Quantitative analysis of tau protein in paired helical filament preparations: Implications for the role of tau protein phosphorylation in PHF assembly in Alzheimer's disease

Neurobiology of Aging, Vol. 16, No. 3, pp. 409-431, 1995 Copyright © 1995 Elsevier Science Ltd. Printed in the USA. All rights reserved 0197-4580/95 $9.50 + .00

Pergamon 0197-4580(95)00037

Quantitative Analysis of Tau Protein in Paired Helical Filament Preparations: Implications for the Role of Tau Protein Phosphorylation in PHF Assembly in Alzheimer's Disease C L A U D E M . W I S C H I K , * I " ¶ 1 P A T R I C I A C . E D W A R D S , * ¶ R O B E R T Y. K. L A I , * ¶ H E R M A N N . - J . G E R T Z , # J O H N H . X U E R E B , * § E U G E N E S. P A Y K E L , t C A R O L B R A Y N E , : ~ F E L I C I A A . H U P P E R T , I" E L I Z A B E T A B. M U K A E T O V A - L A D I N S K A , * ¶ RAI]L MENA, MARTIN ROTH*$ A N D C H A R L E S R. H A R R I N G T O N * ¶

*Cambridge Brain Bank Laboratory, t Departments of Psychiatry, ~Community Medicine, §Pathology, University of Cambridge Clinical School, and ¶MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, United Kingdom #Psychiatrische Klinik und Poliklinik, Abtailung far Gerontopsychiatrie, Universi!tatsklinikum Rudolf Virchow, Freie Universitiit Berlin, Berlin, Germany STrinity College, Cambridge, United Kingdom Department of Physiology and Neuroscience, CINVESTAV-IPN, Mexico WISCHIK, C. M., P. C. EDWARDS, R. Y. K. LAI, H. N.-J. GERTZ, J. H. XUEREB, E. S. PAYKEL, C. BRAYNE, F. A. HUPPERT, E. B. MUKAETOVA-LADINSKA, R. MENA, M. ROTH AND C. R. HARRINGTON. Quantitativeanalysisof

tau protein in pairedhelicalfilamentpreparations: Implicationsfor the role of tau proteinphosphorylation in PHFassembly in Alzheimer'sdisease'.NEUROBIOL AGING 16(3) 409-43 l, 1995. -- In Alzheimer's disease, there is a major redistribution of the tau protein pool from soluble to PHF-bound forms. PHF-bound tau can be distinguished from normal tau by acid reversible occlusion of a generic tau epitope in the tandem repeat region and characteristic sedimentation in the if-II protocol developed in this laboratory. We show that 85°70 of tau bound in the PHF-like configuration can be recovered in the if-II PHF-fraction. Less than 1070of this material was phosphorylated at the mAb AT8 site in aged clinical controls or in cases with minimal or mild dementia. Of tau phosphorylated at the mAb AT8 site, only 12070was found to co-sediment with PHFs. These low levels could not be explained by postmortem dephosphorylation. As more than 95070 of PHF-tau is not phosphorylated, even at early stages of pathology, it is misleading to use the terms "PHF-tau" and "phosphorylated tau" as though they were synonymous, particularly as this implies a pathogenetic role which phosphorylation need not have. Alzheimer's disease

Paired helical filaments (PHFs)

Tau protein

INTRODUCTION

Phosphorylation

A molecular mechanism which has been proposed to explain this redistribution of the tau protein pool is aberrant phosphorylation (7,9,19,22,38). It has been known for some time that some neurofibrillary tangles (NFTs) and dystrophic neuropil threads in Alzheimer's disease contain tau protein which is abnormally phosphorylated (12,29; reviewed in 32). It has also been shown that P H F s can be isolated from A D brain tissues which contain phosphorylation-dependent epitopes (4,16) and that tau proteins can be extracted from such P H F preparations which show characteristic phosphorylation-dependent shifts in gel mobility (6,10,11). However, normal tau protein is also partially phosphorylated at many of the same sites as those found in

A L Z H E I M E R ' S disease (AD) is associated with a m a j o r redistribution o f the tau protein pool from the soluble form to a form which is assembled into paired helical filaments. In frontal cortex, for example, there is a 95°7o loss of normal tau, and in temporo-parietal cortices 1:here is a 45-fold increase in tau protein incorporated into P H F ' s relative to controls (26,27). This redistribution of a protein which is essential for maintaining axonal microtubules in a polyrnerised state may underly the loss of synaptic connectivity in cortico-cortical association circuits and thus produce clinical dementia.

