the journal of biological vol. 268, no. 32, issue of ......the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Moleculal ’ Biology, Inc.

Vol. 268, No. 32, Issue of November 15, pp. 24374-24384,1993 Printed in U.S.A.

Microtubule-associated Protein Tau ABNORMAL PHOSPHORYLATION OF A NON-PAIRED HELICAL FILAMENT POOL IN ALZHEIMER DISEASE*

(Received for publication, May 3, 1993, and in revised form, July 16, 1993)

Evelyn Kopke$, Yunn-Chyn Tung, Sadia Shaikh, Alejandra del C. Alonso, Khalid Iqbal, and Inge Grundke-Iqbals From the New York State Institute for Basic Research in Deuelopmental Disabilities, Staten Island, New York 10314

The major protein subunit of the paired helical filaments (PHF) of Alzheimer disease (AD) is the microtubule-associated protein tau. Tau is a family of phos- phopolypeptides that are abnormally phosphorylated in PHF. In this study, a non-PHF pool of tau abnormally phosphorylated at Ser-199/202, and tau not phosphorylated at this site (AD P-tau and AD tau, respectively) were isolated from the 27,000 X g to 200,000 X g fraction of AD brain homogenate by extraction in 8 M urea, followed by dialysis against Tris buffer. AD P-tau and AD tau were further purified and separated from each other by acid precipitation, glial fibrillary acidic protein affinity chromatography, and phosphocellulose chromatography. The resulting AD P-tau and AD tau preparations were free of cyto- skeletal proteins, ubiquitin, and &amyloid peptide. Im- munochemical and morphological analysis of AD P-tau preparations revealed that most of the protein was of non-PHF origin. The AD P-tau was about 3-4-fold (-8 mol P04/mol protein, M, 4 1,3 18) more phosphorylated than cytosolic tau from AD and control brains. Unlike PHF, the AD P-tau lacked ubiquitin. In AD brain the levels of cytosolic tau were about half of those in control aged cases. These findings suggest that the abnormal phosphorylation of tau in AD occurs in the cytosol.

Neurofibrillary changes in the form of PHF’ intermixed

* This study was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, National Institutes of Health Fellowship F32 AGO 5541 (to E. K.) and National Institutes of Health Grants NS 18105, AG 04220, AG 05892, and AG 08076, a grant from the Alzheimer Disease Research Program of the American Health Assistance Foundation (Rockville, MD), and Zenith Award (to K. I.) from Alzheimer’s Association. Parts of the data were presented at the Second and Third International Confer- ence on Alzheimer’s Disease and Related Disorders in Toronto, Canada, July 15-20, 1990 and in Abano Terme, Italy, July 12-17, 1992. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10591. $ Present address: Regeneron Pharmacology Inc., Tarrytown, NY

To whom correspondence should be addressed New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5263; Fax:

’ The abbreviations used are: PHF, paired helical filaments; AD, Alzheimer disease; AD tau, tau from AD brain sedimenting at 27,000 to 200,000 X g and reactive with mAb Tau-1 without prior dephosphorylation; AD P-tau, abnormally phosphorylated tau from AD brain sedimenting at 27,000 to 200,000 X g and reactive with mAb Tau-1 only after dephosphorylation; c-tau, cytosolic tau that does not

718-494-5269.

with 2.1-nm tau filaments (1, 2) are one of the most characteristic neuropathological lesions in AD. PHF accumulate as large bundles in the form of neurofibrillary tangles in the neuronal cell body and the dendrite and extend through the neuronal processes far into the neuropil, forming a dense network of the so-called neuropil threads (3). PHF also accumulate in the dystrophic neurites that surround the extracellular amyloid in neuritic (senile) plaques, another histo- pathological hallmark of AD. Many of these neurons with neurofibrillary changes degenerate, leaving behind what are called tombstones or ghost tangles in the extracellular space. Because of the @-pleated sheet conformation of the polypeptides in PHF (4) these tangles are apparently not degraded by the proteases in the tissue.

One population of PHF, the PHF I, are readily soluble in 1% SDS at room temperature (5-7), whereas others, the PHF 11, including the ghost tangles, only dissolve in SDS on boiling, and then only after repeated extractions with SDS or after ultrasonication ( 5 ) . The major protein subunit of PHF is the microtubule-associated protein tau ( 5 , 6, 8, 9). In addition to tau, small amounts of ubiquitin are also present in PHF I1 (10-12).

Tau consists of at least six isoforms that have been gener- ated through alternate mRNA splicing from a single gene (13, 14). Tau promotes microtubule assembly and contributes to the stability of microtubules, the major component of the axoplasmic transport system of neurons (15-17). Tau in PHF is abnormally phosphorylated (9, 18-22). In the AD brain the abnormal phosphorylation of tau can already be detected immunocytochemically in the earliest stages of tangle formation. These so-called stage “0” tangles contain mostly amorphous tau-positive material intermixed with occasional short filaments/PHF (23). Non-PHF tau in neurons with neurofibrillary changes is apparently associated with ribosomes (24). Biochemically, abnormal tau has been found in non-PHF form, co-sedimenting with tubulin aggregates isolated from AD brains (25). It is of crucial importance to isolate the abnormal tau that is not yet polymerized into PHF and to separate it from normal tau for further biochemical studies on the nature of its alteration and on its role in the pathogenesis of the neurofibrillary degeneration in AD.

The term abnormally phosphorylated tau was originally coined by us for the AD brain protein that was phosphorylated at the mAb Tau-1 site and that only reacted with this antibody after treatment with alkaline phosphatase (18). Since then, in addition to the Tau-1 site (Ser-199/202), seven additional

sediment at 200,000 X g and does not require dephosphorylation to react with Tau-1; GFAP, glial fibrillary acidic protein; mAb, monoclonal antibody; PC, phosphocellulose; MES, 4-morpholineethane- sulfonic acid.

24374

Unpolymerized Abnormal Tau from Alzheimer Disease Brain 24375

abnormal phosphorylation sites have been detected in PHF tau (9, 18-22, 26). Furthermore, a 68-kilodalton polypeptide, A68 recognized by a monoclonal antibody Alz-50, originally reported to be unique to AD brain (27), has been subsequently shown to be the same as the abnormally phosphorylated PHF tau (9, 28). Tau phosphorylated at Ser-199/202 has also been observed in brains of rats that had been perfused with large concentrations of phosphate (29), possibly inhibiting a variety of endogenous phosphatases. This form of tau, however, seems to be transitory since it is not normally observed in the adult rat or human brain.

The present study describes the bulk isolation of a pool of non-PHF tau, which comprises both abnormally phosphorylated (AD P-tau) and apparently normal species (AD tau), with sedimentation properties different from those of cytosolic tau (c-tau). The resulting AD P-tau and AD tau are readily soluble in buffer, and AD P-tau has a higher total phosphate content than the c tau from either AD or control human brains. Like PHF-tau, AD P-tau is abnormally phosphorylated at several sites. However, unlike PHF, AD P-tau does not contain any ubiquitin immunoreactivity.

