117
Neuroscience Letters, 128 (1991) 117-120 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100342J
NSL 07860
Alzheimer patients: preamyloid deposits are immunoreactive with antibodies to extracellular domains of the amyloid precursor protein Fabrizio Tagliavini 1, Giorgio Giaccone 1, Laura Verga *, Jorge Ghiso 2, Blas Frangione 2 and Orso Bugiani 1 ~Istituto Neurologico Carlo Besta, Milan (Italy) and 2New York University School of Medicine, New York, N Y (U.S.A.)
(Received 18 February 1991; Revised version received 9 April 1991; Accepted 10 April 1991) Key words:
Preamyloid deposit; Amyloid; Amyloid precursor protein; Alzheimer's disease; Down's syndrome
In patients with Alzheimer's disease, in patients with Down's syndrome and in aged non-demented individuals, anti-t-protein antibodies label not only the fibrillary amyloid, but also preamyloid deposits. The latter are made up of amorphous material lacking the tinctorial, optical and ultrastructural properties of amyloid fibrils. To investigate the antigenic profile of preamyloid deposits, we have carried out an immunohistochemical study on specimens of cerebral cortex from 4 Alzheimer patients and two non-demented individuals, using antibodies to the t-protein (anti-SP28), the C-terminal region of the amyloid precursor protein (APP) (anti-SP20) and an APP extracellular epitope between residues 50 and 100 (anti-preA4). Anti-preA4 and anti-SP28 immunoreactivity was found to be present in preamyloid deposits, whereas anti-SP20 immunoreactivity was not. These findings suggest that an extracellular portion of APP, close to the N-terminus of the molecule, participates with ]/-protein in the composition of preamyloid deposits.
Amyloid deposits in the neuropil and cerebral vessel walls are a hallmark of Alzheimer's disease (AD), Down's syndrome and brain aging. The main component of this amyloid is a 39--42 residue self-aggregating peptide (t-protein or A4) [6, 13, 17], which is a fragment of membrane-associated glycoproteins (amyloid precursor proteins, APP) of 695, 714, 751 or 770 amino acids, r e s p e c t i v e l y (APP695,714,751,770) [7, 11, 12, 16, 18, 21]. Immunohistochemical studies with anti-t-protein antibodies on brains from patients with AD, Down's syndrome, and from non-demented individuals have revealed a greater number of immunoreactive deposits than have conventional methods for amyloid [1, 5, 8, 19, 25]. This difference is due to the ability of anti-t-protein antibodies to label not only amyloid fibrils, but also deposits of extracellular material that differ from the former in being non-birefringent after Congo red staining, non-fluorescent after thioflavine S treatment and nonfibriUary [19, 22]. In Down patients of different ages such deposits were found to develop 10-20 years earlier than amyloid fibrils, neurofibrillary tangles and degenerating neurites with paired helical filaments [5]. Accordingly, they were designated as preamyloid deposits and the hypothesis was put forward that they were constituted of Correspondence: F. Tagliavini, Istituto Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy.
either APP or APP fragments including the t-protein region or unpolymerized t-protein [1, 5, 19, 22]. To better define the antigenic profile of preamyloid deposits, we carried out an immunohistochemical, light and electronmicroscopic (EM) study using an antiserum to the t-protein and antibodies to intra- and extracellular domains of APP. These were: (1) anti-SP28, a rabbit antiserum to a synthetic peptide corresponding to residues 597-624 of the sequence deduced from APP695 cDNA [2]; this antiserum recognizes an epitope located at the carboxyl end of peptide SP28 (J. Ghiso and B. Frangione, unpublished observation); (2) anti-SP20, a monoclonal antibody raised against a 20-residue synthetic peptide homologous to the C~terminal segment of APP [4]; (3) anti-preA4 (Boehringer Mannheim), a monoclonal antibody to a purified APP fusion protein [23] that recognizes an epitope between residue 50 and 100 (U. Moenning and K. Beyreuther, personal communication). Light microscopy study was performed on 4 % paraformaldehyde-fixed, paraplast-embedded blocks of autopsy cortical specimens obtained from two non-demented individuals aged 48 and 61 years, respectively, having cortical preamyloid deposits but no senile plaques or neurofibrillary tangles, and from 4 Alzheimer patients aged 66-86, in whom preamyloid deposits could be compared with amyloid deposits and senile plaques. Serial 8-/~m-thick sections were alternately immuno-
118 stained with anti-SP28 (after formic acid treatment), anti-SP20 and anti-preA4 (1:100). To differentiate preamyloid deposits from amyloid deposits and senile plaques, additional sections were treated with thioflavine S or incubated with Alz50 (1:10), a monoclonal antibody that recognizes a 68-kDa protein in brains from Alzheimer patients and immunoreacts with paired helical filaments [24]. Anti-SP28 was evidenced by the peroxidase-antiperoxidase (PAP) method with swine antirabbit immunoglobulins, PAP complex (Dako) and 3,3'diaminobenzidine (DAB). Anti-SP20, anti-preA4 and Alz50 were revealed by an avidin-biotin system with biotinylated anti-mouse serum raised in sheep followed either by streptavidin-alkaline phosphatase (Amersham)