1Requests for reprints should be addressed to C. M. Wischik, Cambridge Brain Bank Laboratory, Department of Psychiatry, MRC Centre, Hills Road, Cambridge, CB2 2QH, United Kingdom. 409

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PHF tau (23), and more extensive phosphorylation is a normal feature of tau protein during neuronal development (30,31,33). Because brain tissues from patients dying with Alzheimer's disease can be distinguished from aged-matched controls by a substantial accumulation of phosphorylated tau (15,18), the inference has been drawn that abnormal phosphorylation of tan plays an important role in causing PHF assembly (7,22). Indeed the terms "PHF," "PHF-tau," and "phosphorylated tan" have become synonymous in many reports. However, in the absence of independent methods for measuring PHF-tau that do not depend on detection of phosphorylated antigens or alternative preparative protocols, it has been impossible to determine how much PHF-tau is phosphorylated or how much phosphorylated tau is in the form of PHFs in Alzheimer's disease. We have compared various ways of measuring pathologic tau accumulations in AD brain tissues using two commonly used preparative protocols for PHFs. The first is the A68 protocol which produces a fraction that is thought to be rich in pathologically phosphorylated tau derived exclusively from PHFs (10,21,38). The second is a protocol that was developed to maximise the total yield of PHFs irrespective of their state of phosphorylation from AD brain tissues ("if-I1 protocol," 35), which has been used extensively this laboratory (13,14,15,17,26,27,28). Two types of immunochemical measure were used for both preparations: (a) Measurement of phosphorylated tau species based either on densitometry of A68 proteins on immunoblots (6) or immunochemical reactivity in ELISA with mAb AT8 (15), an antibody which recognises tau phosphorylated at Ser-199/202 (1,8); (b) Measurements of two tau epitopes associated with the core structure of the PHK The first is an epitope located in the tandem repeat region of tau that is detected using mAb 7.51. This epitope is occluded when tan is bound in a PHF-like configuration but can be released after formic acid treatment (13,14,26,27). The second is a measure of tau C-terminally truncated at Glu-391 (detected by mAb 423, 28) which is characteristic of some of the tau found within the inner core of the PHF (13,14,26,27,28,39). METHOD

Brain Tissue Preparation Brain tissue was derived either from patients with a neuropathological diagnosis of AD at the time of postmortem examination (40) or from unselected cases coming to postmortem from a prospective epidemiological study of cognitive decline and normal aging (41). In all the experiments, tissue was thawed at room temperature and then homogenised in 0.32 M sucrose. Fifty g were homogenized in 100 ml for the bulk protocol or 2 g in 4 ml for the analytical protocol. The homogenate was divided into two equal portions, each equivalent to half the starting brain tissue. One portion was processed through the A68 protocol and the other through the if-II protocol. The A68 Protocol. This is shown in Fig. 1. An equal volume of buffer A containing 2 M NaC1, 1 mM MgC12, 2 mM EGTA, 0.32 M sucrose, 200 mM MES (pH 6.5), was added to one portion of brain homogenate. The homogenate was centrifuged in a Beckman L7 SW28 ultracentrifuge SW28 rotor at 100,000 g for 15 min (bulk protocol) or in a Beckman TLI00 ultracentrifuge TL100.3 rotor at 25,000 g for 15 min at 4°C (analytical protocol). The supernatant was removed and retained on ice. The pellet was reextracted once more with an equal volume of 0.32 M sucrose and buffer A, followed by centrifugation as above. The second supernatant was pooled with the first and sarkosyl was added to a final concentration of 1%; the mixture was incubated with gentle rotation at 25°C for 1 h. It was then centrifuged at 200,000 g for 30 min at 4°C. The A68 Supernatant 1 (Fig. 1) was

discarded. The pellet was suspended in an equivalent volume of 0.5 M sucrose and centrifuged at 200,000 g for 30 min at 4°C. The resulting A68 Supernatant 2 (Fig. 1) was retained for immunoassay and the pellet suspended with 200/A of NH4HCO 3 (50 mM; pH 8.0). The latter fraction is called the A68-tau fraction. mAb AT8 and 7.51 immunoreactivities were measured in this fraction and in A68 Supernatant 2 by competitive ELISA. The initial pellet from this procedure (normally discarded in the A68 protocol) was processed through the if-II protocol (see below) to prepare a PHF fraction. The if-IIProtocol. This is shown in Fig. 1. An equivalent volume of 0.32 M sucrose was added to 50 ml (bulk protocol) or 2 ml (analytical protocol) portions of brain homogenate in 0.32 M sucrose. The homogenate was then centrifuged in a Beckman ultracentrifuge SW 28 (bulk protocol) or SW50 (analytical protocol) rotor at 100,000 g for 1.5 h (bulk protocol) or 233,000 g for 22 min (analytical protocol) at 15°C. The supernatant, referred to as the S1 fraction, was used to prepare normal tau (14,26,27). The pellet was suspended in 0.5 M sucrose (bulk protocol) or 1 M sucrose (analytical protocol) and centrifuged at 200,000 g for 2 h (bulk protocol) or 233,000 g for 1 h (analytical protocol) at 15°C. The Supernatant 1 (Fig. 1) was retained for immunoassay. The pellet was resuspended in 0.5 M sucrose and divided into two equal portions. Pronase (2/zg/ml, final concentration) was added to half the preparation which was incubated for 1 h at 35°C; the other half was stored during this time at 4°C. The mixtures were then centrifuged at 200,000 g for 3 h (bulk protocol) or 233,000 g for 1 h (analytical protocol) at 15°C. The Supernatant 2 (Fig. 1) was retained for immunoassay, and the pellet suspended with 500/zl (bulk protocol) or 200/zl (analytical protocol) of NH4HCO 3 (50 mM, pH 8.0). This fraction was called the PHF fraction, mAb AT8 and 7.51 immunoreactivities were measured in the PHF fractions prepared from both the if-II protocol and from the initial pellet of the sarkosyl protocol as well as the Supernatants 1 and 2.