MATERIALS AND METHODS

Human Tissue-Brains from 15 histopathologically confirmed AD cases and eight non-AD controls were used in this study. Autopsy was performed within 2-9 h (4.2 f S.E. 0.5) for the AD cases and within 3-7 h (5.0 f 0.6) for the controls. The age of the AD cases ranged from 60 to 89 years and for the control cases from 39 to 73 years. Immediately after autopsy, the brains were frozen; they were stored at -75 "C until used.

Antibodies-mAb Tau-1 (ascites, a gift from Dr. L. Binder, Ref. 30) antiserum 92e (12) to bovine tau and mAb PHF-1 (culture supernatant, a gift from Dr. S. Greenberg, Ref. 28) were used at a dilution of 1:50,000, 1:5,000, and 1:100, respectively. Immunoaffinity purified rabbit antibody 102c which reacts with amino acid residues 42-49 of human tau (20) was used at a concentration of 0.5 pg/ml. Other antibodies used were mAb SMI 31 and SMI 34 (ascites, Sternberger Monoclonals, Inc. MD) to neurofilament subunits H and M at a dilution of 1:100), mAbs 5-25 and 3-39 (ascites, Refs. 31, 32) to PHF-ubiquitin at dilutions of 1:50,000 and 1:20,000, respectively; mAb 2F9 (ascites, Ref. 33) to amyloid (3 peptide at a dilution of 1:5,000; mAbs AP14 and AP18 to MAP2, undiluted culture supernatants (a gift from Dr. L. I. Binder); mAb NF68 (Boehringer Mann- heim) to neurofilament L subunit at a dilution of 1:20; mAb YL 1/2 (ascites, Ref. 34) to a-tubulin (Accurate Chemicals, Westbury, NY) at a dilution of 1:500; and an antiserum to glial fibrillary acidic protein (GFAP) (DAKOPATTS, Denmark) at a dilution of 1:1,000.

Protein Profiles on SDS-Polyacrylamide Gels and Immunoblots- Protein samples were electrophoresed on slab gels (80 X 60 X 0.75 mm), using a linear acrylamide gradient (5-15%) and the Laemmli buffer system (35). After electrophoresis, polypeptides and prestained protein standards (Bethesda Research Laboratories) were transferred to Immobilon membrane (0.45 pm; Millipore, MA) at 105 V for 1 h and immunostained as previously described (8). Blots were dephosphorylated with alkaline phosphatase (Boehringer Mannheim) at 43 pg/ml (18). In some cases, blots were stained with 0.25% Coomassie Blue in methanol/water/acidic acid (45:45:10, v/v/v).

Protein concentrations were determined by the modified Lowry assay of Bensadoun and Weinstein (36).

Levels of tau and ubiquitin were determined by immunoslot blot analysis using rabbit anti-tau serum 92e or mAb Tau-1 with or without dephosphorylation (37) and a mixture of mAb 5-25 and 3- 39 to PHF-ubiquitin (31). In the case of antiserum 92e, the blots were developed with alkaline phosphatase-conjugated anti-rabbit IgG (Bio- Rad) and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as substrate. In the case of tau determinations with mAb Tau- 1, the bound antibody was detected with lZ5I-labeled anti-mouse IgG, Amersham Corp. (37). At least three dilutions of the respective samples were applied in triplicates to the nitrocellulose membrane using a slot-blot apparatus (both from Schleicher & Schuell). Purified bovine tau (8) or purified ubiquitin (Sigma) in amounts ranging from 2 to 20 ng were used for the standard curves. The immunoreactivity on the individual slots was measured by scintillation counting or densitometry (Shimadzu CS 9000, Japan). In some cases, units of

total tau were calculated from the OD units of the scans of the immunoblots that had been dephosphorylated prior to incubation with Tau-1. The ratios of AD P-tau to AD tau were estimated by densitometry of immunoblots that had been developed with mAb Tau-1 with or without pretreatment with alkaline phosphatase.

Isolation of PHF-Neurofibrillary Tangle-PHF were prepared from neuronal cell bodies by treatment with 1% SDS at room temperature, absorption of blood vessels on glass beads, and discontinuous sucrose gradient fractionation as previously described (5). These preparations contain almost exclusively the late stages of tangle formation, the PHF 11, which are only soluble in SDS-(3-mercaptoethanol at 95 "C after ultrasonication.

Zsolation of AD P-tau and AD Tau-AD P-tau and AD tau were isolated from a 5% homogenate (w/v) of cerebral cortex from AD brains in a phosphatase and protease inhibitors mixture buffer modified from Saitoh and Dobkins (38): 20 mM Tris, pH 8.0, 0.32 M sucrose, 10 mM (3-mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 5 mM MgS04, 5 mM benzamidine, 10 mM glycerophosphate, 6 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 1 mM sodium ortho- vanadate, 0.1 mM chloroquine, 10 mM soybean trypsin inhibitor, 0.1 mg/ml tosyl-arginine-methionine, 5 pg/ml leupeptin, 1.5 pg/ml pep- statin, and 2 pg/ml of aprotinine. After removal of meninges and white matter of the partially thawed tissue, the gray matter was homogenized in a motor-driven glass-Teflon homogenizer. The Teflon pestle was moved up and down 10 times at 2,500 revolutions/min for a total of approximately 4 min. All isolation procedures were performed at 4 "C, unless otherwise indicated. Cellular debris and neurofibrillary tangles were pelleted at 27,000 X g for 30 min. The supernatant was then centrifuged at 200,000 X g for 45 min. The resulting pellet was extracted with 8 M urea at room temperature (22 "C) for 60 min and then spun at 334,000 X g for 45 min at 22 "C. The supernatant was first dialyzed for 18 h with three changes against a 200-fold volume of 10 mM Tris, pH 7.6, and then acid precipitated by dialysis for 19 h against 100 mM MES, 0.5 mM MgCl,, 1 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 0.75 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, and 50 mM NaF, pH 2.7. The precipitated proteins were removed by centrifugation at 200,000 X g for 45 min. The 200,000 X g supernatant was dialyzed against 25 mM MES, pH 6.4, 0.5 mM MgClZ, 0.1 mM EDTA, and 1 mM dithiothreitol (PC buffer) and subsequently fractionated on a PC column (Cellulose Phosphate P11; Whatman), which was equilibrated with the same buffer. The PC column was loaded with 2.2 mg of protein/ml PC at protein concentrations of 0.06 to 0.28 mg/ml. Proteins were eluted with a 0-1 M NaCl linear gradient in PC buffer, using 10-fold the column volume. AD P-tau eluted with 0.1 to 0.28 M and AD tau with 0.31 to 0.8 M NaCl (see Fig. 5, a-c). The tau content of each fraction was estimated on slot-blots (see below), using antiserum 92e to tau. In one case prior to PC chromatography, GFAP was removed from the acid-soluble protein fraction by affinity chromatography on a Protein A-Sepharose column to which antibodies to GFAP had been covalently linked with dimethylpimelinidate (39).

Isolation of Cytosolic Tau from Normal and AD Brain-Homoge- nates and supernatants were prepared as above. Solid ammonium sulfate was added to the 200,000 X g supernatants to 35% saturation, stirred for 30 min, and centrifuged at 60,000 X g for 20 min. A second ammonium sulfate cut of the supernatant to 45% saturation was performed, and the suspension was centrifuged as above. The 35- 45% pellet was suspended in 10 mM Tris, pH 7.6, and processed for purification of tau as described above for AD P-tau.

