHF-tau were digested with NTCB, and the digests were separated on 12% SDS-PAGE gels followed by transfer to nitrocellulose. The MAbs used to probe the tau digests are shown. Tau indicates the presence of undigested tau, N marks the position of the amino-terminal fragments, whereas C labels the carboxyl-terminal fragments. 12E8 only binds to the amino-terminal fragments of tau.



Effect of Phosphorylation of Ser on the Microtubule Binding Competence of Tau

To determine the consequence of the phosphorylation of Ser on the binding affinity of tau to endogenous MTs, we performed MT binding assays on high speed homogenates of biopsy-brain samples (Fig.5). Fig. 5C shows that MT BI and BC biopsy-derived adult tau phosphorylated at Ser were recovered from supernatant and pellet fractions. Under the conditions described here, approximately 75% of total tau was recovered in the MT pellet (Fig.5A). Since 55% of the BC tau proteins were 12E8 positive, the phosphorylation of Ser does not appear to play an essential role in eliminating the binding of tau to MTs (Fig.5C). Similarly, phosphorylation at Ser/Ser or Ser/Thr did not eliminate binding of tau to MTs (Fig.5, B and D, respectively).


Figure 5: Western blot analysis of BI-tau (supernatant) and BC-tau (pellet). BI-tau (in lanes marked S) and BC-tau (in lanes marked P) were subjected to immunoblot analysis (panelsA-D). S(1)P(1) and S(2)P(2) denote BI and BC-tau from two different biopsies. The amount of protein loaded for detection by T14/46 (panelA) in the supernatant (S) fraction was three times that for the pellet (P) fraction. To obtain comparable levels of T14/46 immunoreactive tau (as shown in E), we loaded 2 µg of PHF (for all antibodies) and 5 µg of A. Approximately 10 µg of B from the pellet fraction were loaded per lane in panelA. The amount of A and B loaded for the antibodies PHF1, 12E8, and AT8 was two times as that for T14/46. PanelsE-H show the quantitative data using a PhosphorImager with I-labeled goat anti-mouse IgG as secondary antibody.



Regulation of Tau Phosphorylation at Ser during Fetal Rat Brain Development, during Postsurgical Delays in Cortical Biopsies, and in the Alzheimer's Disease Brain

To further characterize the phosphorylation of Ser, rat brain tau obtained at different developmental stages was probed with MAb 12E8 (Fig.6). The brain samples came from rats at embryonic day 18 (E18), postnatal days 7, 11, 19, and 25 (i.e. P7-25), and from 3- and 20-month-old rats. These studies showed that Ser was heavily phosphorylated in ``fetal'' tau at E18 and that the extent of phosphorylation progressively declined postnatally as the rodent brain matured (Fig.6, B and D). These observations suggest that during embryogenesis and in the early postnatal period, tau is heavily phosphorylated at Ser, but by P11 and beyond, progressively fewer phosphate residues remain at this site. Indeed, only about 10% of tau from 20-month-old rat brain was phosphorylated at Ser.


Figure 6: Western blot analysis of fetal, postnatal, and adult rat tau. Nitrocellulose replicas were prepared from 7.5% SDS-PAGE gels, and representative Western blots are shown in panelsA and B. To obtain comparable signals of T46/49 immunoreactive tau, we loaded in lane1, embryonic day 18 (E18, 15 µg); lane2, post-natal day 7 (P7, 15 µg); lane3, postnatal day 11 (P11, 25 µg); lane4, postnatal day 19 (P19, 25 µg); lane5, postnatal day 25 (P25, 40 µg); lane6, adult 3-month-old (3 M, 40 µg); and lane7, adult 20-month-old (20 M, 40 µg). The antibodies used here are labeled above each blot. The amount of protein loaded for the antibody 12E8 was three times that mentioned above for T46/49. PanelsC and D show quantitative data from PhosphorImager analysis of the immunoblots shown in panelsA and B. The decrease in phosphorylation was normalized to 100% for E18. Standard deviations were generated from quadruplicate samples, and the quantitation was generated using I-labeled goat anti-mouse IgG.



Since many phosphorylation sites in adult tau are rapidly dephosphorylated by endogenous brain phosphatases during postmortem or postsurgical delays(23) , we simulated such conditions by allowing freshly excised human brain biopsy samples to remain at room temperature for varying lengths of time as described(23) . The tau proteins isolated from these samples were then analyzed by immunoblotting using many of the anti-tau antibodies described above (Fig.7). Fig. 7A shows the profile of tau proteins in these samples labeled with the T14/46 mixture of MAbs. Consistent with previous studies(23) , Fig.7, C and G, shows that biopsy-derived adult human brain tau was rapidly dephosphorylated at Ser at room temperature. In fact, after only 1 h of simulated postsurgical delay, the extent of phosphorylation of tau at Ser was reduced by over 90% (Fig.7G). Notably, Ser was much more rapidly dephosphorylated than Ser/Ser or Ser/Thr. After only a 5-min postsurgical delay, the intensity of the immunobands detected by 12E8 was reduced to a far greater extent (i.e. by about 70%) than that produced by other phosphate-dependent MAbs (i.e. compare Fig.7B, 7D, 7F, and 7H with 7C and 7G).