Competitive Immunoassays mAb AT8. mAb AT8 immunoreactivity was measured by competitive ELISA as in Harrington et al. (15). AT8 was used at a final concentration of 5 ng/ml. A68-tau prepared from the brain of a patient with severe AD (FD112) was used as the solid phase antigen. Neocortical grey matter tissue (10 g) was used to prepare A68-tau that was used to coat ELISA plates at a dilution of 1/200. Recombinant tau (T40, 6) was phosphorylated with rat brain kinase extract, according to Biernat et al. (1), and used as a standard for mAb AT8 immunoreactivity. The relative AT8 immunoreactivity of the brain tissue, determined by competitive ELISA, is expressed as pmoi of tau per gram (wet weight) of tissue. mAbs 7.51 and 423. Brain fractions were assayed for 7.51 immunoreactivity with or without formic acid treatment according to Mukaetova-Ladinska et al. (26,27). Recombinant human tau (dGAE; 28) was coated as the solid phase antigen at 1/xg/ml and mAb 7.51 (hybridoma supernatant) was used at 1/500. Immunoreactivity is expressed as pmol/g (wet weight) using recombinant tau (dGAE) as a standard, mAb 423 was measured in Pronase-resistant PHF-tau preparations as described in Harrington et al. (14). All results are normalised for an equivalent amount of each fraction obtained from a corresponding quantity of brain tissue. RESULTS

Preparative Protocols Two basic preparative protocols have been used to isolate tau and PHF fractions (Fig. 1). In the first we have used the if-II

M E A S U R E M E N T O F T A U IN P H F s

411

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A68 PROTOCOL

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1

PHF Fr~actionI

FIG. 1. Two basic preparative protocols have been used in this study. The first is a simplified version of the if-II protocol which has been used extensively in our laboratory as a means of preparing PHFs. The main simplifications consist in using isopycnic density (0.5 or 1.0 M sucrose) rather than density gradient (0.4-1.4 M sucrose) centrifugations (35), and collection of fractions as pellets rather than from density interfaces. The standard A68 protocol has been modified to permit comparisons to be made with the if-II protocol. The first modification consists c f addition of a further 0.5 M sucrose centrifugation step to permit Pronase digestion to be carried out in some experiments. The second modification consists of processing the material normally discarded after the first centrifugation step of the A68 protocol, and using this to generate the standard PHF fraction of the if-II protocol. Both bulk protocols were scaled down as discussed in the Method section for analytical studies on small tissue aliquots.

p r o t o c o l to p r e p a r e b o t h n o r m a l t a u in the "S1 f r a c t i o n " a n d a f r a c t i o n enriched in P H F s ( " P H F - f r a c t i o n " ) . In the second, we have p r e p a r e d a n " A 6 8 - t a u f r a c t i o n " t h a t fails to sediment f r o m b r a i n h o m o g e n a t e after b r i e f c e n t r i f u g a t i o n b u t is insoluble in sarkosyl. In addition, a P H F fraction was also p r e p a r e d f r o m the material t h a t sediments at the start o f the A68 prepar a t i o n b u t which is n o r m a l l y discarded in the A68 protocol.

P H F s a n d insoluble tau f r o m all these protocols were sedimented finally t h r o u g h 0.5 M sucrose with or w i t h o u t a P r o n a s e treatm e n t (0 or 2/~g/ml). The latter yields tau protein which is associated with the P r o n a s e - r e s i s t a n t core o f the P H F . T h e initial investigations o f these protocols were conducted in a large scale f o r m a t , referred to as the " b u l k p r o t o c o l " in the M e t h o d section, before being applied to cases originating f r o m a prospec-

412

WISCHIK ET AL.

tive clinico-pathological correlation study in a modified "analytical protocol".