and naphthol AS-MX phosphate/Fast blue BB salt or by streptavidin-horseradish peroxidase (Amersham) and DAB. Some sections were subjected to sequential labeling with anti-preA4 and anti-SP28. For this procedure, sections were first immunostained with anti-preA4 evidenced by the alkaline phosphatase method and photographed, then destained in xylene, immunostained with anti-SP28 evidenced by the PAP system, and rephotographed. Photographs of the same fields were subjected to a computer-assisted image analysis (IBAS, Kontron Zeiss). The EM study was carried out on 4% paraformaldehyde-fixed autopsy specimens of frontal cortex from one Alzheimer patient and one non-demented individual. Following a pre-embedding method described in de-
Fig. 1. Preamyloiddeposits in the frontal cortex of a 48-year-oldnon-demented individual: same field of a single section subjected to sequential immunolabeling with anti-preA4 antibody (a,c: streptavidin-alkalinephosphatase with naphthol-AS-MX-phosphate/Fast blue BB salt) and antiSP28 antibody (b,d: peroxidase-antiperoxidasewith diaminobenzidine).Preamyloiddepositsare labeled with identicalpattern and intensityby either antibody, a,b: × 95; c,d: x 475, Nomarski optic.
119
Fig. 2. Electrondense, extracellular amorphous material of preamyloid deposits. Double immunogold labeling with anti-preA4 (5-nm particles) and anti-SP28 (10-nm particles) antibodies. Pre-embedding immunostaining method, x 110,000.
tail elsewhere [22], double immunogold labeling with anti-preA4 (1:20) and anti-SP28 (1: 50) was performed by means of goat anti-mouse immunoglobulins conjugated with 5-nm colloidal gold particles (Biocell, 1:20) and goat anti-rabbit immunoglobulins conjugated with 10nm colloidal gold particles (Biocell, 1:20). Specificity of immunoreactions was verified with normal rabbit or mouse serum as first-step reagent, or by absorption of anti-SP20 and anti-SP28 antibodies with peptides SP20 and SP28, respectively, as described previously [20]. Results were as follows. Preamytoid deposits were strongly labeled by anti-SP28 and anti-preA4, not by anti-SP20. Automated analysis of photographs of the same field from sections subjected to sequential immunostaining showed that all preamyloid deposits revealed by anti-SP28 were revealed also by anti-preA4 (Fig. la,b). Further, preamyloid deposits displayed the same morphology and pattern of immunoreactivity with either antibody (Fig. lc,d). Immuno-EM showed that
/ " ~ ~ i~i
/ ~I~ ~:~
extracellular amorphous material of preamyloid deposits [22] was decorated by anti-SP28 and anti-preA4 antibodies at once (Fig. 2). Unlike preamyloid deposits, amyloid deposits and the core of senile plaques were labeled by anti-SP28, but not (or weakly on some occasions) by anti-preA4. On the other hand, the senile plaque crowns contained a number of 1-15/lm, round and rod-shaped structures immunoreactive with either anti-preA4 or anti-SP20 or Alz50 (Fig. 3). Immunoreactivity was absent with normal rabbit or mouse serum as first-step reagent and was abolished by absorption of anti-SP28 and anti-SP20 with related peptides. These results rule out the possibility that preamyloid deposits are made up of either whole APP molecules or merely unpolymerized r-protein. In fact, they were not immunoreactive with anti-SP20, an antibody that recognizes an epitope on the intracellular domain of APP, while they were immunolabeled by anti-preA4, which recognizes an epitope on the APP extracellular domain far from the r-protein region. On the other hand, immunolabeling of preamyloid deposits with both anti-SP28 and anti-preA4 indicates that two distinct epitopes are present and suggests that preamyloid deposits are made up of at least two APP fragments or of a single fragment including both epitopes. Further, immunolabeling of preamyloid deposits with anti-SP28 suggests that these fragments do not derive from soluble forms of APP; in fact, these forms, whose C-terminus corresponds to residue 611 of APP695 [3], lack the r-protein epitope recognized by anti-SP28. Senile plaques were found to differ, from preamyloid deposits in containing anti-SP20 immunolabeled material and in having a pattern of immunoreactivity with antipreA4 antibody that was related to round or rod-shaped profiles rather than to amorphous and diffuse material. According to this pattern and to Alz50 immunoreactivity, several of such round and rod-shaped profiles might well have been cell processes. In this regard, our results
•
Fig. 3. Senile plaques in the frontal cortex of a 75-year-old Alzheimer patient: round and rod-shaped profiles are immunoreactive with either antiSP20 antibody (a) or anti-preA4 antibody (b) or Alz50 antibody (c). a,b,c: peroxidase-antiperoxidase with diaminobenzidine, × 475, Nomarski optic.
