THE JOURNAL 0 1986 by The American Society of Biological Chemists, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 261, No. 13, Issue of May 5, pp. 6084-6089,1986 Printed in U.S.A.

Microtubule-associated Protein Tau A COMPONENT OF ALZHEIMER PAIRED HELICAL FILAMENTS*

(Received for publication, November 27, 1985)

Inge Grundke-Iqbal, Khalid Iqbal, Maureen Quinlan, Yunn-Chyn Tung, Masooma S. Zaidi, and Henryk M. Wisniewski From the New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phospho- cellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with per- chloric acid. All of these tau preparations reacted spe- cifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypep- tides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results sug- gest that tau is a major component of Alzheimer paired helical filaments.

Paired helical filaments (PHF’) are the most characteristic cytoskeletal alteration affecting numerous neurons in Alz- heimer disease (senile dementia of the Alzheimer type). PHF form neurofibrillary tangles in neuronal perikaryon and in dystrophic neurites of the neuritic (senile) plaques in Alz- heimer cerebrum. These histopathological lesions are also seen in small numbers in the brain of non-demented, normal, aged persons around age 70 and above. Morphologically, PHF are unique. They are unlike any of the normal neurofibers and have not been found in any aged animal or experimental animal model (for review, see Ref. 1). Although recently PHF have been isolated (2, 3) and their polypeptide patterns on SDS-polyacrylamide gels determined (2, 4), the question of their biochemical origin has remained unanswered.

The first demonstration of immunocross-reactivity of PHF with normal brain proteins was with normal brain microtu- bules assembled in vitro (5,6). Antisera to brain microtubules have been shown to label (i) isolated PHF which had been repeatedly washed with SDS and (ii) Alzheimer neurofibril- lary tangles and plaque neurites, but not amyloid, in tissue sections (7). The immunolabeling of SDS-treated PHF which, unlike the fibrils in situ are free from the fuzzy coat around

Grants NS 18105, AG 05892, and PO1 AG/NS 04220. The costs of * This work was supported in part by National Institutes of Health

publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PHF, paired helical filaments; SDS, sodium dodecyl sulfate; Mes, 2-(N-morpholino)ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; MAP, mi- crotubule-associated proteins; MT, microtubules; anti-MT (PHF) serum, PHF-reactive antiserum to normal MT.

them (2,8), suggests that the anti-microtubule sera react with these fibrils and not any adventitious polypeptides. Since the original observation, a number of normal polypeptides, i.e. neurofilament polypeptides (9-13), vimentin (14), high mo- lecular weight microtubule-associated proteins (MAP) (15), somatostatin (16), and rabbit IgG (17) have been reported to cross-react with PHF immunocytochemically, but none of these polypeptides has been shown to be present in PHF. The immunocytochemical cross-reactivities may not necessarily reflect biochemical, functional, or pathogenetic relationships. The size of an antigenic site detected by an antibody is relatively small, and identical or closely related antigenic sites comprised of a few amino acids have been found on molecules which are otherwise unrelated, e.g., between the transforming protein of Rous sarcoma virus and tubulin, myosin, and vimentin (18) and between thymus and neuronal cell surface proteins (19). Furthermore, it is not possible to determine with immunocytochemical techniques alone whether cross- reactivities may be due to similarities in primary amino acid sequence or due to antigenic sites created from folding of different regions of a polypeptide chain (20). Using a com- bined immunocytochemical and biochemical approach, stud- ies reported in this paper demonstrate for the first time that the microtubule-associated protein tau, a normal brain cyto- skeletal protein, is a component of the PHF.

MATERIALS AND METHODS

Isolation of PHF and Microtubules PHF were prepared using the long procedure of Iqbal et al. (2) from

the brains of nine Alzheimer patients which had been frozen 5-12 h after death. Microtubules were prepared from histopathologically normal human and animal brains by assembly-disassembly i n vitro (21). The human brains for microtubule preparations were obtained not later than 3 h after death. Calf brains were obtained from a local slaughterhouse. Both human and calf brains were placed in ice-cold disassembly buffer (21) before transporting to our laboratory. Other animal brains were dissected immediately after killing.

Isolation of Tau Tau was prepared from calf brain microtubules by the following

five different methods. The yield and the purity of the tau prepara- tions are shown in Table I.