Correlation Between Phosphorylated and Core-PHF Tau in A D Brain Tissue The quantity of protease-resistant PHFs in AD brain tissue can be measured immunochemicallyin the PHF-fraction using either a monoclonal antibody which selectively detects tau that is truncated at Glu-391 (mAb 423) or a monoclonal antibody (mAb 7.51) which detects a generic tau epitope in the the tandem repeat region that is only exposed after formic acid treatment. We have determined both types of immunoreactivity in 27 large-scale if-II preparations derived from 9 neuropathologically confirmed AD cases (Fig. 2). There was a high degree of correlation between these two measures of tau protein which is intrinsic to the core structure of the PHF (r = 0.955, p < 0.001). In order to relate the quantities of phosphorylated tau in the A68-tau fraction and protease-resistant PHF-tau in the PHF fraction, we have undertaken a comparative study in 24 preparations from 12 neuropathologically confirmed cases of AD in which the brain homogenate was split and processed according to the two different protocols, mAb 423 immunoreactivity was used as the measure PHF-tau in the core PHF fraction. Three different measures of phosphorylated tau in the A68 preparation were used: densitometry of SDS-soluble A68-proteins detected by immunoblot (6), direct and competitive ELISA of tau phosphorylated at Ser-199/202 detected by mAb AT8 (15).

3

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The mAb AT8 immunoreactivities detected in the A68 fraction by direct or competitive ELISA were highly correlated with each other (r = 0.79, p < 0.001). Both of these parameters were also found to be highly correlated with mAb 423 immunoreactivity in the corresponding PHF fraction (r = 0.84, p < 0.001 in both instances; Fig. 3A). By contrast, densitometry of SDSsoluble A68 proteins which could be visualised by immunoblot was correlated neither with ELISA measures of mAb AT8 immunoreactivity in the A68 fraction (r = 0.36 and 0.26, for direct and competitive ELISA, respectively) nor with mAb 423 immunoreactivityin the PHF fraction (r -- 0.26; Fig. 3B). Thus, whereas the quantity of phosphorylated tau in the A68 preparation was highly correlated with core PHF-tau content of the same brain preparation, the yield of SDS-soluble A68 proteins was quantitatively related to neither. This suggests that the SDSsolubility of phosphorylated tau in the A68 preparation is not quantitative.

Preparative Distribution of Phosphorylated Tau in A68 and If-H Protocols In order to examine this discrepancy further, we analysed the distribution of mAb AT8 immunoreactivity in all of the intermediate fractions generated in the course of the two protocols in 2 cases with mild AD pathology. The relative quantity of phosphorylated tau (measured by competitive immunoassay using mAb ATS) obtained at various stages of the A68 protocol are shown in Table 1. Essentially, all of the mAb AT8 immunoreactivity was extracted into the first low-speed supernatant and recovered in the sarkosyl-insoluble pellet. All mAb AT8 immunoreactivity was abolished following digestion with Pronase (2 #g/ml). When the same starting material was processed via the if-II protocol, the overall recovery of mAb AT8 immunoreactivity was 8007o of that recovered in the A68 protocol. Of this, 4407o was recovered in the $1 fraction, and a similar quantity was recovered in the Supernatant 1 fraction in Fig. I. When examined by electronmicroscopy, neither of these fractions was found to contain PHFs. Only 12% was recovered in the PHF fraction, having sedimentation properties characteristic of PHFs. Thus although the overall recovery of mAb AT8 immunoreactivity in the if-II protocol was comparable to the sarkosyl protocol, about 88070had sedimentation properties characteristic for either normal tau or for non-PHF aggregates of tau, and only 12% was recovered in the fraction where PHFs are known to sediment. This implies that the bulk of mAb AT8 immunoreactivity recovered in the A68 fraction is not in the form of PHFs.

.

Distribution of Bound Tau in A68 and If-H Preparative Protocols

0 0

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mAb 7.51 FIG. 2. The data for mAb 423 and formic acid-dependent mAb 7.51 immunoreactivity from 27 large scale Pronase-if-II preparations from 9 neuropathologically confirmed cases of AD are compared in this figure. The log¢ transforms of the data in arbitrary units are plotted. The correlation coefficient was 0.955. Because the Glu-391 truncation site is present in only some of the tau isoforms found in the core PHF preparation (28), whereas all tau isoforms are recognised by mAb 7.51, the high correlation betweenthese two independent measures impliesa high degree of constancy in the stoichiometryof truncated tau isoforms whithin the PHF core.

