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Brain Research, 597 (1992) 227-232 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18301
Role of tau in the polymerization of peptides from/3-amyloid precursor protein C l a u d i a B. C a p u t o , L i n d a A . Sygowski, C l a y W S c o t t a n d I r e n e R. E v a n g e l i s t a S o b e l Pharmacology Department, ICi Pharmaceuticals Group, LW-2, ICI Americas, Wilmbrgton, DE 19897 (USA) (Accepted 30 June 1992)
Key words: AIzheimer's disease; Tau protein; Microtubule-associated protein; Amyloid; fl-Amyloid precursor protein; Fibrilogenesis; Amyloidogenesis; Paired helical filament
The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from ~-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A ~" fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from t h e / 3 / A 4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of AIzheimer's disease.
INTRODUCTION Paired helical filaments (PHFs) are amyloid fibrils that are present in both of the hallmark pathological lesions of AIzhiemer's disease, senile plaques and neurofibrillary tangles. They accumulate intracellularly in tangles and in the dystrophic neurites that surround the central core of senile plaques. The composition of PHFs is not fully known. A fragment of tau protein has been demonstrated to be an integral constituent of PHFs 3"22'24"4t.Some tau which co-purifies with PHFs is thought to be ubiquitinated 29'31 and aberrantly phosphorylated t9.42. The PHF structure is highly insoluble, which leaves the possibility that other proteins are also present in PHFs but are not amenable to solubilisation and are not antigenic or accessible to antibodies. In addition tau does not readily form fibrils in vitro 28 a~d has not been reported to form fibrils with amyloid properties. This situation contrasts with that of other amyloidogenie proteins which readily form amyloid fibrils in vitro. For example, the other type of amyloid fibril in
Alzheimer lesions is comprised of /3/A4 peptide 27, which readily forms amyloid fibrils in vitro 'J. This peptide is a fragment of up to 43 amino acids from ~-amyloid protein precursor 2~ (APP). The amino acid sequence of APP resembles that of transmembrane proteins 21. /3/A4 spans the C-terminal region of the extracellular domain and the adjacent N-terminal region of the transmembrane domain of APP. Other proteins have been suggested to be present in PHFs including /3/A4 26, MAP2 2a, neurofilament a7, actin aS, tropomyosin 14, and tubulin 32. In general antibodies to these proteins label tangles, although these antibodies do not react with isolated PHFs and the proteins themselves do not form fibrils that resemble PHFs. Thus their contributions to the PHF structure remain undefined. The C-terminus of APP is thought to be located inside the cell 2t, where PHFs form. Recent immunological data provides evidence that a C-terminal fragment of APP may be associated with PHFs in both tangles t2 and the dystrophic neurites of senile plaques 2°'35 as well as with isolated PHFs even after
Correspondence: C.B. Caputo, Pharmacology Department, ICl Pharmaceuticals Group, LW-2, ICI Americas, Wilmington, DE 19897, USA. Fax: (1) (302) 886-2766.
228 pronase t r e a t m e n t ~''"'4". A n amino acid s e q u e n c e derived from the C-terminal region o f A P P has also been detected in P H F s ~'4°. A synthetic peptide, C - A P P , which corresponds to C-terminal a m i n o acids 676-695 o f APP695, polymerizes into amyloid fibrils but these fibrils lack the characteristic m o r p h o l o g y o f P H F s , that o f twisted fibrils with a regular periodicity 4. T h e r e f o r e we investigated w h e t h e r the C-terminus o f A P P can form fibrils that resemble P H F s morphologically and interact with the only confirmed c o m p o n e n t o f PHFs, tau protein. Identification o f the proteins that are required for P H F s to assemble may help to elucidate the pathobiological processes that lead to P H F formation. PHFs in t u r n p r o b a b l y c o n t r i b u t e to the dem~.-~tia o f AIzheimer's disease 5. Recent evidence is now suggesting.that the n u m b e r s and locations o f the P H F - b e a r i n g tangles but not o f f l / A 4 plaques correlates with the severity of dementia-'. Thus an u n d e r s t a n d i n g o f P H F formation may be useful in the d e v e l o p m e n t o f effective strategies for designing therapies for Alzheimer's disease.
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MATERI,a, LS AND METHODS
Protein,v and pt,ptides PHFs were isolated as the ifll fraction from Alzheimer brain and treated with pronase as previously described41, Bovine tau was prepared as described previously =~. Recombinant ht, man tau isoforms =r' with no inserts (T3), with one insert in the tuhulin-hinding region (~-4). and with all inserts (~'4.~) and amino acids 2t,4-386 of Ihe tuhulin-binding region of T4 were expressed in 1:;. coil as previously describedTM.
Pcptides were synthesized which correspond to APP695 := amino acids 572-587, 585-f~0i1, 597-610, 611-624, 621-(~31,649-(~f~8, f~fi2681, and 676-695 (C-APP). P~plides were synthesized corresponding to amino ackls from the tuhulin.binding region of tau. These peptides included HQPGGGKVQIVYKPVDLSKV (BRI), SKVTSKCGSLGNIHHKPGGG (BR2), PGGGQVEVKSEKLDFKDRVQ (BR3), DRVQSKIGSLDNITHVPGGG (BR4), and PGGGNKKIETHKLTFRENAK (BRS). The structures of all synthetic peptides were confirmed by mass spectroscopy. The /~/A4 (I-40) peptide corresponding to amino acids 597-636 of APP695 and adrenocorticolropic hormone (I-24) were purchased from Bachem.