120
are in agreement with previous immunohistochemical findings also pointing to a relationship between APP and nerve cell processes in senile plaques [9, 10, 14, 15, 18, 20]. In conclusion, this immunohistochemical study suggests that an extracellular portion of APP, close to the N-terminus of the molecule, participates with fl-protein in the composition of preamyloid deposits, and confirms that epitopes of both extra- and intracellular domains of APP are related to senile plaques. The authors are grateful to Drs. P.D. Gorevic and J.E. Gardella, State University of New York at Stony Brook (New York, U.S.A.), for providing anti-SP20 antibody, to Dr. P. Davies, Albert Einstein College of Medicine (New York, U.S.A.), for providing Alz50 antibody, and to Mr. L. Frigerio for skillful technical assistance. This work was supported in part by the Italian Ministry of Health, Department of Social Services, and the U.S. National Institutes of Health (Grants AG05891 and AG08721 to B.F.). 1 Bugiani, O., Giaccone, G., Frangione, B., Ghetti, B. and Tagliavini, F., Alzheimer patients: preamyloid deposits are more widely distributed than senile plaques throughout the central nervous system, Neurosci. Lett., 103 (1989) 263-268. 2 Castafio, E.M., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease fl-protein, Biochem. Biophys. Res. Commun., 141 (1986) 782789. 3 Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J., Cleavage of amyloid fl peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. 4 Gardella, J.E., Ghiso, J., Gorgone, G.A., Marratta, D., Kaplan, A.P., Frangione, B. and Gorevic, P.D., Intact Alzheimer amyloid precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA, Biochem. Biophys. Res. Commun., 173 (1990) 1292-1298. 5 Giaccone, G., Tagliavini, F., Linoli, G., Bouras, C., Frigerio, L., Frangione, B. and Bugiani, O., Down patients: extracellular preamyloid deposits precede neuritic degeneration and senile plaques, Neurosci. Lett., 97 (1989) 232-238. 6 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 7 Golde, T.E., Estus, S., Younkin, L.H. and Younkin, S.G., PCR amplification of reverse transcribed RNA reveals a fourth alternatively spliced flAPP mRNA, Soc. Neurosci. Abstr., 15 (1989) 648. 8 Ikeda, S., Allsop, D. and Glenner, G.G., Morphology and distribution of plaque and related deposits in the brains of Alzheimer's disease and control cases, Lab. Invest., 60 (1989) 113-122. 9 Ishii, T., Kametani, F., Haga, S. and Sato, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease, Neuropathol. Appl. Neurobiol., 15 (1989) 135 147. 10 Joachim, C., Games, D., Morris, J., Ward, P., Frenkel, D. and Sel-
koe, D., Antibodies to non-fl regions of the fl-amyloid precursor protein detect a subset of senile plaques, Am. J. Pathol., 138 (1991) 373-384. 11 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and MiillerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. 12 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530-532. 13 Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K., Amyloid plaque core protein in Alzheimer disease and Down syndrome, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 42454249. 14 Palmert, M.R., Podlisny, M.B., Witker, D.S., Oltersdorf, T., Younkin, L.H., Selkoe, D.J. and Younkin, S.G., Antisera to an aminoterminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques, Biochem. Biophys. Res. Commun., 156 (1988) 432-437. 15 Perry, G., Lipphardt, S., Mulvihill, P., Kancherla, M., Mijares, M., Gambetti, P., Sharma, S., Maggiora, L., Cornette, J., Lobl, T. and Greenberg, B., Amyloid precursor protein is in senile plaques of Alzheimer disease, Lancet, 2 (1988) 746. 