As noted, the mAb 7.51 epitope is occluded in tan which is bound within the PHF via the tandem repeat region, but it can be exposed after formic acid treatment. By contrast, mAb 7.51 immunoreactivity can be detected in free or microtubule-bound tau without formic acid treatment. In the if-II protocol, 98°70 of non formic acid dependent mAb 7.51 immunoreactivityrecovered was found in the S1 fraction. The total nonformic acid dependent mAb 7.51 immunoreactivity detected in any of the fractions produced by the A68 protocol represented less than 4°7o of that found in the S1 fraction. Thus the bulk of normal soluble tau is discarded in the A68 protocol (A68 Supernatant 1 in Fig. 1), and of that tau which is retained, the bulk is occluded at the mAb 7.51 site. Tan protein in A68 Supernatant I consists

MEASUREMENT OF TAU ]IN PHFs

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of normal tau protein as demonstrated by its electrophoretic mobility (bands of 55-65 kD) and absence of reactivity with mAb AT8 on immunoblots (not shown). After formic acid treatment, increases in mAb 7.51 immunoreactivity were observed in the A68-tau fraction, and in the PHF fractions produced either by the standard if-II protocol, or from the material sedimenting at the start of the A68 protocol which is normally discarded (Table 1). Formic acid-dependent mAb 7.51 immunoreactivity provides a selective measure of tau abnormally bound at the tandem repeat region. Of the total bound tau recovered in the A68 protocol, only 9% was found in the A68-tau fraction, the remainder being found in a PHF fraction obtained from material which is normally discarded in this protocol. Thus, the bound tau extracted into the A68-tau fraction during the A68 protocol represents a small proportion of the total tau bound in a PHF-like configuration which is present in the tissue. In contrast to mAb AT8 immunoreactivity, there was no significant loss of formic acid-dependent mAb 7.51 immunoreactivity in either A68-tau or PHF fractions after digestion with Pronase (2/zg/ml).

Proportion of Bound Tau Which Is Phosphorylated in A68-Tau and PHF Fractions

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FIG. 3. Abnormally sedimentingtau proteins prepared via the A68 and if-If protocols were measured by several different immunochemicalmeasures in 24 large scale preparations from 12 cases with neuropathologically confirmed AD. Phosphorylated tau was measured in the A68-tau fraction either by competitive immunoassay using mAb AT8 (A) or by densitometry of SDS-soluble tau proteins with the characteristic gel mobility of A68 proteins (B). Bo;ththese measures were compared with mAb 423 immunoreactivity in the protease-resistant PHF fraction prepared from the same brain tissue via the if-II protocol. Quantities are expressed in arbitrary units. There is a high correlation (r = 0.842) between phosphorylated tau in the A68 tau fraction prepared via the A68 protocol measured by competitive immunoassay using mAb AT8 and mAb 423 immunoreactivity in the protease-resistant PHF fraction prepared via the if-II protocol. By contrast, no significantcorrelation could be demonstrated between SDS-solubletau proteins with the characteristic electrophoretic mobility of "A68 proteins" and mAb 423 immunoreactivity in the corresponding PHF fraction (r = 0.257). There was likewise no significant correlation between this densitometric measure of phosphorylated A68 tau protein and mAb AT8 immunoreactivity measured either by direct or competitive immunoassay of the A68 tau fraction (r = 0.36 and 0.26, respectively).

Having determined the comparative distribution of phosphorylated and bound tau in the A68 and if-II protocols, we undertook a more extensive study of mAb AT8 and postformic acid mAb 7.51 immunoreactivity in the A68 and PHF fractions in 60 preparations from 10 cases spanning a range of clinical severity of AD pathology. These cases came from a prospective clinico-pathological study of cognitive decline in the aging population (41). Clinical assessments were undertaken using the Cambridge Examination for Mental Disorders in the Elderly (CAMDEX, 42), the last assessment having been undertaken within 12 months of death. Three of the cases were clinically and neuropathologically normal. Of the 7 cases with a CAMDEX diagnosis of probable AD at last assessment, the severities were: 3 minimal, 3 mild, 1 severe. The mean ages at death did not differ significantly in the two groups (normals, 92.7 _+ 3.0; AD cases, 89.3 _+0.9; p = 0.373). In each case, 6 brain regions were analysed, and for each brain region, the following biochemical parameters were determined: normal tau (nonformic acid-dependent mAb 7.51 immunoreactivity in the S1 fraction), phosphorylated tau in the A68-tau fraction (mAb AT8 immunoreactivity), total tau in the A68-tau fraction (formic acid-dependent mAb 7.51 immunoreactivity, non-Pronase), phosphorylated tau in the PHF fraction of the if-II protocol (mAb AT8 immunoreactivity), PHF-tau in the PHF fraction of the if-II protocol (formic acid-dependent mAb 7.51 immunoreactivity, non-Pronase). As indicated above, the protocols in this part of the study were carried out in a modified analytical format. In addition to scaling-down the protocol, the first centrifugation of the A68 protocol was reduced from 100,000 g to 25,000 g to enhance recovery of tau protein in the A68-tau fraction. The distribution of tau protein in these preparative fractions is shown in Fig. 4. Analysis of variance (ANOVA) of biochemical data showed that the effect of clinical diagnosis was significant. In the control cases (Fig. 4A), bound tau protein was detectable in the PHF fraction although the amount was less than the level of normal tau. The total amount of bound tau in the A68 fraction was 35.4070 of that found in the PHF faction. As expected, this proportion was about 4-fold higher than recovery obtained found in the earlier large scale studies, due to the 4-fold reduction in the speed of the first centrifugation step. Of this, 13.1070 was immunoreactive with mAb AT8. This represented 4.6°70 relative to total PHF- tau. Tau in the PHF frac-