Electron microscopy of fibrils C-APP peptide was routinely dissolved in water or 2.5 mM Tris-buffer, pH 7, heated at 37°C or fi0°C for ! h, applied to carbon and formvar-eoated grids, partially dried by blotting and stained with 2r~ uranyl acetate. Samples were examined with a Zeiss EM 10A electron microscope with an accelerating voltage of 60 kV. Where indicated C-APP was prepared in 10-10(} mM NaCI or KCI or in 0.01 N tlCI. Other APP peptides, except the ,8/A4 peptide, were dissolved in 2.5 mM Tris-HCI at 12.5 mg/ml, heated at 6iPC for I h, and examined by electron microscopy as described for C-APP. The fl/A4 peptide was dissolved at 12.9 mg/ml in 35% acetonitrile and 0.1% trifluoroacetic acid, heated at 6O°Cand examined as described for C-APP. Tau proteins and peplides at 1.5 mg/ml were added to (}.15-5.0 mg/ml C-APP or/~/A4 peplide prior to incubation. ConIrol samples, containing tau protein or peptide in the absence of an APP peptide, were also examined in each experiment. PHFs were applied to carbon and formvar-coated grids, partially dried by blotling, and stained with I% lithium phosphotungstic acid.
Fig. I. C-APP peptide at a concentration of 15 mg/ml in water was incubated at 611°CCA)or 37~C (B) for I h or 10 mg/ml was incubated at 37°C for I h (C), applied to grids, stained with 2% uranyl acetate and examined by electron microscopy. Magnification, A, C: 25,000×; B: 40,1}(N1x .
Ct,tttrffugation atul other analyses of C-APPfibrils C-APP peptide and PHFs were prepared in !% SDS, I% Triton X-t00, 20 mM CAPS, or 2-6 M guanidine-HCl: C-APP and PHF samples were also prepared in 6 M guanidine-SCN in 50 mM Tris-HCI, pH 8.4 with 5 mM EDTA, 2 mM dithiothreitol, and 6 mM iodoacetie acid (Wischik et al., manuscript in preparation). Samples were examined by electron microscopy as described above. Samples were also centrifuged at 160,000x g to separate fibrils in the pellet from non-polymerized peptide in the supernatant. The protein con. lents of the pellets and supernatants were measured 3~. Samples of 1.5 or 12.5 mg/ml C-APP and 1.5 mg/ml bovine tau were also centrifuged at 160,000× g. The tau content of the supernatants and pellets were determined by ELISA7 using tau.14 anti. body 2"~,generously provided by Dr. Virginia Lee. RESULTS W h e n 10-15 m g / m l C - A P P was dissolved in either 2.5-12.5 m M Tris, p H 7, o r water and i n c u b a t e d at 60°C for 60 min, twisted fibrils w e r e o b s e r v e d (Fig. 1A). Most or all o f the fibrils lacked twists w h e n the peptide was incubated at lower t e m p e r a t u r e s o r for s h o r t e r periods o f time (Fig. 1B,C).
229
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Fig. 2. PHFs (A), C-APP peptide dissolvedin water (B-D) or in 10tl mM NaCI(E, F) at 15 mg/ml, or a syntheticvcptide corresponding to aminoacids 1-40 of/3/A4 (G) were incubatedat 60°Cfor 1 h and examined by electron microscopy.PHFs were stained with lithium phosphotungstic acid and peptide samples were stained with uranyl acetate. Samples in panels (B) and (C) were also stainedbrieflywith 0.25% AIsecblue after the sample was blotted. Magnification,A, C, F, G: 25,000x ; B, D, E: 4l),00ll. The morphology of the fibrils is shown in more detail in Fig. 2. The longitudinal striations described for PHFs (Wischik et al., 1985) are present (Fig. 2B,C). The lengths of the periods for the fibrils in Fig. 2B and 2C are 41 and 53 nm, respectively, compared to 67 nm for the PHFs in Fig. 2A. The C-APP fibrils are 8-13 nm wide at the widest part of the fibril compared to 13 nm for the PHFs. Occasionally a few twisted fibrils were observed which showed longer periodicities (83116 nm) and were 17 nm wide (Figs. 1B, 2D, 2E). in these cases the fibrils displayed the twisted ribbon morphology described for PHFs ~. Analogous to C-APP fibrils, neurofibrillary tangles are also known to contain filaments with these types of morphological variations, even though they are composed of the same subunit H. The morphology of C-APP fibrils was affected by
the presence of monovalent salts. Incubating the peptide in water plus 10-100 mM NaCI or KCI resulted in the formation of non-twisted fibrils. However with 100 mM NaCI, fibrils with the wide periodicity of PHFs were also occasionally observed (Fig. 2E). Fibrils with an alternate morphology which resembled interlocking twisted fibrils were also observed (Fig. 2F). These effects of NaCI on C-APP morphology did not occur in the presence of 0.01 N HCI. Various other peptides were subjected to the same conditions that generated twisted C-APP fibrils and were examined for fibril formation. Most of these peptides did not lbrm fibrils or formed fibrils that did not resemble PHFs. These peptides included those corresponding to amino acids 572-587, 585-600, 597610, 611-624, 621-631, 649-668, and 662-681 of APP695. However one peptide, amino acids 1-40 of /3/A4 (597-636 of APP695), at a concentration of 0.5 m g / m l or greater formed fibrils with a twisted morphology (Fig. 2G). The period length and the width of these fibrils are 107 nm and 13 nm, respectively. Another