Evaluation of the Contribution of PHF Toward AD P-Tau-To evaluate the contribution of PHF toward the isolated AD P-tau, the 27,000 to 200,000 X g fraction of AD brain homogenates employed for the isolation of tau was subjected to sucrose density gradient centrifugation as follows: a 0.7-ml sample was layered on a discontinuous sucrose gradient containing 0.7 ml each of 2.50, 2.25, 2.00, 1.75, 1.50, and 1.25 M sucrose, and was centrifuged for 16 h at 225,000 X g at 4 "C (9). The sample was distributed in seven distinct layers (Table 111, with layer 1 being at the top and layer 7 at the bottom of the gradient. These layers were separated by gently aspirating each layer with a bent-tip Pasteur pipette, and each fraction was analyzed for protein concentration, tau immunoreactivity, and the presence of PHF. PHF, 5 p1 of each fraction at a 1:l dilution, were examined by negative stain electron microscopy (see below), and PHF twists were counted in a standard field of electron micrographs. Because PHF are twisted every 80 nm and filaments differing in length were isolated, counting the number of twists rather than the number of fibrils provides a better estimate of the PHF mass in the preparations.

To determine how much tau could have been extracted from PHF

24376 Unpolymerized Abnormal Tau from Alzheimer Disease Brain with urea, the sucrose gradient fractions 5 and 7, which contained most of the PHF, were combined and dialyzed overnight against the tissue homogenization buffer (see above). After dialysis, the sample was divided into two equal parts. Urea was added to one part to a final concentration of 8 M and an equal volume of distilled deionized water to the other part. The samples were stirred for 1 h at room temperature (22 "C) and subsequently centrifuged at 334,000 X g for 20 min. The resulting pellets were suspended in 100 pl of 10 mM Tris, pH 7.6, and the supernatants dialyzed overnight against the same buffer. Both pellets and supernatants were analyzed by negative stain electron microscopy for the presence of PHF and by slot-blot and immunoblots for ubiquitin and tau levels.

Electron Microscopic Analysis-To study the presence of PHF in the 27,000 to 200,000 X g fraction and the urea- and water-insoluble pellets, a drop of each sample at dilutions of 1:5, 1:50, and 1:lOO in 100 mM sodium phosphate, pH 7.4, was applied on 100 mesh carbon- coated copper grids, which had been in an atmosphere of 25% glutar- aldehyde for 2 h before sample application. After 1 min, excess sample fluid was removed with a wedged filter paper and the sample stained with one drop of 2% uranylacetate, pH 3.8, for 1 min. Grids were allowed to dry and viewed at 75 kV in a Hitachi 7000 electron microscope at a magnification of X 50,000.

Samples, from the sucrose gradient experiment a t a 1:l (v/v) dilution in 100 mM sodium phosphate, pH 7.4, were negatively stained with 2% phosphotungstic acid, as previously described (40), and viewed at a magnification of X 50,000. Six grids were prepared of each sample, and for each grid a t least four different areas with intact carbon coating and uniform distribution of negatively stained structures were examined. PHF twists were counted from a standard area of micrographs at a final magnification of X 135,000. A control experiment showed that under these conditions the relationship between the numbers of PHF twists counted and the protein concentration of the sample was linear (Fig. 3).

Total Phosphate Analyses-Tau samples were precipitated and washed either with 80% cold ethanol or acetone, hydrolyzed with 6 N HC1 for 1 h at 150 "C and extracted with 400 p1 of water. Of this extract, 300 pl was used for the total phosphate analysis, and 100 pl for protein determination by amino acid analysis. All analyses were performed on triplicate samples.

The total phosphate analysis was modified after Hess and Derr (41), whereby 50 p1 of 5% magnesium nitrate in 95% ethanol was added to the dried samples, placed in a heating block for 20-40 min at maximum setting, and ashed over an open flame until a white powder formed. The ash was suspended in 50 pl of 1.2 N HCI, using a Vortex mixer, and 150 pl of dye mix (0.045% malachite green hydrochloride, 4.2% ammonium molybdate in 4 N HCI, 3:1, mixed with 100 pl of 2% Tween-20 and 50 ~ 1 2 % SDS/5 ml) was added and the color allowed to develop for a t least 45 min before reading the OD of the samples a t 660 nm in a Carey 1 Spectrophotometer using 50-pl microcuvettes. The total phosphate of the samples was determined by means of a standard curve containing 0.05-1.0 nmol of phosphate.

Amino acid residues were numbered according to the largest tau isoform, tau 441 (14).

RESULTS

The Sedimentable Non-PHF Tau from AD Brain Contains Both Abnormally Phosphorylated and Apparently Normal Spe- cies-There are four pools of tau in AD brain, the PHF, the AD P-tau, the AD tau, and the c-tau. Both PHF and AD P- tau are abnormally phosphorylated at Ser-199/202 so that they require dephosphorylation to react with Tau-1 antibody; the AD P-tau is soluble and not polymerized into PHF. In contrast AD tau and c-tau are not phosphorylated at Ser-199/ 202 and are labeled by this antibody without any pretreatment of the protein with alkaline phosphatase. Most of the PHF are present as neurofibrillary tangles and sediment at 27,000 x g, whereas AD P-tau and AD tau sediment at 27,000 to 200,000 x g. The c-tau does not sediment even at 200,000 X g. The abnormally phosphorylated tau migrates slower than normal tau on SDS-polyacrylamide gels. A major considera- tion in isolation of non-PHF tau from AD brain is to avoid contamination with PHF, the neurofibrillary tangle pool of abnormally phosphorylated tau. Therefore, to minimize dis-

ruption and fragmentation of the tangles and neuropil threads, the tissue was homogenized with a Teflon glass homogenizer to create minimal shear forces. As a result, most tangles and neuropil threads in the homogenate were sedimented at 27,000 x g, as tested by light microscopy of the pellet and the supernatant after staining with Congo Red (data not shown); Congo Red-stained tangles produce green birefringence in polarized light. On further sedimentation of the 27,000 X g supernatant at 200,000 x g, congophilic material in the pellet was undetectable. The electron microscopic analysis of the negatively stained 200,000 X g pellet at different dilutions showed mostly membranes and amorphous granular material and only a few PHF or PHF fragments (data not shown). However, analysis of the 200,000 X g pellets on immunoblots with antiserum 92e to tau revealed significant levels of tau in the pellet. A similar staining pattern was also observed with mAb Tau-1 to tau; however, in the case of this antibody, maximal staining was achieved only after dephosphorylation of the blots with alkaline phosphatase before application of the antibody (Fig. la) , indicating the presence of abnormally phosphorylated non-PHF tau (AD P-tau).