414

W I S C H I K ET AL. TABLE 1 DISTRIBUTION OF TAU PROTEIN 1N A68 AND ifII PREPARATIVE PROTOCOLS Relative Immunoreactivity* Phosphorylated tau (mAb AT8) A) A68 protocol A68-tau A68 supernatant 2 Supernatant 1 Supernatant 2 PHF fraction Total B) iflI protocol S1 fraction Supernatant 1 Supernatant 2 PHF fraction Total

Free tau (mAb 7.51; nonFA)

Bound tau (mAb 7.51; FA)

[100] 0.01 0.24 0.06 0.04 100.35

0.03 0.03 1.69 0.42 1.58 3.75

1.06 0.05 1.2 0.3 I 1.67 14.28

35.35 35.22 0.06 9.46 80.09

[100] 0.66 0.2 1.02 101.88

[100] 1.96 0.3 17.03 119.29

*The relative immunoreactivity in individual fractions is expressed as a percentage of that found in A68-tau (for AT8) or in the $1 fraction (for 7.51), as indicated in parentheses. For details of different fractions, see Method Section and Fig. 1. FA = formic acid.

tion which was immunoreactive with m A b AT8 represented 0.7°70 o f total P H F tau. The A D cases in this sample were characterised b o t h by a 36070 loss o f normal tau ( p = 0.078), and an approximate 10-fold

8 m

A.

increase in total b o u n d tau in the A68 fraction ( p = 0.002), phosphorylated tau in the A68 fraction ( p = 0.004), total b o u n d tau in the P H F fraction ( p -- 0.03) and p h o s p h o r y l a t e d tau in the P H F fraction ( p < 0.001). The largest pool o f abnormal tau

Controls

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FIG. 4. Tau protein was measured in brain tissue from 18 brain tissue specimens from 3 cases diagnosed prospectively as normal by CAMDEX within 12 months of death (A), and 42 brain tissue specimens from 7 cases diagnosed as probable Alzheimer's disease by CAMDEX within 12 months of death. The following biochemical parameters (from left to right) were determined: normal tau (nonformic acid mAb 7.51 immunoreactivity in the $1 fraction), phosphorylated tau in the A68-tau fraction (mAb AT8 immunoreactivity), phosphorylated tau in the PHF-fraction (mAb AT8 immunoreactivity), total tau bound in a PHF-like configuration in the A68-fraction (formic acid-dependent mAb 7.51 immunoreactivity, no protease), total tau bound in a PHF-like configuration in the PHF-fraction (formic acid-dependent mAb 7.51 immunoreactivity, no protease). There was approximately a 10-fold increase in all measures of abnormally sedimenting tau in the tissue specimens from probable AD cases, and all pairwise comparisons between controls and AD cases were statistically significant.

MEASUREMENT OF TAU i[N PHFs was the bound tau in the PHF fraction, i.e., PHF-tau. The total amount of bound tau found in the A68-tau fraction represented 27.7070 of that found in the PHF fraction. This proportion did not differ significantly from that seen in the control cases (p = 0.544). Of this, 14.5070was phosphorylated at the mAb AT8 site. This represented 4.0°70 relative to total PHF-tau, also not different from controls (p = 0.823). Tau in the PHF fraction immunoreactive with mAb AT8 represented 0.9°70 of total PHFtau. This also did not differ from the proportion seen in controls (p = 0.385). Thus, although AD cases differed from controls by a 10-fold increase in abnormally sedimenting tau species, this increase was not associated with any overall change in the proportions of phosphorylated forms of tau relative to total PHF-tau. Overall, there was a 16-fold difference in the the proportion of bound tau that was phosphorylated in the A68-tau and PHF fractions, indicating that much more of the tau which is recovered in the A68-tau fraction is phosphorylated. There was a high correlation between mAb AT8 immunoreactivity and formic acid-dependent mAb 7.51 immunoreactivity in both fractions (r = 0.939 and r = 0.955 in A68-tau and PHF fractions, respectively, p < 0.001 in both). There was no evidence of any significant relationship between postmortem interval and any of the parameters examined (0.042 < r < 0.099, 0.354 < p < 0.692), except for formic-acid dependent mAb 7.51 immunoreactivity in the A68-tau fraction. In this case, there was a low positive correlation with postmortem delay (r = 0.199, p = 0.06), indicating a weak tendency for more tau to be found in this fraction at longer postmortem intervals. DISCUSSION

