Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2853-2857, April 1989 Medical Sciences protein and neurofibrillary tangles coexist in the same in Alzheimer disease (amyloid 13 peptide/A4 peptide/intraneuronal protein/paired helical rdaments/senile plaques) INGE GRUNDKE-IQBAL*, KHALID IQBAL, LALU GEORGE, YUNN-CHYN TUNG, KWANG Soo KIM, AND HENRY M. WISNIEWSKI New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 Communicated by Philip Siekevitz, January 3, 1989 (receivedfor review July 27, 1988)

ABSTRACT In Alzheimer disease, paired helical filaments tants of mAb to amyloid at dilutions of 1:2-1:20 were used accumulate in the neuron, and amyloid fibers are found in the (IgG subclass in parentheses): 4G5 (IgGi), 2B8 (IgGl), 2F9 extracellular space in the neuropil and vessels. Amyloid (IgGl), 4E11 (IgG2a), 4G8 (IgG2b). mAb tau-1 to tau (IgG2a, and paired helical filaments are morphologically distinct. ascites, 1:50,000) was the generous gift of L. I. Binder Although messenger RNA that encodes the amyloid has also (University of Alabama, Birmingham). Mouse ascites fluid been shown in several tissues, including brain, the intracellular from plasmacytoma line MOPC21 was purchased from Litton expression of the protein has not been observed. By using Bionetics. Reagents for the avidin-biotin complex technique monoclonal antibodies to a synthetic amyloid (3 peptide, the were purchased from Vector Laboratories, and reagents for present study demonstrates that amyloid reactivity is present in the peroxidase-anti-peroxidase technique were from Stern- both Alzheimer patients and normal individuals in different bergerMeyer Immunocytochemicals (Jarrettsville, MD). Al- types ofneurons, including the with the neurofibrillary kaline phosphatase-conjugated antibody to mouse IgG and tangles, but not in the tangle itself. the alkaline phosphatase color development reagents 5- bromo4-chloro-3-indolyl phosphate (p-toluidine salt) and Alzheimer nitroblue tetrazolium chloride were from Bio-Rad. Alzheimer disease/senile of the type Immunoblots. Electrophoresis on NaDodSO4/polyacryl- (AD/SDAT) are characterized neuropathologically by the amide gels (80 x 60 x 75 mm slab) was performed according accumulation of two types of aberrant fibrils, the paired to Laemmli (23). Proteins were transferred to Immobilon helical filaments (PHF) and the amyloid. PHF are intraneu- (Millipore) in 25 mM Tris/0.7 M glycine/20% (vol/vol) ronal and accumulate in bundles of parallel fibrils, called methanol (24, 25) at 105 V for 1 hr at room temperature. tangles, in the neuronal cell bodies, their dendrites, and the Residual protein binding sites were blocked with 5% (wt/vol) dystrophic neurites of the neuritic (senile) plaques. In con- defatted dry milk in 10 mM phosphate buffer (pH 7.4) trast, amyloid is not found within the neurons and their containing 0.15 M NaCl for at least 1.5 hr at 22TC. The blots processes but occurs extracellularly in association with the were then incubated for 18 hr at 220C with antibodies diluted plaques either as wisps in the neuropil among the plaque in the blocking solution. Bound IgG was detected by using neurites or as a concentrated mass in the center forming the either alkaline phosphatase-conjugated antibody to mouse plaque core (1). Frequently accumulations of amyloid are IgG (1:2000) or the peroxidase-anti-peroxidase technique also found in the neuropil around the vessels and within the (26) with 5-bromo-4-chloro-indolyl phosphate/nitroblue tet- vessel walls. Amyloid is morphologically readily distinguish- razolium chloride or diaminobenzidine substrates, respec- able from the PHF by the smaller dimensions ofits flibrils and tively. their organization in irregular clusters. Plaque and cere- Immunocytochemistry. Blocks of hippocampus, cerebrum, brovascular amyloid are primarily made up of a 40- to cerebellum, and spinal cord from five patients with histo- 42-amino acid residue peptide, called the P3 peptide or A4 pathologically confirmed AD/SDAT (56-84 years of age, peptide (2-4), whereas mainly