Comparison of the 200,000 x g supernatants and pellets on immunoblots revealed that AD P-tau was almost entirely in the pellet, whereas most of the apparently normal tau was in

I; 97.4- ? 8,

vi 68-

' a

43-

25.7- w

1 a.4- *- 14.3-

a Tau-1 b *- Tau-1 FIG. 1. Immunoblot analysis of the 27,000 to 200,000 X g

pellet and supernatant of AD brain homogenate and comparison of the urea-soluble and -insoluble fractions of the pellet with PHF. The protein samples were electrophoresed on a 80 X 60 X 0.75-mm SDS-polyacrylamide gel (5-15% acrylamide gradient) and transferred to Immobilon. The blot was developed with mAb Tau-1 and the avidin-biotin complex (ABC) system from Vetor, CA. 200K S, 200,000 X g supernatant (16.5 pg); 200K P, 27,000 to 200,000 X g pellet (5 pg); urea S, urea-soluble fraction of the 200K pellet (1.5 pg); urea P , urea-insoluble pellet (6 pg). *, alkaline phosphatase treatment of the immunoblot before antibody application. a, the abnormally phosphorylated tau (AD P-tau) is found in the 200,000 X g pellet and is soluble in 8 M urea. Tau that is not phosphorylated at the Tau-1 epitope is also present in the 27,000 to 200,000 X g pellet and the 200,000 X g supernatant. The slight increase of the tau staining in the supernatant after dephosphorylation seen in the figure was ex- perimental variation and was not reproducible. b, the urea-pellet fraction shows Tau-1 immunoreactivity similar to the PHF pattern, with tau at the top of the gel and in the spacer gel, whereas the urea- soluble material is devoid of these large tau aggregates. The level of urea-insoluble Tau-1 resctivity was variable from AD brain to brain. Numbers at the left indicate the positions of prestained M , markers from top to bottom. Phosphorylase b,97,400 (100,600); bovine serum albumin, 68,000 (71,200); ovalbumin, 43,000 (43,500); cu-chymotrypsinogen 25,700 (27,000); 8-lactoglobulin, 18,400 (18,300); lysozyme, 14,300 (15,000). Numbers in parenthesis represent the apparent M , of these prestained proteins determined by the manufacturer.


the supernatant (Fig. la). In addition, scans of the untreated and dephosphorylated immunoblots developed with mAb Tau-1 revealed that some preparations also contained varying amounts of sedimentable tau that did not require dephosphorylation to be recognized by mAb Tau-1 (AD tau). Initially, this procedure was based on a 27,000 to 100,000 X g centrifugation step. In subsequent studies, the l ~ , O O O x g spin was increased to 2 ~ , 0 0 0 X g because the higher centrifugal force increased the yield of the pelleted AD P-tau by approximately 60% (data not shown). The presence of AD tau in the pellets varied widely from case to case and also from area to area, ranging from undetectable in two cases (three preparations) to a maximum of 40% of the total tau in one case.

Analysis of the 2 7 , O ~ to 2 ~ , 0 0 0 X g pellets of 15 AD and eight control cases on immunoblots revealed that in 14 of the AD cases the pellets contained AD P-tau whereas no AD P- tau and only traces of AD tau were detected in the identically processed tissue from the control cases.

Radioimmunoassays of the 200,000 x g cytosols from five AD and three control cases showed that this fraction from AD brains contained on the average about half as much tau as in the identically treated control brains (Table I).

AD P-tau and AD Tau Are Soluble in Urea and Can Be Renatured in Buffer-The sedimentation o f tau from AD brain in the 200,000 x g pellet suggested that the protein in this fraction might be in large aggregates. On extraction of the pellet with 8 M urea, most of the protein went into solution. Analysis on Western blots of the urea-soluble and -insoluble material showed that both fractions contained abnormally phosphorylated tau; however, whereas the urea- soluble tau fraction consisted mostly of the characteristic tau bands, a significant portion of the tau reactivity of the insoluble fraction was at the top of the spacer and the resolving gels, reminiscent of the tau immunoreactivity pattern of PHF (Fig. Ib). Furthermore, ubiquitin immunoreactivity was only detected in the urea-insoluble pellet (data not shown). Nega- tive stain electron microscopy revealed that the urea-insoluble sample contained membranes and some PHF, whereas the urea-soluble fraction was devoid of any structural components (data not shown). After dialysis of the urea-soluble fraction against 10 mM Tris, pH 7.6, appro~imately 70% of the protein precipitated, whereas most of the tau remained in solution (Table 11).

How Much of the AD P-tau in the 27,000 to 200,000 X g Fraction Is Derived from PHF?-To investigate the contribution of PHF to the AD P-tau fraction, the 27,000 to 200,000 X g fraction of AD cerebral cortex was separated on a discontinuous 1.25-2.5 M sucrose gradient into seven distinct layers. Approximately 80% of the total protein applied was recovered from the sucrose gradient. Analysis by negative stain electron microscopy revealed that the major structures were mem-

TABLE I Tau levels in cytosol of AD and control brain h o m o g e ~ ~ e s

The 200,000 X g supernatant of brain homogenate was assayed by radioimmunoslot blot using mAb Tau-1 as described under “Materials and Methods.” Protein amounts between 20-100 ng were analyzed in triplicates.

AD Control ng ~ a ~ ~ ~ p ~ o t e ; n

5 36 13 11 8 25

18 43 15 38

Mean f S.D. 12 2 5” 31 f 13“ ‘ p < 0.02 (two-tailed t test).

TABLE 11 Purifica~~on of AD P-tau and AD tau from cerebral cortex

The protein values are stated as mean k S.E. Numbers in paren- theses indicate the number of brains used.

AD P-tau fraction Protein yield ”-

mg f 100 g tissue

Homogenate“ 6,992 Jt 939 (6) 27,000 X g supernatant* 2,148 f 292 (6) 27,000-200,000 X g pellet‘ 181 f 75 (3) UrealTris-solubled 49 f 8 (8) pH 2.7-soluble‘ 17 f 9 (5) PC, AD P-tau’ 0.8 f 0.3 (5) PC, AD tau 2.1 (1)

a Brains were homogenized in the presence of protease, kinase, and phosphatase inhibitors as described under “Materials and Methods.”

‘The cell debris, tangles, and plaque amyloid were removed by centrifugation at 27,000 X g for 20 min.

The 27,000 X g supernatant was centrifuged at 200,000 X g for 45 min. Almost all the AD P-tau sedimented as the pellet.

The 27,000 to 200,000 X g pellet was extracted with 8 M urea at room temperature for 1 h, renatured by dialysis against 10 mM Tris, pH 7.6, and centrifuged at 334,000 X g. Almost all tau reactivity remained in the supernatant.

e The Tris-soluble fraction was dialyzed overnight against pH 2.7 buffer and centrifuged at 334,000 X g for 45 min. Most tau reactivity remained in the supernatant.

’AD P-tau and AD tau were further purified and separated from each other by chromatography on PC (see Fig. 5, a and b) .

branes (mainly in layers 1 and 2), amorphous granular material (in layers 3-7), and short PHF with one to three twists (Fig. 2). Counts of PHF observed by negative stain electron microscopy (Fig. 3) revealed that most of the PHF were present in layer 5 at the interphase of 1.75 and 2.0 M sucrose and in layer 7 at the bottom of the gradient (57 and 25%, respectively, of the total PHF mass) (Table 111). However, only about 35% of the total abnormally phosphorylated tau was present in these layers, as shown by densitometric meas- urements on immunoblots developed with the mAb Tau-1 with and without prior dephosphorylation of the blots. In contrast, 54% of the abnormal tau was in sucrose layers 2-4, which on the ultrastructural level contained mainly amorphous material and membranes and only a small proportion of the PHF (11%).