Method 1: Preparation of M A P by Fractionation on Phosphocellu- lose (22)”A thrice-cycled microtubule pellet was resuspended to 1.25 mg of protein/ml in cold Mes-EDTA buffer (25 mM Mes, 0.5 mM MgCl,, 1 mM dithiothreitol, 0.1 mM EDTA, pH 6.4) and centrifuged at 3200 X g for 5 min, and 1.2 ml of the supernatant were fractionated on a 2.5-ml phosphocellulose column (Cellulose Phosphate P11, What- man). Tubulin was eluted with Mes-EDTA buffer, and the remaining protein was eluted with 15 ml of a 0-1.0 M NaCl gradient. Tau and high molecular weight MAP started to elute together at about 0.4 M NaCl with a maximum at 0.7 M NaC1. The total protein of this fraction was 180 fig.

Method 2: Preparation of Heat-stable M A P at pH 6.4 (23)”Two

6084

Tau in Alzheimer Paired Helical Filaments 6085

ml (32.5 mg of protein) of thrice-cycled microtubule pellet were suspended in 1.2 ml of buffer containing 100 mM Mes, 0.5 mM MgClz, 1 mM EGTA, 0.1 M EDTA, 0.75 M NaCl, 0.1 mM GTP, and 3 mM dithiothreitol, pH 6.4, and kept on ice for 1 h. The suspension was then heated at 95 "C in a boiling water bath for 5 min and centrifuged at 25,000 X g and 4 "C for 30 min. The resulting supernatant (480 pg of protein) contained both tau and high molecular weight MAP.

Method 3: Preparation of Heat-stable Tau at pH 2.7-One volume of thrice-cycled microtubule pellet (10-20 mg/ml of assembly buffer) was suspended in 3 volumes of buffer containing 100 mM Mes, 0.5 mM MgC12, 1 mM EGTA, 0.1 mM EDTA, 0.75 mM NaC1, 2 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, pH 2.7, and processed identically as for Method 2. The yield was 15.1 f 7.0 mg of protein/kg of brain. When tau was prepared from once- or twice-cycled microtubules, the yield increased respectively to 105 and 65 mg/kg of brain (78 mg/kg from human brain). The resulting tau fractions were, however, less pure than tau prepared from thrice- cycled microtubules.

Method 4: Purification of Tau from Once-cycled Microtubules by Extraction with Perchloric Acid-Tau (5 mg) prepared from once- cycled microtubules according to Method 3 was dialyzed against 20 mM Mes, 1 mM MgClZ, 2 mM EGTA, 0.1 mM EDTA, 80 mM NaCl, 1 mM 0-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, pH 6.75, and brought to 2.5% perchloric acid, and the proteins in the supernatant were precipitated with 20% trichloroacetic acid (24). The precipitate was pelleted at 15,000 X g, washed with ethanol, and dissolved in above buffer, and tau (0.23 mg) precipitated at 25% glycerol concentration. Substitution of the perchloric acid extraction step with precipitation at 35-45% ammonium sulfate saturation resulted in about half the yield but without any apparent increase in the purity of tau.

Method 5: Purification of Tau from Microtubules by Ammonium Sulfate Precipitation and Extraction with Perchloric Acid (24)"Mi- crotubules were prepared by one or three assembly-disassembly cycles in the presence of 1 mM each GTP and ATP and processed according to Method I11 of Lindwall and Cole (24). The yield was 0.2 and 0.045 mg of protein/kg of brain from once- and thrice-cycled microtubules, respectively.

TABLE I Yield and purity of tau prepared by different methods

Estimates of the purity were obtained by densitometry of the Coomassie Blue-stained gels. Tau were identified by Western blots developed with monoclonal antibody to tau.

Method Yield" Puritf

mg proteinlkg brain % 1 55 79 2 9.2 62 3 15.1 96 3 (1 cycle of MT) 105 90 4 4.9 92 5 0.2 96

Mean of two preparations except Method 3, which is the mean of nine preparations.

Protein Determination Protein concentrations were estimated by the assay of Lowry et al.

(25) and by amino acid analysis and verified on Coomassie Blue- stained SDS-polyacrylamide gels.

Antibodies PHF-reactive anti-MT sera (anti-MT (PHF) sera) and the other

primary antibodies employed in this study and their characteristics are listed in Table 11. Peroxidase-anti-peroxidase complex produced in rabbits was purchased from CooperBiomedical, Malvern, PA and avidin-biotin complex kit from Vector Laboratories, Burlingame, CA.