ubiquitin (5, 6) and microtu- autopsy at 2-24 hr postmortem) and from eight non- bule-associated protein tau (7-15) have been shown in PHF. Alzheimer individuals (ages 38-81 years, autopsy at 4-14 hr Tau, a family of closely related polypeptides with an average postmortem) were fixed in neutral formaldehyde for an molecular mass of 58 kDa, is abnormally phosphorylated in average of 5 days. Alternatively, some tissue was fixed in PHF and probably also contains other alterations (16-18). Perfix for 2 days. Perfix is a commercial fixative (Fisher) Sequences of amyloid P peptide have also been reported in containing 16.8% isopropyl alcohol, 2.0% (wt/vol) trichloro- preparations enriched in PHF (19-21). However, it has not acetic acid, 4.1% (wt/vol) paraformaldehyde, and 2.0% been resolved as yet whether the amyloid peptide is actually (wt/vol) zinc chloride. In the developing nervous system, it a component of the PHF or a contaminant. has been shown to preserve the antigenic activity of glial fibrillary protein better than formaldehyde- or glutaralde- MATERIALS AND METHODS hyde-containing fixatives (27). The tissue was embedded in Paraplast, and 6-,um serial sections were prepared. Neuronal Antibodies and Immunological Reagents. Monoclonal anti- cell bodies were isolated from AD/SDAT frontal cortex as bodies (mAbs) to a synthetic peptide corresponding to the described previously (28), and cell smears were dried onto first 24 amino acids of the ,B peptide were generated as gelatin-coated slides and either fixed with 10% (vol/vol) previously described (22). All antibodies reacted with the 17- neutral formalin or with 10o neutral formalin containing 5% 24 amino acid sequence fragment of 83 peptide in ELISA and (vol/vol) dimethyl sulfoxide for 10 min at room temperature with plaque and vascular amyloid by immunocytochemistry (29) or fixed with -20°C acetone for 3 min. Some sections (22). For the studies in the present paper, culture superna- Abbreviations: AD/SDAT, Alzheimer disease/senile dementia of The publication costs of this article were defrayed in part by page charge the Alzheimer type; PHF, paired helical filaments; mAb, monoclonal payment. This article must therefore be hereby marked "advertisement" antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2853 Downloaded by guest on September 29, 2021 2854 Medical Sciences: Grundke-Iqbal et al. Proc. Natl. Acad Sci. USA 86 (1989)

were treated with 86% (vol/vol) formic aciid for 5-60 min of about 93, 55 (a doublet), 40, and 31 kDa. In calf brain, the prior to immunostaining according to the met! hod of Kitamoto 93-kDa band instead of the 125-kDa doublet was most et al. (30). Immunocytochemistry was perft )rmed on tissue prominently labeled. The major bands (i.e., the 125-kDa sections or cell smears by using the avidin--biotin complex doublet in human brain and the 93-kDa band in calf brain) technique (29). In some cases, immunostainitng was followed were easily visible when as little as 10 ug of protein was by staining with 1% aqueous solution of tLhioflavine S to applied (Fig. 1). In some other cases, an additional doublet of visualize immunonegative tangles and amylloid. The slides about 220 kDa was stained. No labeling was seen with as were photographed immediately after the Ithioflavine step much as 50 ttg of homogenate per lane when mouse IgG from because thioflavine-positive fluorescence raptidly faded when myeloma MOPC21 at a concentration similar to that of the used together with diaminobenzidine immunoostaining. mAb to amyloid was used as a control antibody. In sections of Alzheimer brain, the mAb reacted strongly with plaque and vascular amyloid (Fig. 2). The latter was RESULTS found as heavy deposits within the walls of the cortical and On Western blots (Fig. 1) all amyloid mAbs Ireacted with the meningeal vessels and in addition as loose fibrous accumu- synthetic 1-24 residue 13 peptide. No cros;sreactivity was lations either perivascular or scattered in the neuropil of the observed with ubiquitin, tau, or the polypelptides