Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3033-3036, May 1987 Medical Sciences is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains (senile /paired helical filament/proteolysis/immunocytochemistry/cytoskeleton) GEORGE PERRY*, ROBERT FRIEDMAN*, GERRY SHAWt, AND VINCENT CHAUO *Division of Neuropathology, Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; and Departments of tNeuroscience and tBiochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610 Communicated by E. R. Stadtman, February 9, 1987

ABSTRACT Neurofibrillary tangles (NFT) and neurites MATERIALS AND METHODS associated with senile plaques (SP) in Alzheimer disease- affected brain tissues were specifically immunostained with Tissue Source. Tissues from seven postmortem Alzheimer affinity-purified preparations directed against ubiq- patients, ranging in age at death from 69 to 82 years, were uitin. In addition, a class of neurites seen in brain regions obtained. In each case, the patient had shown a definite containing NFT and SP were also specifically stained. Cross- history of dementia and met the National Institute on Aging reactivity of the ubiquitin antisera for tau , neurofila- guidelines for Alzheimer disease (12). A biopsy sample from ment , and high molecular weight -associ- another Alzheimer patient was obtained, with tissue being ated proteins (MAPs) were ruled out by (i) the inability of the removed from the frontal cortex of a 69-year-old patient. ubiquitin antisera to stain these proteins in immunoblotting Control brains from non-Alzheimer subjects were from a and the of 101-year-old individual, and from four individuals between 36 experiments (ii) inability tau, , and and 69 years old. MAP preparations, when preincubated with the ubiquitin Immunostaining. Tissue was fixed in 10% formalin or antisera, to inhibit the selective neurofibrillar staining ob- Bouin's fixative for 1-2 days and embedded in paraffin, and served. Our results are consistent with the suggestion that 5-,4m sections were cut. Sections were stained by the ubiquitin is covalently associated with the insoluble neurofibril- peroxidase-antiperoxidase technique (13) with 3',3'-diamino- lary material of NFT and SP. We propose that the ubiquitin- benzidine as the reaction cosubstrate. In some cases, stained mediated degradative pathway may be ineffective in removing sections were treated with 1% osmium tetroxide for 30 s to these fibrillar structures in Alzheimer disease brain. slightly enhance positive staining. Stained sections were mounted in Permount. Alternatively, 10-pim sections of Neurofibrillary tangles (NFT) and senile plaques (SP) are two unfixed frozen tissues were cut on a cryostat and processed prominent histologically defined lesions found in distinct for immunofluorescence microscopy. regions of brain tissue in patients affected with Alzheimer . Antibodies to ubiquitin were prepared as de- disease (1). NFT and SP can be visualized at the light- scribed (14). Purified ubiquitin was covalently bound to microscope level by a variety ofchemical staining procedures keyhole limpet hemocyanin as described by Haas and Bright (2). At the electron-microscope level, the fibrillary structures (15). The conjugate was injected into two rabbits, and are seen to be composed predominantly of paired helical immune sera were collected. Both sera stained ubiquitin and filaments in addition to components Direct ubiquitin-protein conjugates in rabbit reticulocyte lysate amorphous (3). (14). One of these antisera was selected for affinity purifica- determination of the protein composition of these fibrillary tion on ubiquitin coupled to Sepharose CL-4B. In the present structures has been difficult because oftheir insoluble nature experiments, we used both the affinity-purified antibody as (4), and for this reason much interest has been focused on well as appropriate dilutions of the crude sera from both immunocytochemical studies. Such studies have implicated rabbits. Both antisera behaved identically in these experi- various