peptide which contains only amino acids 34-42 of / I / A 4 was previously reported to form amyloid fibrils, some of which showed a twisted morphology even without incubation ~3. Therefore peptides with several different sequences from APP can polymerize into twisted amyloid fibrils. Tau protein had a dramatic effect on C-APP fibril formation. C-APP at 1.5 mg/ml in water formed almost no fibrils alone, whereas in the presence of bovine tau massive numbers of fibrils were present uniformly across each grid (Fig. 3, Table I). No fibrils were ever observed in any samples containing solely tau. Other proteins such as bovine serum albumin and adrenocorticotropic hormone did not alter C-APP fibril formation. Bovine tau protein at 1.5 mg/ml had no significant effect on the number of fibrils formed with 0.5 m g / m l fl/A4. Almost no fibrils formed with 0.15 mg/ml in the absence or presence of 1.5 mg/ml tau protein. Three isoforms of nonphosphorylated recombinant human tau expressed in E. coil also induced massive C-APP fibril formation at low C-APP concentrations. The shortest and Iohgest isoforms of tau were equally effective, as was an expressed construct containing only the repeat region of the longest isoform (Fig. 3, Table 1). Several peptides (BR1-BR5) with sequences from the repeat region of tau were ineffective. The isoelectric points of BR1, BR2, and BR5 were 10.5, 10.7 and 10.9, respectively. These values are similar to the isoelectric point of 10.5 for the repeat region, suggesting that charge alone is not the basis for induction of C-APP fibril formation.
230 TABLE I
C-APP fibril formation C-APP fibril formation was assessed in the presence of 1.5 m g / m l tau protein in 2.5 mM Tris-buffer, pH 7, or in water. The mean n u m b e r +S.D. of fibrils per microscope field (at 25,000x magnification) is shown. Soh'ent
C-APP conch. (mg / ml)
Acerage number of fibrils per field (n) C-APP C-APP + tau
Type of tau c
"Iris
5.0
16_+ 7 (7)
5.0 0.5
8_+ 6 (8) 3_+ 2(10)
100+ a (5) 100+ a (6) 100+ a (7) 100 + a (6) 77+12(15) 6 + 4(11)
human(~-3) human (74) human (¢448) human (repeat region) bovine bovine
1.5 0.15
10+_ 12 (8) 1 (l) b
lO0+ a (19) 40+_19 (4)
bovine bovine
Water
In fields with over 100 fibrils present an accurate count could not be made as individual fibrils are not easily discerned from each other at this density. t, Only one fibril was found per grid. 73. tau isoform with no inserts: ~'4, isoform with one insert in the tubulin-binding region; ¢4ss, isoform with all inserts present; repeat region, fragment of tau that contains only the tubulin-binding region with insert (amino acids 264-386 of ¢45s).
The solubility properties of C-APP fibrils were compared to those of PHFs. When observed by electron microscopy, C-APP fibrils and PHFs both appeared to be stable to treatment with 2 M guanidine-HCl (although most C-APP fibrils lacked twists). No C-APP fibrils remained after treatment with 6 M guanidineSCN, which also solubilizes PHFs (Wischik et al., manuscript in preparation). The proportion of C-APP protein recovered in the pellet after ¢entrifugation was also reduced, from 15.8-19.6% to 1.7%, with 6 N guanidine-SCN treatment but was not reduced after 1% SDS, 1% Triton X-100, 6 M guanidine-HCI or 20 mM CAPS treatment, conditions that also do not solubilize PHFs. Tau protein appears to bind to C-APP fibrils. When bovine tau was added to C-APP, most of the tau was recovered in the pellet. From 59.6 :t: 2.2% to 63.4:1: 2.1% of tau immunoreactivity was recovered in the pellet when tau was combined with 1.5 and 12.'5 mg/ml C-APP, respectively. In the absence of C-APP only 6.7% of tau was recovered in the pellet. DISCUSSION
The present results demonstrate that both /3/A4 (amino acids 1-40) and C-APP can form fibrils that closely resemble PHFs morphologically. However only C-APP fibrils were induced to form by tau protein. The presence of only a fragment of tau was adequate to induce C-APP fibril formation. This fragment contained the tubulin-binding region of tau and closely resembles the fragment of tau that remains tightly bound to PHFs even after pronase treatment 4~. Our data raise the possibility that C-APP or a peptide with
a related sequence may be an important factor in PHF formation, which along with tau and other PHF components, may be involved in nucleation events or copolymerization. The immunological6'8't2'2°'35 and biochemical s'4° evidence suggesting that the C-terminus of APP is present in PHFs substantiates this possibility. Validation of a roIe for the C-terminus of APP in PHF formation would provide a biochemical link between the etiologies of both neurofibriilary and fl/A4 pathology. Specifically, proteolytic processing of APP may play a part in generating both lesions. The presumed proteolytic event that leads to the release of a fragment from the C-terminal region of APP that associates with PHF-tau has not been defined. Lysosomes may play a role in the proteolytic generation of both /3/A4 and the C-terminus of APP. Lysosomes have already been suggested to be important in the processing