in micro- most affected areas. Similarly, in the of non-demented tubule and neurofilament preparations. Hoowever, in total aged individuals, the sparsely occurring plaque and vascular brain homogenate, several polypeptides w,ere labeled. In amyloid was also stained strongly. On treatment of the homogenates from an Alzheimer and an ageed control indi- sections with formic acid prior to application of the antibod- vidual, the mAb prominently labeled a polypeptide doublet of ies, a dramatic increase in the number and intensity of 125 kDa and, in addition, weakly labeled polypeptidebands immunostained plaque, vascular, and amorphous amyloid deposits in the neuropil was observed (data not shown). 1 2 3 4 5 6 7 In addition to the extraneuronal amyloid, dark labeling of accumulations of intracellular granules in various types of 8 9 neurons in cerebral and cerebellar cortices and spinal cord was observed (Figs. 3 and 4). Dilutions of the culture supernatants for optimal staining ranged from 1:10 to 1:50 depending upon the myeloma line and was the same in each case for both intracellular and extracellular amyloid staining. The immunostaining was not affected when the salt concen- tration was increased from 0.15 M to 0.3 M and 0.05% Tween-20 was added to the antibodies. Like the extraneu- _ ronal amyloid, the immunoreactivity of this intraneuronal -ir~.materialX increased in the formic acid-treated tissue sections (data not shown). The intraneuronal amyloid immunoreac- tivity appeared to be expressed in cerebellum and cerebrum _ s of AD/SDAT as well as of the non-Alzheimer control group - irrespective of age or postmortem time investigated. In the cerebellum, the labeling was most conspicuous in the cytoplasm ofthe Purkinje cells, which were almost all stained in the cases studied ranging from a 38-year-old control individual to an 81-year-old Alzheimer patient (Fig. 3 a-d). Sometimes the immunopositive grains were seen along al- most the whole length of the apical dendrite extending into the molecular layer (Fig. 3b). On parallel sections that had been stained for polysaccharides with periodic acid/Schiff reagent, accumulations of periodic acid/Schiff reagent- positive material were also seen in the perikarya of the Purkinje cells (Fig. 3e). Amyloid immunostaining was also FIG. 1. Reactivity of mAb 2B8 to amyloid perptide and to tau on found in the cells of the granular layer (Fig. 3c) and occa- immunoblots. mAb 2B8 (and similarly mAbs 4G55, 2F9, 4E11, and sionally in the neurons of the molecular layer. Large motor 4G8, but not shown here) reacts with synthetic 24. -residue /3 peptide neurons of the spinal cord were also strongly positive (data (lane 1, 4 u~g; lane 2, 2 pg). In addition to the lovw molecular mass not shown). material, diffuse labeling of some not completeely dissociated 3 In hippocampus, the amyloid immunoreactivity was pres- peptide, in the 40- to 60-kDa area; is sometimes observed. The mAb ent in the cytoplasm of both granular and pyramidal neurons to amnyloid does not react with ubiquitin (lane 3, ppg), tau (lane 4, (Fig. 4 a, b, and d-g). Most striking was the immunolabeling 2 .ug), in vitro-assembled (31, 32) microtubule poll 10 t~g), and neurofilament polypeptides (lane 6, 30;pLg), whereas mAb pattern in the Alzheimer hippocampus where the amyloid- tau-1 to tau (lane 7) (33) strongly labels the ttau bands of the positive granules were seen as a heavy dark mass, reminis- microtubule preparation. In homogenates of an Akznheimer (lane 8, 10 cent of tangles in the perikarya and apical dendrites of the .&g) and calf (lane 9, 10 ;Lg) brain, a doublet of 125 and 93 kDa, pyramidal cells (Fig. 4 a and b). However, immunostaining of respectively, are predominantly labeled by the annyloid antibodies. the adjacent sections with antibodies to tau or PHF (18) (These molecular masses were estimated from a polyacrylamide gel revealed that the tangles were less frequently present in these with a 5-15% linear acrylamide gradient.) Proteins were electro- neurons than the amyloid-positive material and were always transferred from NaDodSO4/polyacrylamide gelIs (5-22% acryl- fibrillar and not globular (Fig. 4c). In tissue sections coun- amide gradient). Bars at left of panels indicate thie positions of the terstained with thioflavine S. the coexistence of __ -- - -_ thioflavine---_ _.