Protein Profiles on SDS-Polyacrylamide Gels, Zmmunoblots, and Dephosphorylation

Samples were electrophoresed on 0.75-mm slab gels using linear acrylamide gradients and the Laemmli (28) buffer system. Sample preparation and immunoblots were carried out as describedpreviously (4). PHF were sonicated prior to dissolution in sample buffer (2). Dephosphorylation of polypeptides on paperblots with alkaline phos- phatase (43 and 120 pg/ml) was performed according to Sternberger and Sternberger (29). Tissue sections were incubated with trypsin prior to the phosphatase treatment as previously described (29).

Affinity Purification of Antibodies to Tau Tau polypeptides prepared from thrice-cycled microtubules by

heating in pH 2.7 buffer (Method 3) were separated by electrophoresis on SDS-polyacrylamide gels (12.5% acrylamide, 16 X 11 cm) and transferred to nitrocellulose paper. The remaining protein-binding sites were blocked by immersing the paper in 10 mM phosphate- buffered saline, pH 7.2, containing 5% defatted dry milk for 2 h at room temperature. Analytical strips were cut from each side of the paper, and immunolabeled with either anti-MT(PHF) serum or monoclonal antibody to tau. The strips were aligned with the remain- ing untreated paperblot and five horizontal preparative strips (I-V) cut out corresponding to the major immunostained polypeptides. The preparative strips were incubated with anti-MT(PHF) serum over- night at room temperature, washed, and sliced into small pieces. The bound antibodies were extracted according to Olmsted (30) and tested on paperblots of PHF and tau and on 6-pm sections of paraffin- embedded, formalin-fixed Alzheimer hippocampus using the peroxi- dase-anti-peroxidase technique (4).

RESULTS

Absorption of Tangle-staining Antibodies with Microtubules from Various Species-Anti-MT(PHF) sera labeled tangles and plaque neurites in Alzheimer brain tissue sections before but not after absorption with twice-cycled microtubules (5 pg of protein/ml of diluted antisera) prepared from the brains of 5-,8-,17-,25-,40-,74-, and 83-year-old persons or from rat, guinea pig, rabbit, or bovine brain. These findings suggested that the protein in brain microtubules that is immunochemi- cally cross-reactive with PHF is neither age-dependent nor unique to human. Therefore, all subsequent studies on the

TABLE I1 Antibodies employed and their reactivity on immunoblots

Antibodies Immunogen Dilution Polypeptides labeled

Polyclonal Anti-MT (PHF) (5-7) Human MT, 2 cycles 1:3,000 Tubulin, tau," high molecular weight MAP

Anti-MT IV (7) Calf MT, 2 cycles 1:1,000 Tubulin. high molecular weight MAP. NF6 triplet, PHF

Anti-NF triplet (4, 7) Bovine NF Anti-tubulin' (7) 6 S tubulin

NF triplet -

1:2,000 NF triplet 1 d m 1 01 and 0 tubulins

Monoclonal Tau-1 (26) Bovine MAP 0.1 pg/ml 5-25 (27) PHF 1:50,000

Tau"

NR4d PHF

NF P68 1 d m 1 NF P68 All molecular species of normal human and bovine brain.

Affinity-purified antibodies purchased from CAABCO, TX. Purchased from Boehringer Mannheim.

' Neurofilaments.

6086 Tau in Alzheimer Paired Helical Filaments

identification of the microtubule protein that is immunochem- ically related t,o PHF were carried out with calf brain micro- tubules.

Co-purification of PHF-Cross-reactivity with Microtu- bules-In order to evaluate whether the PHF-cross-reactive antigen(s) in the microtubule fraction were a microtubule protein or a contaminant, enrichment of PHF-reactivity in microtubules prepared from calf brain by one, two, or three assembly-disassembly cycles was estimated by determining the amount of protein needed for absorption (4) of an anti- MT( PHF) serum to eliminate staining of tangles and plaque in tissue sections. By this assay the thrice-cycled microtubule fraction, the purest of the three preparations, was found to be most enriched in PHF-reactivity (Table 111).