16 Ponte, P., Gonzalez-De Whitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F. and Cordell, B., A new A4-amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature, 331 (1988) 525527. 17 Prelli, F., Castafio, E.M., Glenner, G.G. and Frangione, B., Differences between vascular and plaque core amyloid in Alzheimer's disease, J. Neurochem., 51 (1988) 648~551. 18 Selkoe, D.J., Podlisny, M.B., Joachim, C.L., Vickers, E.A., Lee, G., Fritz, L.C. and Oltersdorf, T., fl-Amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membraneassociated proteins in neural and nonneural tissue, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7341-7345. 19 Tagliavini, F., Giaccone, G., Frangione, B. and Bugiani, O., Preamyloid deposits in the cerebral cortex of patients with Alzheimer's disease and nondemented individuals, Neurosci. Lett., 93 (1988) 191-196. 20 Tagliavini, F., Ghiso, J., Timmers, W.F., Giaccone, G., Bugiani, O. and Frangione, B., Coexistence of Alzheimer's amyloid precursor protein and amyloid protein in cerebral vessel walls, Lab. Invest., 62 (1990) 761-767. 21 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J.F. and Neve, R.L., Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease, Nature, 331 (1988) 528 530. 22 Verga, L., Frangione, B., Tagliavini, F., Giaccone, G., Migheli, A. and Bugiani, O., Alzheimer patients and Down patients: cerebral preamyloid deposits differ ultrastructurally and histochemically from the amyloid of senile plaques, Neurosci. Lett., 105 (1989) 294~ 299. 23 Weidemann, A., K6nig, G., Bunke, D., Fisher, P. Salbaum, J.M., Masters, C.L. and Beyreuther, K., Identification, biogenesis and localization of precursors of Alzheimer's disease A4 amyloid protein, Cell, 57 (1989) 115-126. 24 Wolozin, B.L., Pruchnicki, A., Dickson, D.W. and Davies, P., A neuronal antigen in the brains of Alzheimer patients, Science, 232 (I 986) 648~i50. 25 Yamaguchi, H., Hirai, S., Morimatsu, M., Shoji, M. and Harigaya, Y., Diffuse type of senile plaques in the brains of Alzheimer-type dementia, Acta Neuropathol., 77 (1988) 113-119.
Neuroscience Letters, 128 (1991) 117-120 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100342J
NSL 07860
Alzheimer patients: preamyloid deposits are immunoreactive with antibodies to extracellular domains of the amyloid precursor protein Fabrizio Tagliavini 1, Giorgio Giaccone 1, Laura Verga *, Jorge Ghiso 2, Blas Frangione 2 and Orso Bugiani 1 ~Istituto Neurologico Carlo Besta, Milan (Italy) and 2New York University School of Medicine, New York, N Y (U.S.A.)
(Received 18 February 1991; Revised version received 9 April 1991; Accepted 10 April 1991) Key words:
Preamyloid deposit; Amyloid; Amyloid precursor protein; Alzheimer's disease; Down's syndrome
In patients with Alzheimer's disease, in patients with Down's syndrome and in aged non-demented individuals, anti-t-protein antibodies label not only the fibrillary amyloid, but also preamyloid deposits. The latter are made up of amorphous material lacking the tinctorial, optical and ultrastructural properties of amyloid fibrils. To investigate the antigenic profile of preamyloid deposits, we have carried out an immunohistochemical study on specimens of cerebral cortex from 4 Alzheimer patients and two non-demented individuals, using antibodies to the t-protein (anti-SP28), the C-terminal region of the amyloid precursor protein (APP) (anti-SP20) and an APP extracellular epitope between residues 50 and 100 (anti-preA4). Anti-preA4 and anti-SP28 immunoreactivity was found to be present in preamyloid deposits, whereas anti-SP20 immunoreactivity was not. These findings suggest that an extracellular portion of APP, close to the N-terminus of the molecule, participates with ]/-protein in the composition of preamyloid deposits.