of t h e / 3 / A 4 region of APP ~°'t7. In addition PHFtau has been localized to granulovacular degeneration complexes t which are related to lysosomes3° and may be the site of initial PHF formation. Additional evidence also suggests that the appearance of plaques and of tangles is linked. For example, PHFs are present in both lesions. Both lesions usually develop slowly with aging and more rapidly as Alzheimer's disease progresses and as patients with Down's syndrome age. It seems unlikely that the co-development of these two lesions is coincidental. A single mutation in the APP gene seems to be sufficient to lead to Alzheimer pathology, including the appearance of both plaques and tangles ~5. Experimental data also suggest a link between the two lesions. When brain grafts of trisomy 16 mice (which are trisomic for the APP gene) were transplanted into normal mice, ira-
231 may provide a biochemical link between both or the amyloid proteins in AIzheimer's disease. Acknowledgements. The authors would like to gratefully acknowledge the technical assistance of Patricia DeFeo, William Brunner and Andrea Klika. PHFs were kindly provided by Dr. Claude Wischik, the tau isoforms by Drs. Peter Barth and David Blowers and the tau fragment by Dr. Mathew Lo. The tau clones were provided by Dr. Michel Goedert. REFERENCES
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Fig. 3. C-APP peptide was dissolved at 5 mg/ml in 2.5 mM Trisbuffer, pH 7, and was incubated for 1 h at 60°C alone (A) or in the presence of 1.5 mg/ml bovine tau (B), or recombinant human tau with no inserts (C), one insert in the tubulin binding region (D) or all inserts (E) or with a fragment with only the tubulin-bindingregion of tau (F). Samples were stained with 2% uranyl acetate and examined by electron microscopy. Magnification, 25,000 x.
munological changes developed in the grafts that were characteristic of PHFs as well as of f l / A 4 3 3 . In conclusion our data raise the possibility that a C-terminal fragment of APP may play an important role in PHF formation, probably in concert with tau protein. Therefore the proteolytic processing of APP
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232 Merit, R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M. and Hardy, J., Segregation L:f a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nau¢w, 349 (1991) 704-706. 16 Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A., Multiple isoforms of human microtubule-associated protein tee: sequences and localization in neurofibrillary t:ang!e~of ,~!-_heimer'sdisease, Neuron, 3 (I 989) 519-526. 17 Golde, T.E., Es~us, S., Younkin, L.H., Selkoe, D.J. and Younkin, S.G., Processing of the amyloid protein precursor to potentially amyloidogenic derivatives, Science, 255 (1992) 728-730. 18 Grundke-lqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, M.S. and Wisniewski, H.M., Microtubule-associated protein tau. A component of Alzheimer paired helical filaments, J. Bml. Chem., 261 (1986) 6084-6089. 19 Grundke-lqbal, 1., lqbal, K., Tung, Y.-C., Quinlan, M., Wisniewski, H.M. and Binder, L.I., Abnormal phosphorylation of the microtubule associated protein tau (r) in AIzheimer cytoskeletal pathology, Prec. Natl. Acad. Set. USA, 83 (1986) 4913-4917. 20 Ishii, T., Kametani, F., Haga, S. and Sate, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4-protein in senile plaques in AIzheimer's disease, Neu~opathol. Appl. Neurobiol., 15 (1989) 135-147. 21 Kang,J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K. and Muller-Hill, B., The precursor of AIzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. 22 Kondo, J., Honda, T., Mort, H., Hamada, Y., Miura, R., Ogawara, M. and Ihara, Y., The carboxyl third of tau is tightly bound to paired helical filaments, Neuron, I (1988) 827-834. 23 Kosik, K.S., Duffy, L.K., Dowling, M.M., Abraham, C., McClaskey, A. and Selkoe, D.,l., Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain ¢pitopes into Alzheimer neurofibrillary tangles, Prec. Nail. Acad. Sci. USA, 81 (1984) 7941-7945. 24 Kosik, K.S., Joachim, C.L, and Selkoe, D.J., Microtubule-assocatied protein tau (~-) is a major antigenic component of paired helical filaments in Alzheimer disease, Prec. Natl. Acad. &'i. USA, 83 (1986) 4044-41148. 25 Kosik, K.S., Orecchio, L.D., Binder, L., Trojanowski, ,l.Q., Lec, V.M.Y. and Lee. G., Epitopes that sp:m the tau molecule are shared with paired helical filaments, Nei~ron, I (1988) 817-825, 26 Masters, C,L., Multhaup, G., Simms, G., Pottgiesser, J,, Martins, R.N. and Beyrcuther, K., Neuronal origin of a cerebral amyloid: ncurofibrillary tangles of AIzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels, EMBO J., 4 (1985) 2757-2763. 27 Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K., Amyloid plaque core protein in AIzheimer disease and Down syndrome, Prec. Natl. Aead. Sci. USA, 82 (1985) 4245-4249. 28 Montejo de Garcini, E., Carrascosa, J.L., Correas, !., Nieto, A. and Avila, ,l., Tau hctor polymers are similar to paired helical filaments of Alzheimer's disease, FEBS Lett., 236 (1988) 150-154.