-- protein standards (BRL). For lanes 1-7, from tc )p to bottom: ov- positive tangles in the same neuron with the immunostained albumin (43.1 kDa), carbonic anhydrase (29.4 WDEa), aolotoglobulin (18.4 kDa), lysozyme (14.3 kDa), bovine trypsin finihibitor (6.2 kDa). amyloid-positive globular material was frequently observed For lanes 8 and 9, from top to bottom: myosin (2( )O kDa), phospho- (Fig. 4 d and e). rylase B (97.4 kDa), bovine serum albumin (8kD)a), ovalbumin (43 Immunostaining of tissue fixed in Perfix instead of formalin kDa). -o-, Top of separating gel. similarly showed the amyloid reactivity in the neurons of the Downloaded by guest on September 29, 2021 Medical Sciences: Grundke-Iqbal et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2855

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FIG. 3. Amyloid-positive material in Purkinje (a-f) and granular layer (c, ar- rows) cells of Alzheimer (d and e) and 38-year-old control cerebellum (a-c and f). Cells were stained with mAb to amy- loid P-peptide (a-d andf) or stained with periodic acid/Schiff reagent for polysac- charides (e). In formalin-fixed tissue (a- d), the intraneuronal amyloid-positive material is granular, whereas it is diffuse in Perfix-fixed tissue (f). (a, b, and f, x400; c-e, x 1000.) Downloaded by guest on September 29, 2021 2856 Medical Sciences: Grundke-Iqbal et al. Proc. NatL Acad. Sci. USA 86 (1989)

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FIG. 4. Immunostaining ofAlzheimer (a-e) and 38-year-old control (f and g) hippocampus with mAb to amyloid (a, b, and d-g) or anti-PHF serum (c). (a) mAb to amyloid stains plaques (arrowhead) and intraneuronal material (arrows), which at low power looks like neurofibril- lary tangles. However, at high magnifi- cation (b), it presents as tightly packed granular material and can be easily dis- tinguished from the tangles, which are fibrillar when stained with anti-PHF se- rum (ref. 5) (c) or thioflavine S (e). (d and e) Amyloid-immunoreactive material and thioflavine S-positive fluorescent tangles frequently occur in the same neuron. (a, x200; b and c, x1000; d and e, x420;f and g, x280.) regardless of the fixative used. In contrast, in both Perfix- acid residue precursor protein with the characteristics of a treated tissue and isolated neurons, Alzheimer tangles were cell surface receptor (4); in vitro translation resulted in a easily recognizable by their characteristic shape and fibrillar 92.5-kDa polypeptide (36). The precursor protein may also appearance when stained with antibodies to PHF or to tau have an insert corresponding to a inhibitor domain (data not shown). (37, 38). The doublet labeled by the amyloid antibodies on immunoblots of Alzheimer brain homogenate may thus represent these two species of precursor polypeptides. That DISCUSSION their molecular masses are higher than those predicted from The typical amyloid fibrils are located extraneuronally in the the cDNA sequences (about 85 kDa and 90 kDa) may be due neuropil and vessels of the brain. Although, by in situ to posttranslational modifications. For instance, the pre- hybridization and Northern blots, messenger RNA that dicted precursor proteins contain two potential N-gly- encodes amyloid precursor protein has been shown recently cosylation sites (4). Closer to the molecular mass of the in several tissues including the neurons of the neocortex (34, hypothetical amyloid precursor protein is the molecular mass 35), the intraneuronal presence of the protein itself has not of the amyloid-positive polypeptide in calf brain homogenate been demonstrated previously. The present study shows that (estimated at 93 kDa). It is not resolved as yet whether this is P3-peptide-reactive protein is indeed expressed