Co-purification of PHF-Cross-reactivity with Tau-To iden- tify the PHF-cross-reacting microtubule protein, microtu- bules were further separated into microtubule subunit protein, tubulin and MAP by several different methods. The latter proteins are composed of high molecular weight MAP and a class of proteins called tau. When a thrice-cycled microtubule preparation was fractionated on phosphocellulose (Method I) , the large majority of the PHF-like reactivity was detected in the fraction enriched in MAP containing both tau and high molecular weight MAP (Fig. 1, lane A ) , whereas the tubulin fraction contained 100-fold less antigen (Table 111). Likewise, when the heat-stable microtubule proteins consisting mostly of tau and high molecular weight MAP were prepared from thrice-cycled microtubules by heating at pH 6.4 in the pres- ence of 0.1 mM GTP (Method 2) (Fig. 1, lane B ) , PHF- reactivity was enriched at approximately the same degree as in the MAP fraction prepared by Method 1. During these studies we discovered that tau could be very effectively puri- fied from high molecular weight MAP when thrice-cycled microtubules were heated at pH 2.7 (Method 3). The resulting heat-stable fraction was highly enriched in tau proteins and besides tau, only contained varying amounts of polypeptides with molecular weights lower t,han that of tau (Fig. 1, lane C). The enrichment of PHF-reactivity in this fraction was about 4-fold higher than in the heat-resistant fraction containing the mixture of tau and high moleculay weight MAP prepared

TABLE I11 Enrichment of PHF activity

Anti-MT(PHF) serum, 100 p1 a t 1:300 dilution, was absorbed with different amounts of each antigen preparation. After absorption the antiserum was brought to a final dilution of 1:3000 and tested on paraffin sections of Alzheimer hippocampus using the peroxidase- anti-peroxidase technique. Microtubules (MT) were prepared from calf cerebrum using predominantly gray matter by assembly-disas- sembly in vitro (21). Methods 1-5 for preparation of tau from calf brain microtubules are described under “Materials and Methods.”

Antigen

Protein needed to absorb

tangles-staining antibodies

Brain homogenate, 72,000 X g supernatant MT, 1 cycle MT, 2 cycles MT, 3 cycles Phosphocellulose-purified tubulin

Tau preparations Method 1, tau and high molecular weight MAP Method 2; tau and high molecular weight MAP Method 3; ( 3 cycles of MT); tau Method 3; (1 cycle of MT); tau Method 4; tau Method 5: tau

!4 6.0 2.0 1.4 0.8 2.0

0.020 0.018 0.005 0.025 0.014 0.007

4

4

4

FIG. 1. Protein patterns of tau prepared from calf brain microtubules by different methods and employed for absorp- tion of the anti-MT(PHF) sera. SDS-polyacrylamide gel electro- phoresis (16 X 11 cm), 5-229; linear acrylamide gradient; gel was stained with Coomassie Blue. Tau prepared by: A ) Method 1. phos- phocellulose chromatography (22) of 3 cycles of microtubules (MT): in this preparation high molecular weight MAP were only visible by silver (not shown); R ) Method 2, heating 3 cycles of M T with pH 6.4 buffer containing GTP (23); C) Method 3, heating 3 cycles of M T with pH 2.7 buffer without GTP: 11) same as in C but using 1 cycle of MT; E ) Method 4, tau purified from I ) by extraction with perchloric acid and precipitation with glycerol (24); F ) Method 5, ammonium- sulfate precipitation and perchloric acid extraction of 1 cycle of MT (24); G shows M , standards from top to bottom: myosin (200,000), phosphorylase b (92,500), bovine serum albumin (68,000), ovalbumin (43,000). tu-chymotrypsinogen (25,700), /j-lactoglobulin (18,4001, cy- tochrome c (12,300).

by Method 2. Heating of only once-cycled microtubule prep- arations with pH 2.7 buffer significantly increased the yield. However, the resulting tau preparation was less pure (Table I and Fig. 1, lane D ) and less enriched in PHF-reactivity (Table 111). Further purification of tau from this preparation by extraction with perchloric acid and precipitation with glycerol (Method 4) (Fig. 1, lane E ) increased PHF-reactivity. Similarly, high enrichment of PHF-cross-reactivity was ob- served when tau was prepared from microtubules by ammo- nium sulfate precipitation and extraction with perchloric acid (Method 5) (Fig. 1, lane F ) . Thus, enrichment of PHF activity corresponded with purification of tau by all the five different methods employed (Table 111).