29 Mort, H., Kondo, J. and lhara, Y., Ubiquilin is a component of paired helical filaments in Alzheimer's disease, Science, 235 (1987) 1641-1644. 30 Okamoto, K., Hirai, S., lizuka, T., Yanagisaja, T. and Watanabe, M., Reexamination of granulovacuolar degeneration, Acta Neuropathol., 82 (1991) 340-345. 31 Perry, G., Friedman, R., Shaw, G. and Chau, V., Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of AIzheimer disease brains, Prec. Natl. Acad. Sci. USA, 84 (1987) 3033-3036. 32 Perry, G., Johnsok~, A.B., Mulvihill, P., Siedlak, S., Galloway, P., Tabaton, M. and Gambetti, P., AIzheimer's disease paired helical filaments contain tubulin, J. Neuropathol. Exp. Neurol., 48 (1989) 354. 33 Richards, S.-J., Waters, J.J., Beyreuther, K., Masters, C.L., Wischik, C.M., Sparkman, D.R., White, C.L., Abraham, C.R. and Dunnett, S.B., Transplants of mouse trisomy-16 hippoeampus provide a model of Alzheimer's disease neuropathology, EMBO J., ll) (1991) 297-303. r 34 Scott, C.W, Blowers, D.P., Barth. P.T., Lo, M.M.S., Salama, A.I. and Caputo, C.B., Differences in the abilities of human tau isoforms to promote microlubule assembly, J. Neurosci. Res., 30 (1991) 154-162. 35 Shoji, M., Hirai, S, Yamaguchi, H., Harigaya, Y. and Kawarabayashi, T., Amyloid /3-protein precursor accumulates in dystrophic neurites of senile plaques in Alzheimer-type dementia, Brain Res, 512 (1990) 164-168. 36 Smith, P.K., Krohn, R.I., Hermanson,~.T., Mallia, A.K., Garb ner, F.H., Provenzano, M.D., FujimotC E.K., Goeke, N.M., OIson, B.J. and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150 (1985) 76-85. 37 Sternberger, N.H., Sternberger, L.A. and Ulrich, `l., Aberrant neurofilament phosphorylation in AIzheimer's disease, Prt~'. Natl. Acad. Sci. USA, 82 (1985) 4274-4276. 38 Vogelsang, G.D,, Zemlan, F.P. and Dean, G.E., Untangling the insoluble: a characterization of Alzheimer's paired helical filaments. In C. Finch and P. Davies (Eds.), The Molecular Biology of AIztwimer's Disease, Cold Spring Harbor Laboratory, New York, It)88, pp. 150-158, 39 Wischik, C.M., Crowther, R.A., Stewart, M. and Roth, M., Subunit structure of paired helical filaments in AIzheimer's disease, J. Cell Biol,, 100 (1985) 1905-1912. 411 Wischik, C.M., Novak, M., `lakes, R,, Edwards, P, and Harrington, C.R., Tau/APP association in AIzheimer neurofibrillary pathology, Kumamoto Med. J., 42 (1991) S!5-S16. 41 Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, P.C., Runswick, M.J., `lakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filament of AIzheimer disease, Prec. Natl. Acad. Sci. USA, 85 0988)4506-4510. 42 Wood, J.G., Mirra, S.S., Pollock, NJ. and Binder, L.I., Neurofibrillary tangles of Alzhcimer disease share antigenic determinants with the axonal microtubule-associated protein tau (~-), Prec. Natl. Acad. Sci. USA, 83 (1986) 4040-4043.
Brain Research, 597 (1992) 227-232 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18301
Role of tau in the polymerization of peptides from/3-amyloid precursor protein C l a u d i a B. C a p u t o , L i n d a A . Sygowski, C l a y W S c o t t a n d I r e n e R. E v a n g e l i s t a S o b e l Pharmacology Department, ICi Pharmaceuticals Group, LW-2, ICI Americas, Wilmbrgton, DE 19897 (USA) (Accepted 30 June 1992)
Key words: AIzheimer's disease; Tau protein; Microtubule-associated protein; Amyloid; fl-Amyloid precursor protein; Fibrilogenesis; Amyloidogenesis; Paired helical filament
The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from ~-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A ~" fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from t h e / 3 / A 4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of AIzheimer's disease.
INTRODUCTION Paired helical filaments (PHFs) are amyloid fibrils that are present in both of the hallmark pathological lesions of AIzhiemer's disease, senile plaques and neurofibrillary tangles. They accumulate intracellularly in tangles and in the dystrophic neurites that surround the central core of senile plaques. The composition of PHFs is not fully known. A fragment of tau protein has been demonstrated to be an integral constituent of PHFs 3"22'24"4t.Some tau which co-purifies with PHFs is thought to be ubiquitinated 29'31 and aberrantly phosphorylated t9.42. The PHF structure is highly insoluble, which leaves the possibility that other proteins are also present in PHFs but are not amenable to solubilisation and are not antigenic or accessible to antibodies. In addition tau does not readily form fibrils in vitro 28 a~d has not been reported to form fibrils with amyloid properties. This situation contrasts with that of other amyloidogenie proteins which readily form amyloid fibrils in vitro. For example, the other type of amyloid fibril in
Alzheimer lesions is comprised of /3/A4 peptide 27, which readily forms amyloid fibrils in vitro 'J. This peptide is a fragment of up to 43 amino acids from ~-amyloid protein precursor 2~ (APP). The amino acid sequence of APP resembles that of transmembrane proteins 21. /3/A4 spans the C-terminal region of the extracellular domain and the adjacent N-terminal region of the transmembrane domain of APP. Other proteins have been suggested to be present in PHFs including /3/A4 26, MAP2 2a, neurofilament a7, actin aS, tropomyosin 14, and tubulin 32. In general antibodies to these proteins label tangles, although these antibodies do not react with isolated PHFs and the proteins themselves do not form fibrils that resemble PHFs. Thus their contributions to the PHF structure remain undefined. The C-terminus of APP is thought to be located inside the cell 2t, where PHFs form. Recent immunological data provides evidence that a C-terminal fragment of APP may be associated with PHFs in both tangles t2 and the dystrophic neurites of senile plaques 2°'35 as well as with isolated PHFs even after
Correspondence: C.B. Caputo, Pharmacology Department, ICl Pharmaceuticals Group, LW-2, ICI Americas, Wilmington, DE 19897, USA. Fax: (1) (302) 886-2766.