in neurons of due to age or species differences. The amyloid-reactive poly- cerebrum, cerebellum, and spinal cord. This protein is seen peptide bands with molecular masses below 93 kDa might in brains ofall ages studied so far, ranging from 38 to 83 years, represent the precursor protein in different metabolic stages. with or without Alzheimer neuropathology, and thus seems Similarly, the intraneuronal amyloid-reactive material ob- to be a normally occurring neuronal protein which might not served by us in tissue sections is most likely the precursor be age dependent. protein at a certain level of processing by the cell. According to sequence analysis ofhuman brain cDNA, the In distilled water or in saline, the synthetic ( peptide 3 peptide has been hypothesized to be part of a 695-amino spontaneously polymerizes into fibrils that are morphologi- Downloaded by guest on September 29, 2021 Medical Sciences: Grundke-Iqbal et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2857

cally very similar to the amyloid fibrils seen in situ (39, 40). 14. Yen, S. H., Dixon, D. W., Crowe, A., Butler, M. & Shelanski, That no such fibrils are observed within the neuron may thus M. L. (1987) Am. J. Pathol. 126, 81-91. be due to the stabilizing effect of the rest of the precursor 15. Wischik, C. M., Novak, M., Thongersen, H. C., Edwards, molecule on its P-peptide part. The amyloid fibrils might be P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., formed only when the ,3 peptide (A4 peptide) is cleaved from Roth, M. & Klug, A. (1988) Proc. Natl. Acad. Sci. USA 85, its precursor extracellularly under certain pathological con- 4506-4510. ditions. 16. Grundke-Iqbal, I., Iqbal, K., Tung, Y.-C., Quinlan, M., Wisniewski, H. M. & Binder, I. (1986) Proc. Natl. Acad. Sci. Although in AD/SDAT the amyloid-reactive material is USA 83, 4913-4917. frequently present in the tangle-containing neuron, it could 17. Iqbal, K., Grundke-Iqbal, I., Zaidi, T., Merz, P. A., Wen, always be easily discriminated from the fibrillar tangle G. Y., Shaikh, S. S., Wisniewski, H. M., Alafuzoff, I. & because of its characteristic granular or uniform appearance Winblad, B. (1986) Lancet li, 421-426. when immunostained and thus seems to be an independent 18. Grundke-Iqbal, I., Vorbrodt, A. W., Iqbal, K., Tung, Y.-C., entity. This is also indicated by the diffuse staining of the Wang, G. P. & Wisniewski, H. M. (1988) Mol. Brain Res. 4, neuronal cytoplasm in Perfix-fixed tissue and by the almost 43-52. complete absence of amyloid-reactive material in the isolated 19. Beyreuther, K., Multhaup, G., Sunins, G., Pottgiesser, J., neuronal cell bodies from Alzheimer brain, many of which Schroeder, W., Martins, R. N. & Masters, C. L. (1986) Disc. contain Neurosci. 3, 68-79. tangles. 20. Masters, C. L., Multhaup, G., Sims, G., Pottgiesser, J., Mar- Recently we found that the granular intraneuronal immu- tins, R. N. & Beyreuther, K. (1985) EMBO J. 4, 2757-2763. nostaining parallels distribution of lipofuscin by histochem- 21. Guiroy, D. C., Miyazaki, M., Multhaup, G., Fischer, P., ical stain and that an amyloid-positive 31-kDa polypeptide- Garruto, R. M., Beyreuther, K., Masters, C. L., Simms, G., also seen in immunoblots of the total brain homogenates-is Gibbs, C. J. & Gajdusek, D. C. (1987) Proc. Natl. Acad. Sci. present in the lipofuscin fraction isolated from human brain USA 84, 2073-2077. (41). Furthermore, like amyloid reactivity, lipofuscin in 22. Kim, K. S., Miller, D. L., Chen, C.-M. J., Sapienza, V. J., brains fixed in Perfix loses its granular appearance. It thus Bai, C., Grundke-Iqbal, I., Currie, J. R. & Wisniewski, H. M. appears that lipofuscin is one ofthe locations associated with (1988) Neurosci. Res. Commun. 2, 121-130. the deposition of catabolized amyloid precursor in the neu- 23. Laemmli, J. K. (1970) Nature (London) 277, 680-685. ron. 24. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. NatI. Acad. Sci. USA 76, 4350-4354. 25. Small, G. M., Imanaka, T. & Lazarow, P. B. (1988) Anal. We thank Drs. G. G. Glenner (National Alzheimer's Disease Brain Biochem. 