microtubule-associated proteins (MAPs) (5-9) and ments. Monospecific ubiquitin antibody was used at a dilu- (9, 10) as elements of these complexes. How tion of 1:500 from a stock solution of 0.8 mg/ml, and crude these proteins become incorporated into abnormal and in- sera were used at dilutions of 1:200. Rabbit antibody specific soluble fibrillary structures is an unresolved issue. Unusual for chicken brain microtubule proteins was obtained from posttranslational modifications of unknown proteins in NFT ICN. Antibody to was obtained from Bio-Yeda and SP may provide important insights into this problem. (Rehovot, Israel). Rabbit antibodies to the neurofilament Ubiquitin is a 76-residue protein of Mr 8565, which can be triplet proteins were a mixture of three crude sera, each one linked covalently by its carboxyl terminus to other proteins. directed against a different neurofilament subunit (16). The Current studies suggest that one of the major functions of second antibody in the immunoblot experiments was goat ubiquitin is its involvement in protein degradation (11). anti-rabbit conjugated to horseradish peroxidase and was Ubiquitinylation of proteins has been shown to target a obtained from Sigma. subset of cellular protein for rapid degradation by an ATP- Protein Preparations. Ubiquitin was prepared by the meth- dependent pathway. In this study, we show that antibodies od of Wilkinson et al. (17) and was used in adsorption directed against ubiquitin strongly stain NFT, neurites in SP, experiments at a concentration of 25 ,g/ml. Bovine tau and a subset of neuronal processes, suggesting that all of proteins were made by glycerol precipitation (18) and were these structures are heavily ubiquitinylated. We discuss our used at a concentration of 0.2 mg/ml. Bovine heat-stable findings in relation to current understanding ofthe posttrans- MAP (19) was used at concentrations of 0.75 mg/ml. A lational conjugation of ubiquitin to proteins. neurofilament-enriched fraction from pig was prepared as described by Delacourte et al. (20) and was used The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: NFT, neurofibrillary tangle(s); SP, senile plaque(s); in accordance with 18 U.S.C. §1734 solely to indicate this fact. MAP, microtubule-associated protein.

Downloaded by guest on September 24, 2021 3033 3034 Medical Sciences: Perry et al. Proc.Proc Nal.Natl. Acad Sci. USA 84 (1987)

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FIG. 1. (Legend appears at the bottom of the opposite page.) Downloaded by guest on September 24, 2021 Medical Sciences: Perry et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3035 at a concentration of 1 mg/ml. This preparation contains large amounts of all three neurofilament subunit proteins as well as a significant quantity of the glial fibrillary acidic protein. Adsorption Experiments. The antibodies to be adsorbed 4- H were mixed with the protein solutions at the concentrations *-M indicated above and incubated for 18 hr at 40C. After this time interval, they were used for staining sections as described.

RESULTS

When Alzheimer brain sections were stained with antibodies directed against ubiquitin, several prominent structures char- 1L acteristic of Alzheimer disease were recognized. Fig. lb shows numerous immunostained flame-shaped structures in Sommer's section of the of an Alzheimer patient. At higher magnification, these flame-shaped struc- tures can be resolved into distinct neurofibrillary tangles (Fig. ic). In addition, we observed the staining of a number of enlarged neurites associated with SP (Fig. ld) and a high density of neurites not obviously associated with SP or NFT (Fig. le). Similar observations were seen with six other 1 ' 3 4 6 Alzheimer patients ranging in age at death from 69 to 82 years. Preliminary studies indicated that these three types of FIG. 2. Immunoblotting of ubiquitin antibodies on neuronal ubiquitin-positive profiles are most abundant in regions cytoskeletal proteins. Lane 1, photograph of a transfer of