228 pronase t r e a t m e n t ~''"'4". A n amino acid s e q u e n c e derived from the C-terminal region o f A P P has also been detected in P H F s ~'4°. A synthetic peptide, C - A P P , which corresponds to C-terminal a m i n o acids 676-695 o f APP695, polymerizes into amyloid fibrils but these fibrils lack the characteristic m o r p h o l o g y o f P H F s , that o f twisted fibrils with a regular periodicity 4. T h e r e f o r e we investigated w h e t h e r the C-terminus o f A P P can form fibrils that resemble P H F s morphologically and interact with the only confirmed c o m p o n e n t o f PHFs, tau protein. Identification o f the proteins that are required for P H F s to assemble may help to elucidate the pathobiological processes that lead to P H F formation. PHFs in t u r n p r o b a b l y c o n t r i b u t e to the dem~.-~tia o f AIzheimer's disease 5. Recent evidence is now suggesting.that the n u m b e r s and locations o f the P H F - b e a r i n g tangles but not o f f l / A 4 plaques correlates with the severity of dementia-'. Thus an u n d e r s t a n d i n g o f P H F formation may be useful in the d e v e l o p m e n t o f effective strategies for designing therapies for Alzheimer's disease.
x !~,~
MATERI,a, LS AND METHODS
Protein,v and pt,ptides PHFs were isolated as the ifll fraction from Alzheimer brain and treated with pronase as previously described41, Bovine tau was prepared as described previously =~. Recombinant ht, man tau isoforms =r' with no inserts (T3), with one insert in the tuhulin-hinding region (~-4). and with all inserts (~'4.~) and amino acids 2t,4-386 of Ihe tuhulin-binding region of T4 were expressed in 1:;. coil as previously describedTM.
Pcptides were synthesized which correspond to APP695 := amino acids 572-587, 585-f~0i1, 597-610, 611-624, 621-(~31,649-(~f~8, f~fi2681, and 676-695 (C-APP). P~plides were synthesized corresponding to amino ackls from the tuhulin.binding region of tau. These peptides included HQPGGGKVQIVYKPVDLSKV (BRI), SKVTSKCGSLGNIHHKPGGG (BR2), PGGGQVEVKSEKLDFKDRVQ (BR3), DRVQSKIGSLDNITHVPGGG (BR4), and PGGGNKKIETHKLTFRENAK (BRS). The structures of all synthetic peptides were confirmed by mass spectroscopy. The /~/A4 (I-40) peptide corresponding to amino acids 597-636 of APP695 and adrenocorticolropic hormone (I-24) were purchased from Bachem.
Electron microscopy of fibrils C-APP peptide was routinely dissolved in water or 2.5 mM Tris-buffer, pH 7, heated at 37°C or fi0°C for ! h, applied to carbon and formvar-eoated grids, partially dried by blotting and stained with 2r~ uranyl acetate. Samples were examined with a Zeiss EM 10A electron microscope with an accelerating voltage of 60 kV. Where indicated C-APP was prepared in 10-10(} mM NaCI or KCI or in 0.01 N tlCI. Other APP peptides, except the ,8/A4 peptide, were dissolved in 2.5 mM Tris-HCI at 12.5 mg/ml, heated at 6iPC for I h, and examined by electron microscopy as described for C-APP. The fl/A4 peptide was dissolved at 12.9 mg/ml in 35% acetonitrile and 0.1% trifluoroacetic acid, heated at 6O°Cand examined as described for C-APP. Tau proteins and peplides at 1.5 mg/ml were added to (}.15-5.0 mg/ml C-APP or/~/A4 peplide prior to incubation. ConIrol samples, containing tau protein or peptide in the absence of an APP peptide, were also examined in each experiment. PHFs were applied to carbon and formvar-coated grids, partially dried by blotling, and stained with I% lithium phosphotungstic acid.
Fig. I. C-APP peptide at a concentration of 15 mg/ml in water was incubated at 611°CCA)or 37~C (B) for I h or 10 mg/ml was incubated at 37°C for I h (C), applied to grids, stained with 2% uranyl acetate and examined by electron microscopy. Magnification, A, C: 25,000×; B: 40,1}(N1x .