Since AD P-tau was isolated from the urea extract of the 27,000 to 200,000 X g fraction of brain homogenate, some of the PHF might have dissolved and become the source of the AD P-tau. In order to estimate this contribution of PHF to the AD P-tau preparation, layers 5 and 7, which contained 82% of the total PHF mass, were combined, extracted with 8 M urea or water, and centrifuged at 334,000 X g. Electron microscopic analysis revealed that the PHF and all other structural components were in the pellet which had been extracted with 8 M urea or water. Slot-blot analysis showed that 68% of the tau immunoreactivity was in the urea extract compared with 8% tau immunoreactivity in the water extract. This indicated that maximally 60% of the total tau of the sucrose layers 5 and 7 might have been derived from PHF. These findings were in agreement with the protein assays, which also revealed 61% more protein in the urea extract than in the water extract of the PHF-enriched fractions. According to estimates based on electron microscopic and immunoblot data, sucrose layers 5 and 7 contained 82% of the total PHF, 27% of the total tau, and 35% of the abnormally phosphorylated tau (Table 111). Accordingly, 100% PHF, i.e. PHF present in layers 1-7 of the gradient, would account for maximally 27 X 100/82 = 33% of the total tau and 35 X 100/82 = 43% of the abnormally phosphorylated tau. However, because only 60% of the PHF dissolved in urea,

24378 Unpolymerized Abnormal Tau from Alzheimer Disease Brain 7 .,-ffm mql 3. 1 ITg- .: "., ~

I

I

0 , I 0 20 40 60 80 100

% sample concentration

FIG. 3. Relationship between sample dilution and PHF twists counted. Several dilutions of layers which contained the highest number of PHF were made in a final concentration of 0.8 M sucrose, 50 mM sodium phosphate, pH 7.4. Two grids each were prepared of each dilution and the PHF twists counted in represent- ative fields ranging from three to seven for each sample. The decrease in percent PHF twists is proportional to sample dilution.

TABLE 111 Separation of tau and PHF by discontinuous sucrose gradient

centrifugation The 27,000 to 200,000 X g pellet of an AD brain homogenate was

suspended in water, layered on a 1.25 to 2.50 M discontinuous sucrose gradient, spun a t 225,000 X g for 16 h (9) and separated into seven fractions. In each of the fractions, protein concentrations, number of PHF twists, total tau, and AD P-tau levels were estimated. The values given in each of the columns represent the percentages of the total sum of the values for fractions 1-7.

Fraction Sucrose Protein % PHF % total % ADP no. mass' taub P-tau' .. . . ~~

M % 1 0 7 2 7 7 2 0-1.25 33 NDd 22 12 3 1.25 40 4 19 16 4 1.25-1.75 12 7 22 26 5 1.75-2.00 4 57 21 28 6 2.00 1 5 3 4 7 2.25-2.50 3 25 6 7

PHF mass was determined by counting the number of PHF twists in a standard area of micrographs a t ~50,000 magnification.

* Levels of total tau were estimated from scans of immunoblots, which had been dephosphorylated before incubation with mAb Tau- 1.

Levels of AD P-tau were estimated from the differences of the densitometry values of samples developed with mAb Tau-1 on the untreated blots and those of the corresponding values obtained with the dephosphorylated blots.

ND, not detected.

FIG. 2. Electron microscopic analysis of sucrose gradient fractions of the 27,000 to 200,000 X g pellet from AD brain homogenate. The 27,000 to 200,000 X g pellet was suspended in water and layered onto a discontinuous sucrose gradient of 1.25-2.5 M sucrose, centrifuged for 16 h at 175,000 X g, and the resulting seven distinct layers aspirated into separate tubes (see Table 111). Samples were negatively stained and viewed at a magnification of X 50,000. The major components in the 200,000 X g pellet were membranes (mainly in layers 1 and 2) and amorphous material (layers 3-7) and some short PHF. The majority of PHF were present in layers 5 and 7. a, layer 2 showing the presence of mostly membranes; b, layer 4 showing amorphous material and membranes; c, sucrose layer 5 showing PHF, amorphous material and some polysomes (arrow- heads).

at most 33 X60/100 = 20% of the total tau and 43 X 60/lOO = 26% of the AD P-tau of the urea extract is calculated to have been derived from PHF. Thus, the remaining 74% of the AD P-tau in the urea extract most probably represents the

non-PHF pool of abnormally phosphorylated tau. Purification and Separation of AD P-tau and AD Tau-AD

P-tau reacts with the mAb Tau-1 only after treatment with alkaline phosphatase whereas AD tau does not require this dephosphorylation step. Thus, the two forms of tau are distin- guished on the basis of their Tau-1 reactivity. The urea- soluble tau fraction was fractionated by PC column chromatography. This step separated the AD tau from the AD P-tau as tested by their reactivity with mAb Tau-1. The AD P-tau preparations were highly enriched, as shown by comparison of the immunoblots with the adjacent Coomassie Blue-stained blots (Fig. 4). Most polypeptides in the AD P-tau fractions reacted with tau antibodies (92e, and Tau-1 after dephosphorylation); the immunoreactivities to amyloid p peptide, neurofilament L subunit, microtubule-associated protein 2, and a-tubulin were negative, as was in most cases, the GFAP immunoreactivity (figure not shown). Only one preparation


c tau AD

ab c = I

c ab

....

FIG. 4. Analysis of Phosphocellulose-purified AD P-tau and c-tau from AD and control brains on blots stained with Coo- massie Blue and with antiserum to tau. Samples were electrophoresed as described in Fig. l a and transferred to Immobilon. Half of the blot was stained with Coomassie Blue (c); the other half was immunolabeled with antiserum 92e to tau (ab) . Not shown in this figure, similar patterns were obtained when blots were dephosphorylated and stained with mAb Tau-1. 8 and 2 r g of protein/lane were applied for the Coomassie Blue and immunostaining, respectively. Panel at right shows the immunoblot pattern of the same tau preparations run on adjacent lanes of the same gel. Lines at the left indicate from top to bottom, the positions of prestained M, markers, myosin (H-chain), 200,000 (208,100), phosphorylase b, bovine serum albumin, ovalbumin, a-chymotrypsinogen, /3-lactoglobulin, and lysozyme. For M, of the markers other than myosin, see legend to Fig. 1. The tau- positive polypeptides of around 75,000 seen in cytosolic tau from control brain are frequently observed, especially in overloaded blots. Not shown in this figure, immunoblots with mAb Tau-1 have revealed that these polypeptides are not abnormally phosphorylated.

contained significant amounts of GFAP-reactive polypeptides which were removed by consecutive affinity chromatography. Fig. 5, a and d, show the elution profile of the PC chromatography and the immunoblot of the corresponding PC fractions developed with mAb Tau-1. In this particular preparation, no tau immunoreactivity was detected in any of the PC fractions on blots that were not dephosphorylated, indicating that practically all tau was in the form of AD P-tau. The elution profile shows a major tau peak between 0.10 M and 0.30 M NaCl and a minor peak between 0.36 M and 0.60 M NaC1, indicating the presence of a wide variety of differently charged tau species that are all abnormally phosphorylated. As demonstrated by the corresponding immunoblot, the first tau polypeptides that elute are the molecular species with the slowest electrophoretic mobility.