heat-stable where SP and NFT are abundant, as defined by Congo red MAPs stained with affinity-purified ubiquitin antiserum. Lane 2, a staining (2). Regions lacking Congo red staining also lack transfer from the same series stained with an antibody directed against microtubule protein (19). The ubiquitin antiserum has no fibrillar ubiquitin staining. Thus, we saw fewer ubiquitin reactivity against any of the bands stained with the microtubule immunoreactive profiles in cortex cerebral than in hip- protein antibody. Lane 3, photograph of a transfer oftau proteins (18) pocampus, and none in cerebellum of Alzheimer patients. stained with ubiquitin antiserum. Lane 4, a similar transfer stained Staining ofcontrol brains in patients between 36 and 69 years with tau antibody. Ubiquitin antibody shows no reactivity for any of old at death revealed no ubiquitin reactivity of this type. A the tau-reactive bands. Lane 5, photograph of a transfer incubated brain from a 101-year-old individual showing no Alzheimer with a neurofilament-rich supernatant (20), which has been stained symptoms revealed a few ubiquitin-positive NFT and other with ubiquitin antisera. Lane 6, similar transfer, which has been neuritic profiles, as would perhaps have been predicted from stained with a mixture of rabbit polyclonal antisera directed against previous studies of normal aging (Fig. la). To make sure that neurofilament H, M, and L proteins (16). Again, no reactivity of the ubiquitin antibody for the neurofilament subunits is detectable. the ubiquitin immunoreactivity was not the result of post- Transfers 1-4 were made from 10% NaDodSO4/polyacrylamide gels, mortem changes, we obtained biopsy specimens from the and transfers 5 and 6 were from 7.5% NaDodSO4/polyacrylamide cerebral cortex of an Alzheimer patient. The biopsy speci- gels. Arrow on left indicates the position of a 68-kDa marker (on a mens revealed prominent staining of SP-associated and other gel run parallel to lanes 1-4), and three arrows on right indicate the ubiquitin-positive neurites similar to those seen in postmor- position of the neurofilament high (H), middle (M), and low (L) tem Alzheimer brains. molecular weight protein (on a gel run parallel to lanes 5 and 6). Neurofibrillary tangles have previously been immuno- stained with several antibodies, including some directed reactivity. In contrast, preincubation of the antibodies with against the proteins MAP-2 and tau as well as neurofilaments purified ubiquitin completely removed the plaque- and tan- (5-10). Immunoblotting experiments revealed that affinity- gle-associated fibrillar staining pattern. purified ubiquitin antibodies had no reactivity with either tau, It is significant that a large number of neurites that are not neurofilaments, or heat-stable MAPs (Fig. 2). Previous stud- obviously associated with NFT and SP were also labeled with ies have shown that these ubiquitin antibodies stain purified antibodies directed against ubiquitin. Such labeled fibers ubiquitin and ubiquitin conjugates on immunoblots under were not observed in the control, non-aged brain tissues, or conditions similar to those described here (14). In parallel to in regions of the brain lacking specific NFT- and SP- the experiments described here, purified ubiquitin dot blotted associated neurofibrillar staining. Whether the conjugated onto nitrocellulose filters was stained strongly and specifi- proteins in these neurites share identity with those in NFT- cally with ubiquitin antibodies. We further tried to ensure the and SP-associated neurites remains to be defined. Similar specificity of the staining observed in Fig. 1 by preincubating neurite staining was also observed by others using antibodies the ubiquitin antibodies in solutions containing these same to paired helical filament material (21) and to tau protein cytoskeletal proteins. Preincubation ofthe ubiquitin antibod- (5-8). Further studies will be necessary to show whether ies with MAPs, tau, and neurofilament preparations had no immunoreactivity for tau and paired helical filament markers effect on the staining pattern, so that the three types of overlaps that for ubiquitin observed here. ubiquitin-positive structures showed undiminished immuno- We could detect no ubiquitin immunoreactivity on either