Ct,tttrffugation atul other analyses of C-APPfibrils C-APP peptide and PHFs were prepared in !% SDS, I% Triton X-t00, 20 mM CAPS, or 2-6 M guanidine-HCl: C-APP and PHF samples were also prepared in 6 M guanidine-SCN in 50 mM Tris-HCI, pH 8.4 with 5 mM EDTA, 2 mM dithiothreitol, and 6 mM iodoacetie acid (Wischik et al., manuscript in preparation). Samples were examined by electron microscopy as described above. Samples were also centrifuged at 160,000x g to separate fibrils in the pellet from non-polymerized peptide in the supernatant. The protein con. lents of the pellets and supernatants were measured 3~. Samples of 1.5 or 12.5 mg/ml C-APP and 1.5 mg/ml bovine tau were also centrifuged at 160,000× g. The tau content of the supernatants and pellets were determined by ELISA7 using tau.14 anti. body 2"~,generously provided by Dr. Virginia Lee. RESULTS W h e n 10-15 m g / m l C - A P P was dissolved in either 2.5-12.5 m M Tris, p H 7, o r water and i n c u b a t e d at 60°C for 60 min, twisted fibrils w e r e o b s e r v e d (Fig. 1A). Most or all o f the fibrils lacked twists w h e n the peptide was incubated at lower t e m p e r a t u r e s o r for s h o r t e r periods o f time (Fig. 1B,C).
229
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',,
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o9,~,
.
,
"'':
.'i.
r,
Fig. 2. PHFs (A), C-APP peptide dissolvedin water (B-D) or in 10tl mM NaCI(E, F) at 15 mg/ml, or a syntheticvcptide corresponding to aminoacids 1-40 of/3/A4 (G) were incubatedat 60°Cfor 1 h and examined by electron microscopy.PHFs were stained with lithium phosphotungstic acid and peptide samples were stained with uranyl acetate. Samples in panels (B) and (C) were also stainedbrieflywith 0.25% AIsecblue after the sample was blotted. Magnification,A, C, F, G: 25,000x ; B, D, E: 4l),00ll. The morphology of the fibrils is shown in more detail in Fig. 2. The longitudinal striations described for PHFs (Wischik et al., 1985) are present (Fig. 2B,C). The lengths of the periods for the fibrils in Fig. 2B and 2C are 41 and 53 nm, respectively, compared to 67 nm for the PHFs in Fig. 2A. The C-APP fibrils are 8-13 nm wide at the widest part of the fibril compared to 13 nm for the PHFs. Occasionally a few twisted fibrils were observed which showed longer periodicities (83116 nm) and were 17 nm wide (Figs. 1B, 2D, 2E). in these cases the fibrils displayed the twisted ribbon morphology described for PHFs ~. Analogous to C-APP fibrils, neurofibrillary tangles are also known to contain filaments with these types of morphological variations, even though they are composed of the same subunit H. The morphology of C-APP fibrils was affected by
the presence of monovalent salts. Incubating the peptide in water plus 10-100 mM NaCI or KCI resulted in the formation of non-twisted fibrils. However with 100 mM NaCI, fibrils with the wide periodicity of PHFs were also occasionally observed (Fig. 2E). Fibrils with an alternate morphology which resembled interlocking twisted fibrils were also observed (Fig. 2F). These effects of NaCI on C-APP morphology did not occur in the presence of 0.01 N HCI. Various other peptides were subjected to the same conditions that generated twisted C-APP fibrils and were examined for fibril formation. Most of these peptides did not lbrm fibrils or formed fibrils that did not resemble PHFs. These peptides included those corresponding to amino acids 572-587, 585-600, 597610, 611-624, 621-631, 649-668, and 662-681 of APP695. However one peptide, amino acids 1-40 of /3/A4 (597-636 of APP695), at a concentration of 0.5 m g / m l or greater formed fibrils with a twisted morphology (Fig. 2G). The period length and the width of these fibrils are 107 nm and 13 nm, respectively. Another peptide which contains only amino acids 34-42 of / I / A 4 was previously reported to form amyloid fibrils, some of which showed a twisted morphology even without incubation ~3. Therefore peptides with several different sequences from APP can polymerize into twisted amyloid fibrils. Tau protein had a dramatic effect on C-APP fibril formation. C-APP at 1.5 mg/ml in water formed almost no fibrils alone, whereas in the presence of bovine tau massive numbers of fibrils were present uniformly across each grid (Fig. 3, Table I). No fibrils were ever observed in any samples containing solely tau. Other proteins such as bovine serum albumin and adrenocorticotropic hormone did not alter C-APP fibril formation. Bovine tau protein at 1.5 mg/ml had no significant effect on the number of fibrils formed with 0.5 m g / m l fl/A4. Almost no fibrils formed with 0.15 mg/ml in the absence or presence of 1.5 mg/ml tau protein. Three isoforms of nonphosphorylated recombinant human tau expressed in E. coil also induced massive C-APP fibril formation at low C-APP concentrations. The shortest and Iohgest isoforms of tau were equally effective, as was an expressed construct containing only the repeat region of the longest isoform (Fig. 3, Table 1). Several peptides (BR1-BR5) with sequences from the repeat region of tau were ineffective. The isoelectric points of BR1, BR2, and BR5 were 10.5, 10.7 and 10.9, respectively. These values are similar to the isoelectric point of 10.5 for the repeat region, suggesting that charge alone is not the basis for induction of C-APP fibril formation.