Amyloid deposits in the neuropil and cerebral vessel walls are a hallmark of Alzheimer's disease (AD), Down's syndrome and brain aging. The main component of this amyloid is a 39--42 residue self-aggregating peptide (t-protein or A4) [6, 13, 17], which is a fragment of membrane-associated glycoproteins (amyloid precursor proteins, APP) of 695, 714, 751 or 770 amino acids, r e s p e c t i v e l y (APP695,714,751,770) [7, 11, 12, 16, 18, 21]. Immunohistochemical studies with anti-t-protein antibodies on brains from patients with AD, Down's syndrome, and from non-demented individuals have revealed a greater number of immunoreactive deposits than have conventional methods for amyloid [1, 5, 8, 19, 25]. This difference is due to the ability of anti-t-protein antibodies to label not only amyloid fibrils, but also deposits of extracellular material that differ from the former in being non-birefringent after Congo red staining, non-fluorescent after thioflavine S treatment and nonfibriUary [19, 22]. In Down patients of different ages such deposits were found to develop 10-20 years earlier than amyloid fibrils, neurofibrillary tangles and degenerating neurites with paired helical filaments [5]. Accordingly, they were designated as preamyloid deposits and the hypothesis was put forward that they were constituted of Correspondence: F. Tagliavini, Istituto Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy.
either APP or APP fragments including the t-protein region or unpolymerized t-protein [1, 5, 19, 22]. To better define the antigenic profile of preamyloid deposits, we carried out an immunohistochemical, light and electronmicroscopic (EM) study using an antiserum to the t-protein and antibodies to intra- and extracellular domains of APP. These were: (1) anti-SP28, a rabbit antiserum to a synthetic peptide corresponding to residues 597-624 of the sequence deduced from APP695 cDNA [2]; this antiserum recognizes an epitope located at the carboxyl end of peptide SP28 (J. Ghiso and B. Frangione, unpublished observation); (2) anti-SP20, a monoclonal antibody raised against a 20-residue synthetic peptide homologous to the C~terminal segment of APP [4]; (3) anti-preA4 (Boehringer Mannheim), a monoclonal antibody to a purified APP fusion protein [23] that recognizes an epitope between residue 50 and 100 (U. Moenning and K. Beyreuther, personal communication). Light microscopy study was performed on 4 % paraformaldehyde-fixed, paraplast-embedded blocks of autopsy cortical specimens obtained from two non-demented individuals aged 48 and 61 years, respectively, having cortical preamyloid deposits but no senile plaques or neurofibrillary tangles, and from 4 Alzheimer patients aged 66-86, in whom preamyloid deposits could be compared with amyloid deposits and senile plaques. Serial 8-/~m-thick sections were alternately immuno-
118 stained with anti-SP28 (after formic acid treatment), anti-SP20 and anti-preA4 (1:100). To differentiate preamyloid deposits from amyloid deposits and senile plaques, additional sections were treated with thioflavine S or incubated with Alz50 (1:10), a monoclonal antibody that recognizes a 68-kDa protein in brains from Alzheimer patients and immunoreacts with paired helical filaments [24]. Anti-SP28 was evidenced by the peroxidase-antiperoxidase (PAP) method with swine antirabbit immunoglobulins, PAP complex (Dako) and 3,3'diaminobenzidine (DAB). Anti-SP20, anti-preA4 and Alz50 were revealed by an avidin-biotin system with biotinylated anti-mouse serum raised in sheep followed either by streptavidin-alkaline phosphatase (Amersham)
and naphthol AS-MX phosphate/Fast blue BB salt or by streptavidin-horseradish peroxidase (Amersham) and DAB. Some sections were subjected to sequential labeling with anti-preA4 and anti-SP28. For this procedure, sections were first immunostained with anti-preA4 evidenced by the alkaline phosphatase method and photographed, then destained in xylene, immunostained with anti-SP28 evidenced by the PAP system, and rephotographed. Photographs of the same fields were subjected to a computer-assisted image analysis (IBAS, Kontron Zeiss). The EM study was carried out on 4% paraformaldehyde-fixed autopsy specimens of frontal cortex from one Alzheimer patient and one non-demented individual. Following a pre-embedding method described in de-
Fig. 1. Preamyloiddeposits in the frontal cortex of a 48-year-oldnon-demented individual: same field of a single section subjected to sequential immunolabeling with anti-preA4 antibody (a,c: streptavidin-alkalinephosphatase with naphthol-AS-MX-phosphate/Fast blue BB salt) and antiSP28 antibody (b,d: peroxidase-antiperoxidasewith diaminobenzidine).Preamyloiddepositsare labeled with identicalpattern and intensityby either antibody, a,b: × 95; c,d: x 475, Nomarski optic.