Identification of PHF Polypeptides as Tau-On Western blots of each of the five different tau preparations, anti- MT(PHF) serum labeled a group of polypeptides with M, of about 55,000-62,000, and in addition, varying numbers of polypeptide bands with M, less than 55,000. Using monoclonal antibody tau-1 to tau, these protein bands were identified as tau (Fig. 2).

In order to compare the immunolabeling pattern of tau and isolated PHF by anti-MT(PHF) serum, tau and PHF poly- peptides were electrophoretically separated in adjacent lanes of a SDS-polyacrylamide slab gel. The PHF preparations used for this experiment were highly purified and neither tubulin, high molecular weight MAP, nor the neurofilament triplet polypeptides were detected using the PHF-negative polyclonal and monoclonal antibodies to microtubule and neurofilament polypeptides listed in Table 11. Tau was prepared from calf and human microtubules by Method 3. The anti-MT(PHF) serum, besides labeling aggregates excluded from the resolving gel, most prominently labeled the major PHF polypeptides in the 55-62 kDa area from nine different Alzheimer cases studied to date (Fig. 3). These pol?.peptides co-migrated with the human tau bands (Fig. 3b) and had slightly different

Tau in Alzheimer Paired Helical Filaments 6087

A 0 B A F B C D D C B F

: "

o.tau a.MT a.tau a.MT

FIG. 2. Immunolabeling on Western blots of same tau prep- arations as shown in Fig. 1. Blots were incubated with anti- MT(PHF) serum at 1:3000 dilution (a.MT) and with monoclonal antibody to tau (a. tau) a t 0.1 pg/ml of antibody concentration. Bound antibodies were visualized using peroxidase-anti-peroxidase and avi- din-biotin complex techniques, respectively; electrotransfers from 5- 15% (left two panels) and 6-15s (right two panels) acrylamide gra- dient gels (8 X 6 cm). Both the tau polypeptides and their degradative products are immunostained.

electrophoretic mobilities from calf brain tau (Fig. 3a). The PHF polypeptides labeled with the anti-MT(PHF) serum were identical to those labeled with monoclonal antibodies to PHF. Furthermore, the tangle-staining antibodies of the anti- ,MT(PHF) serum were readily absorbed with PHF isolated from all the nine Alzheimer cases studied to date (2.5 pg of PHF protein/lOC) pl of 1:300 antiserum).

Reactivity of Anti-MT(PHF) Serum with PHF Is Not Af- fected by Dephosphorylation-Recent immunocytochemical studies have shown that Alzheimer neurofibrillary tangles share a certain phosphorylated epitope with the M, 150,000 and 200,000 neurofilament polypeptides (31). To study whether the reactivity of anti-MT(PHF) serum with PHF and tau might be likewise determined by a phosphate group, Alzheimer hippocampus sections and paperblots of tau and PHF polypeptides were dephosphorylated before application of the anti-MT(PHF) serum. A comparison of either the phosphate-treated and untreated sections (data not shown) or the immunoblots (Fig. 3b) did not reveal any differences in the staining patterns or intensity, suggesting that the reactiv- ity of the antibodies with PHF is not limited to phosphoryl- ated determinants.

Cross-reactivity between PHF Polypeptides and Different Molecular Species of Tau-To study the relationship of the different molecular species of tau with PHF, Western blots of tau were incubated with anti-MT(PHF) serum, and the bound antibodies were isolated from tau polypeptide bands of five different electrophoretic mobilities. Each of these five affinity-purified antibodies to tau stained Alzheimer tangles and plaque neurites and revealed identical immunolabeling patterns on Western blots of tau and PHF (Fig. 4).

DISCUSSION

Previously we have shown that the PHF polypeptides have tau-like profiles on SDS-polyacryalmide gels and are labeled both by antibodies to isolated PHF and by PHF-reactive antisera to normal microtubules (2,4, 7, 32) . Cross-reactivity of tau with PHF on tissue sections and on Western blots and co-migration of human brain tau with PHF polypeptides on SDS-polyacrylamide gels shown in the present study estab- lishes that tau is a major component of PHF.