In preparations that also contained AD tau in addition to AD P-tau, the two forms of tau were separated by PC chromatography, whereby the AD P-tau eluted first, with a peak between 0.10 M and 0.28 M NaCl, and the AD tau last, with a peak between 0.31 M and 0.80 M NaCl (Fig. 5, b and e). Again, the elution profile showed several tau peaks over a wide range of NaCl concentration in the eluting buffer, indicating a large variety of differently charged AD tau species. The AD P-tau eluting before the AD tau was not cross-contaminated with AD tau. The AD tau fractions contained some contaminants below 40 kDa, most of which were removed by heat treatment at pH 2.7, and some unidentified high M , material (data not shown).

On Western blots treated with alkaline phosphatase (Fig. 5e), a decrease in immunostaining of AD tau with Tau-1 as

compared to the untreated blots was consistently observed with each preparation, but the degree of the difference varied depending on the lot of phosphatase. This effect is most probably the result of proteases contaminating the phosphatase preparations. In contrast, the Tau-1 staining intensity of AD P-tau increased.

Cytosolic Tau (c-tau) from A D and Control Brains Is More Homogeneous in Charge Than AD P-tau and AD Tau-Cy- tosolic tau (c-tau) was purified from the 200,000 x g supernatants of brain homogenates (Table IV) and eluted from the PC column between 0.25 to 0.50 M NaCl (Fig. 5c), the same ionic strength as that for AD tau. Interestingly, in contrast to AD P-tau, the tau polypeptides with a faster electrophoretic mobility eluted before the tau species with a slower mobility. Furthermore, only one tau peak a t 0.35 M NaCl was detected, indicating that the tau species of c-tau are less variable than those of AD P-tau and AD tau, with respect to charge. The corresponding immunoblot (Fig. Sf) shows no abnormally phosphorylated tau species. A 70-kDa polypeptide present in some preparations as a contaminant could be removed by heating the tau-enriched fraction at pH 2.7 (data not shown). Tau isolated from the 200,000 X g supernatants of AD brain homogenates was electrophoretically and immunochemically indistinguishable from the cytosolic tau from control brains (Fig. 4).

Most AD P-tau and AD tau preparations as well as the cytosolic tau from AD and control brains also contained besides the major tau bands in the 45-70 kDa region, varying amounts of a number of tau-reactive bands in areas corresponding to M , lower than 45,000 and as well as higher than 70,000. These bands co-purified with the major tau polypeptides but varied both in number and in relative amounts from brain to brain (compare Figs. 4-6).

AD P-tau Is Hyperphosphorylated-The total phosphate analysis revealed about 3-4-fold as much phosphate for the AD P-tau as for the cytosolic tau (Table V). The phosphate levels of the cytosolic tau from AD and control brains were approximately the same.

The position of hyperphosphorylation sites on AD P-tau in addition to those a t Ser-199/202 at the Tau-1 epitope (26) were probed with antibodies 102c and SMI 31. Rabbit antibody 102c was raised to peptide l a of bovine tau (20) which has a continuous sequence homology to amino acid residues 42-49 of human tau and preferentially reacts with tau that is not phosphorylated a t Ser-46. mAb SMI 31 is an antibody to phosphorylated neurofilament subunit polypeptides P200 and P150 and cross-reacts with tau polypeptides phosphorylated a t both Ser-396 and Ser-404, but not with normal tau (43). On immunoblots of AD P-tau, antibody 102c labeled the two slowest moving polypeptides of approximately 67 & 1 kDa (mean & S.E., three blots) and 70 & 1 kDa (four blots) (Fig. 6). Like in the case of PHF, the immunostaining was strongly enhanced when the blot was dephosphorylated prior to immunolabeling. None of the other Tau-1-positive tau species present in the AD P-tau and PHF preparations were labeled by this antibody. On normal human and bovine tau, the immunolabeling pattern of antibody 102c was very similar to that of mAb Tau-1 (data not shown here, see Ref. 19). mAb SMI 31, like Tau-1 and 102c, labeled both AD P-tau and PHF polypeptides. Like mAb Tau-1, antibody SMI 31 seemed to label all tau isoforms. This antibody did not label normal tau (data not shown). Not shown here, AD P-tau and PHF are also strongly labeled on immunoblots with mAb PHF-1 which recognizes the phosphorylation site at Ser-396 (44) and mAb SMI 34 which recognizes a phosphorylation-dependent con-

24380 Unpolymerized Abnormal

I

rl! 3

a

I T g! s rl! 3

b

.04

4000

.02

.o 1

0 0 0 .2 .4 .6 .8

NaCl [MI lo000

El ADP-tau 0.25

0 I

e

0 .2 .4 .6 .8

Tau from Alzheimer Disease Brain

M NaCl

0.12 0.27 "

0.4 1 kDa *

68-

43- 25.7-

68-

43-

25.7-

d

M NaCl

0.17 0.28 0.40 I I

kDa L

~ i ~ - r ,r- .=- - .

68--1 -3t.

43 - 25.7 -

68 - 43 -

25.7 - e

M NaCl

0.28 0.35 0.42

I

rl! 3

t 0

kDa -

+e 68 - 43 -

25.7 - 68 - 43 -

25.7-

C 0 .2 .4 .6 .8

NaCl [MI

FIG. 5

U ~ ~ o ~ y m e r ~ z e ~ A b ~ o r ~ a l Tau from A ~ z ~ e i m e r Disease Bruin 24381

TABLE Iv Purification of cytosolic tau (c-tau) from control cerebral cortex

Fraction Protein ~~~~~g tissue

Homogenate” 8,040 + 1,222 (4) 27,000 X g supernatantb 2,019 rt 635 (2) 200,000 X g supernatant‘ 1,920 + 152 (3) 3545% (NH4)2S04 pelletd 409 (2) PC“ 7 + 4 (3) aH 2.7.95 “C soluble‘ 3 (1)

a Brains were homogenized in buffer containing proteases, kinases, and phosphatase inhibitors.

Debris was removed by centri~gation at 27,000 X g for 20 min, as described under “Materials and Methods.”

The 27,000 X g supernatant was centrifuged at 200,000 X g for 45 min.

Most of the tau reactivity was obtained in the 35-45% (NHa)2S04 fraction of the 2 ~ , 0 # X g supernatant.

e The ammonium sulfate fraction, after analysis was subjected to PC chromatography. The pool of the fractions eluting from the column between 0.25 and 0.50 M NaCl contained most of the tau activity.

’The PC tau fraction was dialyzed against pH 2.7 buffer (8), and heated at 95 “C for 5 min.