We present evidence to show that the amount of A68-protein released after SDS treatment of the sarkosyl-insoluble fraction is not correlated with the amount of phosphorylated tau protein measured in the same frzction by either direct or competitive immunoassays using mAb AT8. One possible explanation of this discrepancy might be that not all of the A68-protein is phosphorylated at Ser 199/2(12, because a variety of phosphorylation sites have been shown to produce the characteristic gel mobility shifts of the A68-proteins. However, mAb AT8 recognises all three bands of the A68 triplet (1,24) and there is as yet no evidence of abnormally p)~osphorylated tau in AD which is not phosphorylated at the AT8 site (1,16,21), although such species may exist. In this study, mAb AT8 immunoreactivity was found to be highly correlated with total bound tau in the both the sarkosyl and bulk PHF preparations and with protease-resistant core PHF-tau. This implies that there are highly significant quantitative relationships between mAb AT8 immunoreactivity and every other measure of abnormal tau accumulation, other than that provided by densitometry of A68 proteins. A more likely explanation for the discrepancy is that the SDS solubility of tau protein in the sarkosyl-insoluble fraction is not quantitative so that not all of the tau protein present in the preparation runs into the gel as A6g proteins. A possible explanation for the nonquantitative solubility in SDS of the phosphorylated tau protein becomes apparent from an analysis of the preparative distribution of mAb AT8 immunoreactivity when the sarkosyl solubilisation step is omitted. When the same material was processed through the if-II protocol, only 1207oof the mAb AT8 immunoreactivity was recovered in the PHF fraction, the remainder being distributed equally between the S1 fraction of the if-lI protocol, where the normal soluble tau is routinely recovered, and an intermediate supernatant fraction. Neither of these fractions contained PHFs visible by

415 electronmicroscopy. By contrast, the main bulk of bound tau (8307o-88°7o) was recovered in the PHF fraction, where the main bulk of PHFs visible by electronmicroscopy are found (35). Thus, most of the phosphorylated tau recovered in the sarkosyl-insolubleA68-tau fraction does not have the sedimentation properties corresponding to PHFs. The precise form that the phosphorylated tau species in these supernatant fractions might take is not known, but they are not in the form of PHFs. It may be that they are aggregates of phosphorylated tau that are more soluble in SDS than the phosphorylated tau which is bound to PHFs. In any case, because the amount of phosphorylated tau recovered in the sarkosyl-insoluble fraction which can be shown to sediment as PHFs is only about 12070, no conclusion can be drawn as to the molecular composition of PHFs in the standard A68 preparation, because the bulk of the SDS-soluble material detected after gel electrophoresis need not originate from PHFs. A quantitative study of the relationship between phosphorylated tau and total bound tau in the sarkosyl-insoluble fraction also casts significant doubt on the proposition that the only tau found in this preparation is phosphorylated. The formic aciddependent mAb 7.51 immunoreactivity provides a measure of all tau in this fraction which has a tandem repeat region, irrespective of its state of phosphorylation or the presence of the N-terminus. Only 14070of tau in this fraction was found to be phosphorylated either in controls or in AD cases. The remaining 86°7ois either not phosphorylated at Ser-199/202 or lacks the N-terminal segment of the tau molecule which contains this epitope. The latter would be consistent with the observations of Morishima-Kawashima et al. (25) who have shown that a substantial amount of the tau in the sarkosyl insoluble fraction is N-terminally truncated. The relative amount of tau phosphorylated at Ser-199/202 in the bulk PHF fraction is even smaller. The if-II protocol used in this and previous studies has been optimised to maximise the total yield of PHFs. In the present study, the standard bulk PHF fraction contained 8307o-8807o (data from A68 and if-II protocols, respectively) of the total yield of formic acid-dependent mAb 7.51 immunoreactivity (i.e., tau bound in a PHF-like configuration). In the absence of Pronase digestion, it is possible to determine how much of the total bound tau which sediments in this fraction is phosphorylated. In both controls and AD cases, the amount was less than 1070.That is, 99070of tau which can be defined operationally as PHF-tau, on the basis of characteristic sedimentation and occlusion of the mAb 7.51 epitope, is not phosphorylated. This is true regardless of the clinical severity of dementia. It is possible to arrive at a similarly low estimate independently from the study of preparative distribution of bound and phosphorylated tau in the large-scale versions of the two protocols. In the A68 protocol, almost all the phosphorylated tau present in the brain tissue could be recovered in the A68 fraction. However, this fraction accounted for less than 9% of the total bound tau which could be recovered after further processing of the discarded pellet to produce the PHF fraction. That is, phosphorylated tau in the A68 fraction could contribute no more than 9% of total PHF-tau. However, only 12070 of the phosphorylated tau detected by mAb AT8 in the A68-tau fraction was found to have the sedimentation properties characteristic of PHFs when the same starting material was processed through the standard if-II protocol. This implies that the phosphorylated PHFs present in the A68-tau fraction could represent at most 1207oof 9% of total PHFs, i.e., 1070of total PHFs. The fact that it is possible to arrive at a similar estimate for the quantity of PHF-tau which is phosphorylated at the mAb AT8 site by these independent analytical routes implies that the low