230 TABLE I
C-APP fibril formation C-APP fibril formation was assessed in the presence of 1.5 m g / m l tau protein in 2.5 mM Tris-buffer, pH 7, or in water. The mean n u m b e r +S.D. of fibrils per microscope field (at 25,000x magnification) is shown. Soh'ent
C-APP conch. (mg / ml)
Acerage number of fibrils per field (n) C-APP C-APP + tau
Type of tau c
"Iris
5.0
16_+ 7 (7)
5.0 0.5
8_+ 6 (8) 3_+ 2(10)
100+ a (5) 100+ a (6) 100+ a (7) 100 + a (6) 77+12(15) 6 + 4(11)
human(~-3) human (74) human (¢448) human (repeat region) bovine bovine
1.5 0.15
10+_ 12 (8) 1 (l) b
lO0+ a (19) 40+_19 (4)
bovine bovine
Water
In fields with over 100 fibrils present an accurate count could not be made as individual fibrils are not easily discerned from each other at this density. t, Only one fibril was found per grid. 73. tau isoform with no inserts: ~'4, isoform with one insert in the tubulin-binding region; ¢4ss, isoform with all inserts present; repeat region, fragment of tau that contains only the tubulin-binding region with insert (amino acids 264-386 of ¢45s).
The solubility properties of C-APP fibrils were compared to those of PHFs. When observed by electron microscopy, C-APP fibrils and PHFs both appeared to be stable to treatment with 2 M guanidine-HCl (although most C-APP fibrils lacked twists). No C-APP fibrils remained after treatment with 6 M guanidineSCN, which also solubilizes PHFs (Wischik et al., manuscript in preparation). The proportion of C-APP protein recovered in the pellet after ¢entrifugation was also reduced, from 15.8-19.6% to 1.7%, with 6 N guanidine-SCN treatment but was not reduced after 1% SDS, 1% Triton X-100, 6 M guanidine-HCI or 20 mM CAPS treatment, conditions that also do not solubilize PHFs. Tau protein appears to bind to C-APP fibrils. When bovine tau was added to C-APP, most of the tau was recovered in the pellet. From 59.6 :t: 2.2% to 63.4:1: 2.1% of tau immunoreactivity was recovered in the pellet when tau was combined with 1.5 and 12.'5 mg/ml C-APP, respectively. In the absence of C-APP only 6.7% of tau was recovered in the pellet. DISCUSSION
The present results demonstrate that both /3/A4 (amino acids 1-40) and C-APP can form fibrils that closely resemble PHFs morphologically. However only C-APP fibrils were induced to form by tau protein. The presence of only a fragment of tau was adequate to induce C-APP fibril formation. This fragment contained the tubulin-binding region of tau and closely resembles the fragment of tau that remains tightly bound to PHFs even after pronase treatment 4~. Our data raise the possibility that C-APP or a peptide with
a related sequence may be an important factor in PHF formation, which along with tau and other PHF components, may be involved in nucleation events or copolymerization. The immunological6'8't2'2°'35 and biochemical s'4° evidence suggesting that the C-terminus of APP is present in PHFs substantiates this possibility. Validation of a roIe for the C-terminus of APP in PHF formation would provide a biochemical link between the etiologies of both neurofibriilary and fl/A4 pathology. Specifically, proteolytic processing of APP may play a part in generating both lesions. The presumed proteolytic event that leads to the release of a fragment from the C-terminal region of APP that associates with PHF-tau has not been defined. Lysosomes may play a role in the proteolytic generation of both /3/A4 and the C-terminus of APP. Lysosomes have already been suggested to be important in the processing of t h e / 3 / A 4 region of APP ~°'t7. In addition PHFtau has been localized to granulovacular degeneration complexes t which are related to lysosomes3° and may be the site of initial PHF formation. Additional evidence also suggests that the appearance of plaques and of tangles is linked. For example, PHFs are present in both lesions. Both lesions usually develop slowly with aging and more rapidly as Alzheimer's disease progresses and as patients with Down's syndrome age. It seems unlikely that the co-development of these two lesions is coincidental. A single mutation in the APP gene seems to be sufficient to lead to Alzheimer pathology, including the appearance of both plaques and tangles ~5. Experimental data also suggest a link between the two lesions. When brain grafts of trisomy 16 mice (which are trisomic for the APP gene) were transplanted into normal mice, ira-
231 may provide a biochemical link between both or the amyloid proteins in AIzheimer's disease. Acknowledgements. The authors would like to gratefully acknowledge the technical assistance of Patricia DeFeo, William Brunner and Andrea Klika. PHFs were kindly provided by Dr. Claude Wischik, the tau isoforms by Drs. Peter Barth and David Blowers and the tau fragment by Dr. Mathew Lo. The tau clones were provided by Dr. Michel Goedert. REFERENCES
I
~,'?.
i x~
,,
Fig. 3. C-APP peptide was dissolved at 5 mg/ml in 2.5 mM Trisbuffer, pH 7, and was incubated for 1 h at 60°C alone (A) or in the presence of 1.5 mg/ml bovine tau (B), or recombinant human tau with no inserts (C), one insert in the tubulin binding region (D) or all inserts (E) or with a fragment with only the tubulin-bindingregion of tau (F). Samples were stained with 2% uranyl acetate and examined by electron microscopy. Magnification, 25,000 x.
munological changes developed in the grafts that were characteristic of PHFs as well as of f l / A 4 3 3 . In conclusion our data raise the possibility that a C-terminal fragment of APP may play an important role in PHF formation, probably in concert with tau protein. Therefore the proteolytic processing of APP
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