FIG. 1 (on opposite page). Light microscopy ofubiquitin immunoreactivity, visualized by the peroxidase-antiperoxidase method, on sections ofcontrol and Alzheimer brain. (a) Section of Sommer's sector of the hippocampus from an individual who exhibited no symptoms ofAlzheimer disease. A few stained profiles can be seen, corresponding to occasional tangles and plaques, as have been reported in normal (x30) (b) Comparable section of the same region stained with ubiquitin antibody from a patient exhibiting classical Alzheimer symptoms. Numerous densely stained profiles can be visualized, in contrast to a. (x30.) (c) Region of a specimen similar to b viewed under greater magnification. A typical flame-like neurofibrillary tangle can be seen. Filamentous substructure is visible in this specimen. (d) Another region of Alzheimer hippocampus, containing a typical senile plaque. Numerous ubiquitin-positive neurites are components of this plaque, although amyloid was not stained. (e) Region of hippocampus containing numerous ubiquitin-positive fibers. (c, x,1800; d, x 1150; e, x750.) Downloaded by guest on September 24, 2021 3036 Medical Sciences: Perry et al. Proc. Natl. Acad. Sci. USA 84 (1987) SP amyloid or on lipofuscin granules, although both inclu- subsequent proteolytic step has not been carried through. sions were very abundant in the Alzheimer brains we exam- Whether ubiquitin in these fibril structures has become ined. These findings indicate a certain degree of specificity of inaccessible to the specific ubiquitin-dependent protease or the ubiquitinylation modification for the group of fibrous whether there is a defect in the proteolytic pathway are structures we have described. clearly important issues to be examined in the future. The results reported above suggest that ubiquitin is linked We would like to thank Dr. W. Ballinger for making available to components ofNFT and SP-associated neurites, as well as biopsy brain specimens, and Drs. C. West, V. Manetto, P. Mulvihill, certain other neurites. We performed an immunoblotting L. Autilio-Gambetti, and P. Gambetti for valuable discussions. This experiment on a preparation of paired helical filaments work was supported by National Institutes of Health Grants prepared from Alzheimer brain by pelleting after extensive AG00795 (to G.P.), NS22695 (to G.S.), and GM 35803 (to V.C.), and sodium dodecyl sulfate extraction (4). Ubiquitin antibodies by the David S. Ingalls, Sr., Fund (to G.P.). stained a region of the nitrocellulose transfer corresponding to material that would barely enter the 4% acrylamide 1. Mann, D. M. A. (1985) Mech. Ageing Dev. 31, 213-255. 2. Glenner, G. G. (1980) N. Engl. J. Med. 302, 1333-1343. stacking gel (data not shown), as was observed with previ- 3. Hirano, A., Dembitzer, H. H., Kurland, L. T. & Zimmer- ously described antibodies raised to paired helical filament mann, H. M. (1968) J. Neuropathol. Exp. Neurol. 27, 167-182. preparations (22). We conclude that ubiquitin is most prob- 4. Selkoe, D. J., Ihara, Y. & Salazar, F. J. (1982) Science 215, ably covalently bound to fibrillar material found in the paired 1243-1245. helical filament fraction. 5. Brion, J. P., Passareiro, H., Nunez, J. & Flament-Durand, J. (1985) Arch. Biol. 95, 229-235. 6. Kosik, K. S., Joachim, C. L. & Selkoe, D. J. (1986) Proc. DISCUSSION Natl. Acad. Sci. USA 83, 4044-4048. 7. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., Zaidi, M. D. & Wisniewski, H. M. (1986) J. Biol. Chem. 261, 6084- The identification of specific posttranslational modifications 6089. on neurofibrillary structures in Alzheimer-affected brain 8. Wood, J. G., Mirra, S. S., Pollock, N. J. & Binder, L. I. tissue may provide important information toward an under- (1986) Proc. Natl. Acad. Sci. USA 83, 4040-4043. standing of the formation of these abnormal and insoluble 9. Perry, G., Rizzuto, N., Autilio-Gambetti, L. & Gambetti, P. fibrils. By using highly specific antibody preparations, we (1985) Proc. Natl. Acad. Sci. USA 82, 3916-3920. have shown that NFT, enlarged neurites associated with SP, 10. Miller, C. C. J., Brion, J. P., Calvert, R., Chin, T. K., Eagles, as well as a large number of other neurites not apparently P. A. M., Downes, M. J., Flament-Durand, J., Haugh, M., associated with NFT and SP, stain with ubiquitin antibodies. Kahn, J., Probst, A., Ulrich, J. & Anderton, B. H. (1986) The simplest interpretation of our results is that these EMBO J. 5, 269-276. 11. Hershko, A. & Ciechanover, A. (1982) Annu. Rev. Biochem. structures contain a high density of covalently conjugated 51, 335-364. ubiquitin. Since the covalent conjugation of ubiquitin to 12. Khachaturian, Z. S. (1985) Arch. Neurol. 42, 1097-1105. proteins carries specific implications based on our current 13, Sternberger, L. A. (1986) Immunocytochemistry (Wiley, New knowledge of this posttranslational modification, we will York), 3rd Ed. discuss our findings in relationship to current understanding 14. Meyer, E. M., West, C. M. & Chau, V. (1986) J. Biol. Chem. in this area. 261, 14365-14368. Ubiquitin is a highly conserved 76- residue 15. Haas, A. L. & Bright, P. M. (1985) J. Biol. Chem. 260, protein found in all eukaryotes (11). This protein is a 12464-12473. component of an unusual protein posttranslational modifica- 16. Shaw, G., Osborn, M. 0. & Weber, K. (1986) Eur. J. CellBiol. 42, 1-9. tion catalyzed by a specific ATP-dependent enzyme pathway 17. Wilkinson, K. D., Urban, M. K. & Haas, A. L. (1980) J. Biol. in which ubiquitin is covalently conjugated to a restricted set Chem. 255, 7525-7528. of cellular proteins. The linkage involves the formation of an 18. Lindwall, G. & Cole, R. D. (1984) J. Biol. Chem. 259, isopeptide or a peptide bond between the COOH-terminal 5301-5315. carboxyl group on ubiquitin with an amino group on lysine or 19. Herzog, W. & Weber, K. (1978) Eur. J. Biochem. 92, 1-8. the NH2-terminal of acceptor proteins (23). Although the 20. Delacourte, A., Filliatreau, G., Boutteau, F., Biserte, G. & substrate specificity of this reaction is still unclear, only a Schrevel, J. (1980) Biochem. J. 191, 543-546. limited number of proteins and lysine residues in a substrate 21. Braak, H., Braak, E., Grundke-Iqbal, I. & Iqbal, K. (1986) protein can serve as acceptors (24, 25). Neurosci. Lett. 65, 351-355. 22. Ihara, Y., Abraham, C. & Selkoe, D. J. (1983) Nature (Lon- The best-characterized function ofprotein ubiquitinylation don) 304, 727-730. is its role in cellular protein turnover. A number of studies 23. Hershko, A. (1983) Cell 34, 11-12. had shown that the conjugation of ubiquitin serves to target 24. Hershko, A., Ciechanover, A., Heller, H., Haas, A. L. & the acceptor proteins for rapid proteolysis by a nonlysosomal Rose, I. A. (1980) Proc. Natl. Acad. Sci. USA 77, 1783-1786. ATP-dependent proteolytic pathway (26-29). Although the 25. Gregori, L., Marriott, D., West, C. M. & Chau, V. (1985) J. nature of the acceptor proteins has yet to be clearly defined, Biol. Chem. 260, 5232-5235. it has been shown that the degradation of cellular abnormal 26. Ciechanover, A., Finley, D. & Varshavsky, A. (1984) Cell 37, and "short-lived" proteins requires a functioning ubiquitin- 57-66. enzyme system (26). One interpretation of our 27. Mita, S., Yasuda, H., Marunouchi, T., Ishiko, S. & Yamada, conjugation M. (1980) Exp. Cell Res. 126, 407-413. finding is that the protein constituents in the NFT and SP that 28. Chin, D. T., Kuehl, L. & Rechsteiner, M. (1982) Proc. Natl. are labeled with ubiquitin are abnormal proteins and are Acad. Sci. USA 79, 5857-5861. recognized by the ubiquitin conjugation enzyme system. 29. Hershko, A., Leshinsky, E., Ganoth, D. & Heller, H. (1984) This, of course, raises the interesting issue of why the Proc. Natl. Acad. Sci. USA 81, 1619-1623. Downloaded by guest on September 24, 2021