TABLE v P ~ s p h a t e content of purified tau from AD and control brains

The abbreviations used are: PMI, postmortem interval; HD, Hun- tington disease; hypox., hypoxia. ND, not determined. ... -

NO. Disease Age Sex PMI Total phosphate‘

c-taub AD P-tau‘ h mol P f mof proteind

1 AD 78 M 6.5 1.78 5.12 2 AD 60 M 1.5 2.27 8.95 3 AD 81 F 3.3 2.07 8.68 4 AD 70 M 4.0 ND 7.15

Alzheimer disease cases

5 AD 83 F 3.8 ND 7.97 Mean f S.D. 2.04 f 0.25 7.57 2 1.54

Control cases 1 HD 64 F 2.3 1.78 2 3

Hypox. 63 M 6.4 3.59 HD 51 M 3.9 2.15

4 HD 59 M 4.0 3.80 Mean f S.D. 2.83 -C 1.04 Each number represents the mean of three assays.

homogenates and eluted from PC at 0.25-0.50 M NaC1. ‘ Cytosolic tau isolated from the 200 ,O~ X g supernatant of brain

AD P-tau eluted from the PC column at 0.09-0.15 mM NaCI. Based on the average M, 41,318 of the six tau isoforms (42).

formational epitope including the regions flanking the microtubule binding repeats (43).

U ~ p Q ~ y r n e ~ ~ z ~ d A ~ n Q r r n a l ~ ~ P ~ ~ p ~ ~ ~ t e d Tau Is Not Ubi- quitinated-All PHF I1 preparations studied by us to date contain ubiquitin-positive polypeptide bands with very similar migration patterns as those of the PHF tau on SDS-polyacrylamide gel electrophoresis (45). In contrast, the AD P-tau

preparations showed no immunoreactivity on Western blots developed with 3-39 and 5-25 to PHF-ubiquitin (figure not shown here). ~ r the rmore , the comparison of AD P-tau and PHF on slot blots, which were manyfold more sensitive than immunoblots, did not show any ubiquitin immunoreactivity in the AD P-tau and AD tau preparations (Fig. 7). Ubiquitin reactivity was, however, detected in the urea-insoluble fraction of some but not all AD brains indicating the presence of some PHF I1 (data not shown).

DISCUSSION

Tau in AD brain is abnormally hyperphosphorylated and it is in this form that tau is a major protein subunit of PHF. One of the most important questions toward understanding the pathogenesis of Alzheimer neurofibrillary degeneration is whether tau becomes h~e~hosphory la t ed prior to or after its pol~erizat ion into PHF. Previously, immunocytochemical studies have identified abnormally phosphorylated tau in stage “0 tangles” (23) suggesting that abnormal phosphorylation of tau might precede the formation of neurofibrillary tangles. The present study demonstrates biochemically, for the first time, that the abnormal phosphorylation of tau occurs in the cytosol in AD brain.

Previously, we had demonstrated the presence of abnormally phosphorylated tau in fresh AD brains processed for temperature-dependent assembly of microtubules (25). The subsequent search by electron microscopy for PHF in the microtubule fraction turned out to be negative. The existence of abnormally phosphorylated tau in unpolymerized form in the AD brain was thus likely. In the present study we observed that practically all abnormally phosphorylated tau and approximately half of the non-hyperphosphorylated tau of AD brain homogenates sedimented at 200,000 x g and that most of the tau of the 27,000 to 200,000 X g fraction could be rendered water soluble by treatment with urea and subsequent dialysis against buffer. The resulting tau remained in solution and could be fractionated by phosphocellulose chromatography into abnormally phosphorylated and apparently normal isoforms. The homogenization conditions were chosen to keep the disruption of tangles and neuronal processes at a mini- mum; these conditions involved minimal shearing forces, low salt concentration, and no re-extraction of the 27,000 X g tangles-containing pellet. This is probably the reason why only relatively few PHF fragments were detected in the 27,000 to 200,000 x g pellet, and less than 30% of the total tau in the AD P-tau preparations was found to be derived from PHF.

The PHF fragments in the tau preparations in the present study might have been derived from neuropil threads or the earlier stages of tangle formation. PHF at this stage are less modified and probably more soluble than the PHF at a more advanced stage of tangle development. Previously we demonstrated that, in addition to the sparingly soluble PHF, the PHF 11, the AD brain contains a population of PHF, the PHF I, that are readily soluble in SDS at room temperature (5). The present study demonstrates that at least a portion of the

FIG. 5. PC chromatography elution profiles of tau from AD and control brains and corresponding Western blots of the individual fractions. Tau-enriched fractions from two different AD brains (panek a and b ) and a control brain (panel c ) were applied to the PC columns at 2-3 mg of protein/ml PC as described under “Materials and Methods.” The bound protein was eluted with a linear 0-1 M NaCl gradient. a-c, levels of tau (0) were analyzed densitometrically in 0.5-1-pl aliquots of the individual fractions by immunoslot blot using anti-tau serum 92e; 0 represents the ODzm of the fractions. d-f, equal amounts of protein from the individual PC fractions were applied to each lane of the corresponding Western blots (d, 0.5 pgjlane; e and f, 1.0 pg/lane). Western blots were developed with mAb Tau-1 with (*) or without prior treatment with alkaline phosphatase ( ~ ~ e r p a n e ~ ) as described in Fig. 1. At the left are indicated the positions of the prestained M, markers: bovine serum albumin, ovalbumin, and ~ - c h y m o t ~ s ~ n . For M, of the markers, see legend to Fig. 1. The decrease in Tau-I immunos~ining of AD tau (panel e ) after pretreatment of the blots with alkaline phosphatase was observed in all AD tau preparations and is most likely due to loss of the polypeptides by digestion with proteases contaminating the phosphatase.

24382 Unpolymerized Abnormal Tau from Alzheimer Disease Brain

PHF I is even soluble in urea and can be renatured by dialysis against Tris buffer. PHF I, in apparently much higher numbers than in the present study, have been previously isolated by others from the 27,200 to 100,000 X g supernatant of A D brain homogenates (6, 9, 46). However, the two approaches might not be directly comparable since in the previous studies the homogenization buffer contained relatively high concentrations of sodium chloride (SO-SO0 mrd) and the 27,000 x g pellets were re-extracted with the homogenization buffer,

kDa

97.4 - 68 -

43 - 257 - * - - -

102c Taw1 SMI 31

FIG. 6. Sites of hyperphosphorylation on AD P-tau and PHF-tau. AD P-tau (2 pg) and PHF (4 pg) were electrophoresed as described in Fig. la, transferred to Immobilon and immunolabeled with antibodies 102c, Tau-1, and SMI 31. * indicates blots that were treated with alkaline phosphatase prior to the incubation with the antibodies. The positions of the prestained M, markers indicated are from top to bottom: phosphorylase b, bovine serum albumin, ovalbumin, a-chymotrypsinogen, P-lactoglobulin, and lysozyme. For M, of the markers, see legend to Fig. 1. Antibody 102c recognizes amino acid sequence 42-49 of human tau and preferentially reacts with tau that is not phosphorylated at Ser-46. Similarly, mAb Tau-1 only reacts with tau that is not phosphorylated at Ser-199/202. Both antibodies react strongly with AD P-tau and PHF tau after dephosphorylation of the blot (*). The mAb SMI 31 labels tau phosphorylated a t Ser-396 and Ser-404 and reacts on blots with AD P-tau and P H F without prior dephosphorylation.