Figure 7: Dephosphorylation of human tau in situ. Western blot analysis of the time course of dephosphorylation of biopsy-derived brain tau during defined post-surgical intervals of 0-60 min as indicated below the lanes in panelsA-D. Biopsy brain samples were left at room temperature for periods of time extending from 5 to 60 min. The antibodies used for immunoblotting are identified above each panel. All the lanes were loaded with F, A, and PHF. PanelsE-H show quantitative data from PhosphorImager analysis of the immunoblots shown in panelsA-D. The decrease in phosphorylation at each site was normalized to time zero (i.e. no post-surgical delay).



The phosphorylation of biopsy-derived tau and PHF-tau at Ser was also demonstrated by immunohistochemistry (Fig.8). Fig. 8A shows intense 12E8 immunoreactivity in processes throughout the cortical neuropil of a biopsy sample while neuronal perikarya stand out as 12E8 negative oval or pyramidal profiles. In sections from postmortem AD neocortex (Fig.8B), 12E8 intensely labeled the PHF-tau accumulations in perikaryal neurofibrillary tangles, neuropil threads, and dystrophic neurites, much like previously described phosphorylation-dependent anti-tau antibodies (e.g. see results obtained with PHF1 in Fig.8C).


Figure 8: Immunohistochemical localization of tau-phosphorylated Ser with 12E8. Immunohistochemical staining of tissue sections obtained from biopsy control (A) and autopsy AD human brain samples. In panelsA and B, sections had been incubated with 12E8, and in panelC, sections had been incubated with PHF1. All brain sections were obtained from temporal lobe. The magnification in panelsA-C is the same, and the bar in panelC = 50 µm.




DISCUSSION

We have used a novel MAb (12E8) to assess the role of phosphorylated Ser in the pathogenesis of PHF-tau. The specificity of 12E8 for tau phosphorylated at Ser was demonstrated by RIA and Western blot analyses. Since the sequences surrounding Ser and Ser are similar, we performed additional studies using recombinant tau proteins harboring Ser to Ala mutations at positions 262 and/or 356, which showed that 12E8 recognizes recombinant tau when phosphorylated at Ser, Ser, or at both positions. Since 12E8 recognized PHF-tau, fetal brain tau, and biopsy-derived adult brain tau, we probed NTCB cleavage products of tau and demonstrate that only Ser is phosphorylated in tau from normal and AD brain.

The recognition of PHF-tau on immunoblots and the intense staining of neurofibrillary lesions in AD brain by immunohistochemistry with 12E8 confirm that Ser is phosphorylated in PHF-tau(18) ; however, contrary to previous findings(18) , we also found that Ser is a site of phosphorylation in normal fetal and adult human and rodent tau. The demonstration here that Ser is rapidly dephosphorylated by brain phosphatases appears to account for the discrepancy between the present and previous studies(18, 19, 20) . Our results indicate that of the phosphorylation sites in brain tau studied to date, Ser is the most labile since 70% of the phosphate was lost within 5 min of postsurgical delay, compared with 10% for Ser/Ser and 50% for Ser/Thr. Further, we also show that the phosphorylation of Ser in normal brain tau is developmentally regulated and that only a fraction of biopsy-derived tau is phosphorylated at Ser compared with PHF-tau. Similar results also have been obtained with anti-tau antibodies AT8, AT180, AT270, T3P, and PHF1, which recognize specific Ser/Thr-Pro sites in tau when phosphorylated(13, 17, 21, 22, 23) . Since 12E8 is the first antibody to a phosphorylation site in tau that is not of the Ser/Thr-Pro type, the present data indicate that proline-directed kinases do not fully account for the normal or the abnormal phosphorylation of tau. Although a novel 35/41-kDa protein kinase partially purified from brain phosphorylates tau at Serin vitro, both cyclic AMP-dependent protein kinase and Ca/calmodulin-dependent protein kinase II phosphorylate recombinant tau at this site in vitro. (^2)Thus, multiple protein kinases may phosphorylate tau at this site in normal and AD brains. Further, reduced phosphatase activity may play an important role in PHF-tau formation(22, 23) . Although the protein phosphatases that act on the Ser site are unknown, preliminary results indicate that protein phosphatase 2A dephosphorylate tau at Ser. (^3)

The findings described here serve to further clarify the pathogenesis of PHF-tau by demonstrating that phosphorylation of Ser is not sufficient to eliminate the binding of tau to MTs. Indeed, they suggest that the simultaneous phosphorylation of multiple sites may be required for the disruption of the interactions between tau and MTs. Thus, the challenge for the future will be to identify the phosphorylation sites that are most critical for regulating the binding of tau to MTs, as well as the protein kinases and protein phosphatases that modulate the state of phosphorylation of tau in both normal and AD brain.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Anatomic Pathology, Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, HUP/Maloney, Rm. A009, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909.

^1
The abbreviations used are: PHF, paired helical filament; AD, Alzheimer's disease; MT, microtubule; BC-tau, binding competent tau; BI-tau, binding incompetent tau; NTCB, 2-nitro-5-thiocyanobenzoic acid; MAb, monoclonal antibody; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; RIA, radioimmunoassay; 4R tau, tau constructs containing 4 MT binding repeats.

^2
J. M. Litersky, G. V. W. Johnson, R. Jakes, M. Goedert, M. Lee, and P. Seubert, submitted for publication.

^3
V. M.-Y. Lee and M. Mawal-Dewan, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. P. Davies and Innogenetics for providing antibodies PHF1 and AT8, respectively. Dr. David Davis is acknowledged for peptide synthesis.


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