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figure cannot be explained by underdetection of phosphorylated P H F s by m A b AT8, since in the bulk protocols, the estimate comes from the preparative distribution of total m A b AT8 immunoreactivity and total bound tau. The overall estimate was not enhanced by diverting more tau into the A68 protocol by further reducing the speed of the first centrifugation step, and in any case this would only increase the estimate for phosphorylated P H F - t a u to 4% o f total PHF-tau. The same considerations apply in trying to explain the low levels of phosphorylated tau in terms of postmortem dephosphorylation (23). If anything, longer postmortem intervals were associated with somewhat higher levels o f tau in the A68-tau fraction. In general, it appears safe to say that across a representative range of A D pathology, more than 95% of P H F - t a u is not phosphorylated at the m A b AT8 site. This would be consistent with the bulk o f P H F pathology occurring in the absence of pathologic phosphorylation. These studies imply that more than 85% of the tau recovered on the basis o f insolubility in 1%o sarkosyl, and more than 95% o f the tau recovered on the basis of PHF-like sedimentation characteristics, is not phosphorylated, and that the amount of phosphorylated tau which can be detected as tau species with an electrophoretic mobility corresponding to the A68 proteins bears no quantitative relationship to any other measure of P H F tau in A D brain tissues. What then is the significance of tau phosphorylation as a pathogenic mechanism responsible for P H F accumulation in A D ? Since it is known that at least some of the pathologic phosphorylation sites seen in A68 proteins are also found in tan sequestered in the somatodendritic compartment for other reasons, including normal neuronal development, it is conceivable that the detection of phosphorylated tau sequestered in the somatodendritic compartment in the course of neurofibrillary degeneration carries no greater pathogenic significance than that the process occurs in the somatodendritic compartment. That is, the underlying mechanism of neurofibrillary degeneration may be quite independent of the balance between kinases and phosphatases, and the appearance of pathologically phosphor-

ylated tau in a minor proportion of P H F s may be simply an epiphenomenon. Even though the absolute quantity of P H F - t a u which is pathologically phosphorylated is extremely low, it could be argued that phosphorylation represents a rate-limiting step which is required for P H F assembly. There has been no quantitative evidence from tau assembly experiments in vitro to support this view (2,3,34). However, it would be possible to address this question objectively by examining the redistribution of tau protein between various pathologic pools in the course of disease progression. It has been proposed specifically that tau protein found in the A68 fraction represents a pool of early/precursor hyperphosphorylated t a u / P H F s which feed into the total P H F pool, and that over time P H F - t a u is subject to proteolysis and dephosphorylation (6). There is also conjecture that these stages correspond histologically to the early appearance of dystrophic neurites (corresponding to A68-tau) and the later appearance of neurofibrillary tangles (corresponding to PHF-tau; 20). Because there is indeed a substantial difference in the ratio of phosphorylated to bound tau in the A68 and P H F fractions, measurement of these quantities in the course of progression of neurofibrillary degeneration would provide a way of testing the predictions of the phosphorylation hypothesis. This is undertaken in an accompanying article. ACKNOWLEDGEMENTS Supported in part by Medical Research Council (UK) grants to C.M.W., C.R.H., J.X., E.S.P., F.H., C.B.; R.L. was recipient of a Wellcome Prize Studentship within the MB-PhD Programme of the University of Cambridge Clinical School. M.R. and E.M.L. were supported by the Leopold Muller Estate. C.R.H. was support by the Newton Trust, Trinity College, Cambridge. We would like to express our gratitude to A. O'Sullivan and R. Hazelman for assistance with brain tissue donation, to R. Hills and J. Hurt for technical assistance, and to S. West for secretarial assistance.

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