FIG. 7. Levels of ubiquitin immunoreactivity in AD P-tau, AD tau, and PHF. P H F I1 preparation (H) and PC-purified AD P-tau (A) and AD tau (0) in amounts ranging from 0.2 to 80 pg were applied to nitrocellulose membranes using a slot-blot apparatus. The blots were incubated with a mixture of mAb 5-25 and 3-39, and bound antibody was detected with alkaline phosphatase- conjugated secondary antibody and 5-bromo-4-chloro-3-indolylphosphate. Since both antibodies preferentially rec- ognize conjugated ubiquitin, levels of ubiquitin were expressed as area units of the scans. Only P H F had ubiquitin. No ubiquitin activity was detected in AD P- tau and AD tau.

conditions which might lead to the enrichment of PHF protein in the supernatant. The percentage of A D P-tau in the previous preparations was not reported.

Analysis of the total phosphate content showed that the A D P-tau preparations contained between 5-9 mol of phosphate/mol of tau. Similar phosphate contents varying between 5-10 mol were also observed in PHF preparations by Ksiezak- Reding et al. (47). Even larger variations between phosphate contents of 3-15 were observed by the same authors in the perchloric acid/mercaptoethanol heat extract of the 27,000 to 200,000 X g pellets of A D brain homogenates (47). However, the absence of Coomassie Blue or silver-stained protein patterns of these preparations make it difficult to evaluate their degree of purification. As of to date, the combined studies from different laboratories indicate that tau derived from PHF I contains a t least 10 mol of phosphate. Seven to eight phosphate molecules have been demonstrated by protein sequence and mass spectrometric analysis (22), two additional phosphorylation sites at Ser-46 and Thr-123 were identified by immunochemical and immunocytochemical analysis (20, 21). It is not known whether the large variations in phosphate content of the A D P-tau preparations are due to individual variations or to differences in the degree of dephosphorylation caused in uitro during fractionation of the tissue. Immunoblot analysis showed common phosphorylation sites between A D P-tau and PHF-tau at the amino- and carboxyl-terminal end regions as well as in the middle part of the tau molecule. In contrast to A D P-tau, tau isolated from the 200,000 X g supernatants of A D and control brains contained approximately 2 and 3 mol of tau, respectively, suggesting that the phosphorylation state of the cytosolic tau from A D and control brain might be similar. The phosphorylation state of cytosolic tau is as yet not fully elucidated. However, one phosphate has been detected by Hasegawa et al. (22) by mass spectrometry in tau from both AD and control brains and found to be located at the COOH-terminal end of tau. Simi- larly, the phosphate content determined by Ksiezak-Reding et al. (47) was 2.6 and 1.9 mol for the cytosolic tau from A D and normal brains, respectively.

That the slow-moving doublet of A D P-tau is of the highest negative charge, possibly due to hyperphosphorylation, is indicated by its elution from PC at only 0.1 M NaCl. The

* AD P-tau - ADtau

.1 1 10 Protein [ng]

Unpolymerized Abnormal Tau from Alzheimer Disease Bruin 24383

other isoforms of AD P-tau, which elute after the doublet at higher ionic strength, but before the AD tau and c-tau, might be of various intermediate phosphorylation states. At this time, we cannot explain the elution pattern on PC of the AD P-tau preparations derived from brains in which no AD tau was present as analyzed with mAb Tau-1 (Fig. sa). In these preparations, a part of the AD P-tau eluted in the range of the tau species with normal phosphate content. This may be a reflection of an array of tau species that are phosphorylated to different degrees. In addition, the influence of other as yet not defined post-translational modifications on tau's charge or hydrophobicity might also affect its elution characteristics.

Previously it had been demonstrated that in AD brain homogenate the levels of tau which was not abnormally phosphorylated at Ser-1991202 were normal (37). The results of the present study showing that the levels of cytosolic tau are decreased in AD, indicate that at least a part of the Tau-1 reactive tau in the homogenate might be the sedimentable AD tau. Both, abnormal phosphorylation in the form of AD P-tau, and reduced levels of cytosolic tau, probably contribute to the breakdown of the microtubule system reported previously in AD brain (25, 48, 49).

The present study has also revealed that unlike normal brain tau, a significant pool of AD brain tau sediments at 200,000 X g, suggesting that tau in affected neurons might be present in an abnormal biophysical state. The phosphate level of one AD tau preparation analyzed was similar (1.7 mol/mol of tau) to that of the cytosolic tau' suggesting that the former might not be h~erphosphorylated. Thus, the presence of AD tau species, which are altered in an as yet unknown way, might be indicated. It remains to be elucidated whether AD tau was derived from AD P-tau through dephosphorylation during postmortem autolysis and homogenization of the tissue or whether it might represent the precursor of AD P-tau. Recently, we have observed that normal tau binds to AD P- tau, and the levels of AD tau in the 27,000 to 200,000 x g pellet correlate directly to the levels of AD P-tau in this f ra~t ion .~ Thus, the elevated AD tau levels in the 200,000 x g pellet observed in the present study might be due to this binding to the AD P-tau. The binding between AD tau and AD P-tau might also occur in affected neurons in situ. Whether the altered se~mentation characteristics of AD P- tau and AD tau might be the result of their binding to each other and/or their association with ribosomes or membra- neous material remains to be determined.

In conclusion, the following line of events occurring in the AD brain might be envisaged. Because of its altered biophysical characteristics, the abnormal tau accumulates within the neurons, however, only the highly phosphorylated AD P-tau might be resistant to proteolysis and form stable deposits. The stabilizing effect of phosphorylation against proteolysis has been shown in the case of a variety of polypeptides, including tau (50-54). Similarly, results in this study show that AD P-tau is more resistant than AD tau to the contaminating proteases of alkaline phosphatase preparations. This is most probably the reason why it is mainly AD P-tau which forms the amorphous deposits of the stage 0 tangles previously observed by immunocytochemical studies (23, 55). However, the step from the amorphous tau deposits to the formation of the PHF remains elusive, and it still must be established whether the AD P-tau directly polymerizes into PHF or requires further modifications or an additional factor or fac-

E. Kopke, Y . 4 . Tung, S. Shaikh, A. del C. Alonso, K. Iqbal, and

A. del C. Alonso, T. Zaidi, I. Grundke-Iqbal, and K. Iqba1, man- I. Grundke-Iqbal, unpublished data.

uscript in preparation.

tors. The presence of ubiquitin in PHF 11, and its absence in AD P-tau, and as well as PHF I suggests that the role of the ubiquitin system in Alzheimer neurofibrillary degeneration is a late event. Ubiquitin does not appear to be involved in the polymerization of AD P-tau into PHF, but probably increases the stability of the neurofibrillary tangles by forming stable complexes with already formed PHF.

Acknowledgments-We thank the Netherlands Brain Bank (NBB), Amsterdam (Coordinator Dr. R. Ravid), the Canadian Brain Tissue Bank, Toronto, Canada, and the Brain Tissue Resource Center, McLean Hospital, Belmont, MA, for supplying the Alzheimer disease and control brains; Dr. L. I. Binder, University of Alabama, Birming- ham, AL, S. Greenberg, W. M. Burke Medical Research Institute, White Plains, NY, and K. S. Kim of the NYS Institute for Basic Research, Staten Island, NY, for generously supplying antibodies Tau-1, AP14, and AP18, PHF-1, and 2F9, respectively; T. Zaidi for isolating PHF; the Biomedical Photography Unit for phetography; M. Stoddard-Marlow for editorial suggestions; J. Maffei, K. Case and J. Lopez for typing the manuscript.

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