b a.MT

FIG. 3. Comparison of human and calf-brain tau and PHF polypeptides. a, Calf brain tau labeled with anti-MT(PHF) serum (a.M'T) (1:3,000) and PHF polypeptides labeled with the same anti- serum (a .MT) and with monoclonal antibody 5-25 to PHF (a. PHF) (1:50,000); b, human brain tau (H- tau) and PHF polypeptides de- phosphorylated (*) or untreated, labeled with anti-MT(PHF) serum ( a . M n . Calf brain tau was prepared by Method 3 from thrice-cycled microtubules (same preparation as in lane C, Fig. 1). Human tau was similarly prepared from twice-cycled microtubules from the brain of a normal 67-year-old patient. Electrotransfer from SDS gel, 5-15',; and 5-10?; acrylamide (8 X 6 cm) is shown in a and b. respectively. For SDS-polyacrylamide gel electrophoresis, the isolated PHF (1 mg/ ml) were solubilized by first sonicating in 0.32 M sucrose for 30 min at setting 1 and 10% pulse with 4 sec on/off cycles using a Branson Model 200 sonifier equipped with a tapered microprobe and then by heating in a boiling water bath for 3 min with SDS and @-mercapto- ethanol a t both 1% final concentration (2). *, The paperblot was treated with alkaline phosphatase (31) before application of anti- MT(PHF) serum. Although not shown in this figure, both the 200- and the 150-kDa neurofilament pol-ypeptides employed as control were dephosphorylated under these conditions.

Tau, in its native state, has a M, of 57,000 (33). On SDS- polyacrylamide gels tau is known to give a complex polypep- tide pattern consisting of 4-8 bands (24) with the M, of the major bands lying between 55,000 and 62,000. Interestingly, the major polypeptide bands in all PHF preparations from the 20 different Alzheimer cases studied are in the same M , range (2) as tau. These PHF polypeptide bands are labeled both by monoclonal and polyclonal antibodies to PHF and by anti-MT(PHF) sera (32) and their affinity purified antibodies to tau (this study). Judged by amino acid composition and

6088 Tau in Alzheimer Paired Helical Filaments

\

a a.MT a.tau r

I b ~ . M T V IV 111 II I I II 111 IV V

C

FIG. 4. Labeling of tau and PHF polypeptides on Western blots and of tangles and plaque neurites on tissue sections with antibodies purified by immunoaffinity from 5 different molecular species of tau. For a, tau (Method 3) was electrophoresed on SDS-polyacrylamide gels (12.5% acrylamide, 16 X 11 cm) and transferred to nitrocellulose paper. Strips were cut from the sides and developed with anti-MT(PHF) serum (a. “T) and monoclonal anti- body to tau (atau). The remaining blot was used for affinity isolation of anti-tau antibodies from the anti-MT(PHF) serum. Roman nu- merals ( I - V ) indicate the areas of tau species from which the anti- bodies were purified. For details see “Materials and Methods.” In b, immunohlots of tau and PHF polypeptides with affinity-purified antibodies from tau polypeptides of areas I-V are shown. Electro- transfer from SDS gel, 7-10% acrylamide (8 X 6 cm). Differences in the staining intensities of the different antibodies are due to small individual variations in the amounts of the samples applied to the gel. c shows immunocytochemical staining of tangles (some of the tangles marked with arrows) and neurites of plaques (marked with

peptide maps, the individual tau polypeptides are closely related (33). It has been shown that i n uitro phosphorylation of the isolated tau can cause a slight decrease in electropho- retic mobility (24, 34). Interestingly, in PHF there is a shift in the intensity of immunostaining to the slow-moving tau species when compared with tau from human microtubules on Western blots (Fig. 36). However, the antibodies to tau in anti-MT(PHF) serum recognize antigenic site(s) common to all tau species, phosphorylated or not. Therefore, it is not possible to conclude whether the shift to the slow-moving tau species in PHF is due to the different state of phosphorylation.

I n uitro, tau is known to promote the assembly of micro- tubules and to interconnect actin filaments, both of which are influenced by the degree of phosphorylation of tau (34, 35). The role of tau in uiuo is as yet largely unknown. The findings in this paper raise the intriguing possibility that, in selected neurons of the Alzheimer brain, certain post-translational modifications of tau such as phosphorylation might occur which would allow it to assemble either alone or together with other components to the characteristic paired helical fila- ments.

Acknowledgments-We thank T. Zaidi for preparing PHF, the biomedical photography unit for photography, and P. Calimano for typing the manuscript. Monoclonal antibody to tau was generously supplied by Drs. L. I. Binder and A. Frankfurter, University of Virginia, Charlottesville, VA.

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