169, 405-409. Bank) and R. Rudelli (New York Instititute for Basic Research) for 26. Sternberger, L. A., Hardy, P. H., Jr., Cuculius, J. J. & Meyer, providing tissue, Dr. Lester I. Binder (University of Alabama, H. G. (1970) J. Histochem. Cytochem. 18, 315-333. Birmingham) for his generous gift of mAb tau-1, Drs. D. Miller and 27. Dixon, R. G. & Eng, L. F. (1981) J. Comp. Neurol. 20, 15-24. C. Bancher (New York State Institute for Basic Research) for 28. Iqbal, K., Zaidi, T., Thompson, C. H., Merz, P. A. & critically reading the manuscript, the Biomedical Photography Unit Wisniewski, H. M. (1984) Acta Neuropathol. 62, 167-177. for photography, and Mrs. L. Antonucci and M. Cappola for typing 29. Grundke-Iqbal, I., Wang, G. P., Iqbal, K., Tung, Y.-C. & the manuscript. This study was supported in part by the New York Wisniewski, H. M. (1985) Acta Neuropathol. 68, 279-283. State Office of Mental Retardation and Developmental Disabilities 30. Kitamoto, T., Ogomori, K., Tateishi, J. & Prusiner, S. B. and National Institutes of Health Grants NS18105, AG05892, (1987) Lab. Invest. 57, 230-236. AG04220. 31. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Natl. Acad. Sci. USA 70, 765-768. 1. Wisniewski, H. M., Sinatra, R. S., Iqbal, K. & Grundke-Iqbal, 32. Iqbal, K., Grundke-Iqbal, I., Merz, P. A., Wisniewski, H. M. I. (1981) in Aging and Cell Structure, ed. Johnson, J. E., Jr. & Zaidi, T. (1987) Mol. Brain Res. 2, 163-172. (Plenum, New York), Vol. 1, pp. 105-142. 33. Binder, L. I., Frankfurter, A. & Rebhun, L. I. (1985) J. Cell 2. Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res. Biol. 101, 1371-1378. Commun. 120, 855-890. 34. Bahmanyar, S., Higgins, G. A., Goldgaber, D., Lewis, D. A., 3. Wong, C. W., Quaranta, V. & Glenner, G. G. (1985) Proc. Morrison, J. H., Wilson, M. C., Shankar, S. K. & Gajdusek, Natl. Acad. Sci. USA 82, 8729-8732. D. C. (1987) Science 237, 77-80. 4. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., 35. Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A. P., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, St. George-Hyslop, P., Van Keuren, M. L., Patterson, D., K. & Muller-Hill, B. (1987) Nature (London) 325, 733-736. Pagan, S., Kurnit, D. M. & Neve, R. L. (1987) Science 235, 5. Mori, H., Kondo, J. & Ihara, Y. (1987) Science 235, 1641-1644. 880-884. 6. Perry, G., Friedman, R., Shaw, G. & Chau, V. (1987) Proc. 36. Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J. M., Natl. Acad. Sci. USA 84, 3033-3036. Lemaire, H.-G., Kang, J., Mueller-Hill, B., Masters, C. L. & 7. Brion, J. P., Passareiro, H., Nunez, J. & Flament-Durand, J. Beyreuther, K. (1988) EMBO J. 7, 949-957. (1985) Arch. Biol. (Brussels) 95, 229-235. 37. Ponte, P., Gonzalez-DeWhitt, P., Schulling, J., Miller, J., Hsu, 8. Delacorte, A. & Defossez, A. (1986) J. Neurol. Sci. 76, 173- D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., 186. Fuller, F. & Cordell, B. (1988) Nature (London) 331, 525-527. 9. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, 38. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Ko- M. S. & Wisniewski, H. M. (1986) J. Biol. Chem. 261, 6084- maroff, L., Gusella, J. F. & Neve, R. L. (1988) Nature 6089. (London) 331, 528-530. 10. Ihara, Y., Nukina, N., Miura, R. & Ogawara, M. (1986) J. 39. Castano, E. M., Ghiso, J., Prelli, F., Gorevic, P. D., Migheli, Biochem. (Tokyo) 99, 1807-1810. A. & Frangione, B. (1986) Biochem. Biophys. Res. Commun. 11. Kosik, K. S., Joachim, C. L. & Selkoe, D. J. (1986) Proc. Natl. 141, 782-789. Acad. Sci. USA 83, 4044-4048. 40. Bobin, S. A., Currie, J. R., Miller, D. L., Styles, J., Walker, 12. Nukina, N. & Ihara, Y. (1986) J. Biochem. (Tokyo) 99, 1541- W. A., Wen, G. Y. & Wisniewski, H. M. (1987) Acta Neuro- 1544. pathol. 74, 313-323. 13. Wood, J. G., Mirra, S. S., Pollock, N. J. & Binder, L. I. (1986) 41. Bancher, C., Grundke-Iqbal, I., Iqbal, K., Kim, K. S. & Proc. Natl. Acad. Sci. USA 83, 4040-4043. Wisniewski, H. M. (1989) Neurobiol. Aging 10, 125-132. Downloaded by guest on September 29, 2021