119
Fig. 2. Electrondense, extracellular amorphous material of preamyloid deposits. Double immunogold labeling with anti-preA4 (5-nm particles) and anti-SP28 (10-nm particles) antibodies. Pre-embedding immunostaining method, x 110,000.
tail elsewhere [22], double immunogold labeling with anti-preA4 (1:20) and anti-SP28 (1: 50) was performed by means of goat anti-mouse immunoglobulins conjugated with 5-nm colloidal gold particles (Biocell, 1:20) and goat anti-rabbit immunoglobulins conjugated with 10nm colloidal gold particles (Biocell, 1:20). Specificity of immunoreactions was verified with normal rabbit or mouse serum as first-step reagent, or by absorption of anti-SP20 and anti-SP28 antibodies with peptides SP20 and SP28, respectively, as described previously [20]. Results were as follows. Preamytoid deposits were strongly labeled by anti-SP28 and anti-preA4, not by anti-SP20. Automated analysis of photographs of the same field from sections subjected to sequential immunostaining showed that all preamyloid deposits revealed by anti-SP28 were revealed also by anti-preA4 (Fig. la,b). Further, preamyloid deposits displayed the same morphology and pattern of immunoreactivity with either antibody (Fig. lc,d). Immuno-EM showed that
/ " ~ ~ i~i
/ ~I~ ~:~
extracellular amorphous material of preamyloid deposits [22] was decorated by anti-SP28 and anti-preA4 antibodies at once (Fig. 2). Unlike preamyloid deposits, amyloid deposits and the core of senile plaques were labeled by anti-SP28, but not (or weakly on some occasions) by anti-preA4. On the other hand, the senile plaque crowns contained a number of 1-15/lm, round and rod-shaped structures immunoreactive with either anti-preA4 or anti-SP20 or Alz50 (Fig. 3). Immunoreactivity was absent with normal rabbit or mouse serum as first-step reagent and was abolished by absorption of anti-SP28 and anti-SP20 with related peptides. These results rule out the possibility that preamyloid deposits are made up of either whole APP molecules or merely unpolymerized r-protein. In fact, they were not immunoreactive with anti-SP20, an antibody that recognizes an epitope on the intracellular domain of APP, while they were immunolabeled by anti-preA4, which recognizes an epitope on the APP extracellular domain far from the r-protein region. On the other hand, immunolabeling of preamyloid deposits with both anti-SP28 and anti-preA4 indicates that two distinct epitopes are present and suggests that preamyloid deposits are made up of at least two APP fragments or of a single fragment including both epitopes. Further, immunolabeling of preamyloid deposits with anti-SP28 suggests that these fragments do not derive from soluble forms of APP; in fact, these forms, whose C-terminus corresponds to residue 611 of APP695 [3], lack the r-protein epitope recognized by anti-SP28. Senile plaques were found to differ, from preamyloid deposits in containing anti-SP20 immunolabeled material and in having a pattern of immunoreactivity with antipreA4 antibody that was related to round or rod-shaped profiles rather than to amorphous and diffuse material. According to this pattern and to Alz50 immunoreactivity, several of such round and rod-shaped profiles might well have been cell processes. In this regard, our results
•
Fig. 3. Senile plaques in the frontal cortex of a 75-year-old Alzheimer patient: round and rod-shaped profiles are immunoreactive with either antiSP20 antibody (a) or anti-preA4 antibody (b) or Alz50 antibody (c). a,b,c: peroxidase-antiperoxidase with diaminobenzidine, × 475, Nomarski optic.
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are in agreement with previous immunohistochemical findings also pointing to a relationship between APP and nerve cell processes in senile plaques [9, 10, 14, 15, 18, 20]. In conclusion, this immunohistochemical study suggests that an extracellular portion of APP, close to the N-terminus of the molecule, participates with fl-protein in the composition of preamyloid deposits, and confirms that epitopes of both extra- and intracellular domains of APP are related to senile plaques. The authors are grateful to Drs. P.D. Gorevic and J.E. Gardella, State University of New York at Stony Brook (New York, U.S.A.), for providing anti-SP20 antibody, to Dr. P. Davies, Albert Einstein College of Medicine (New York, U.S.A.), for providing Alz50 antibody, and to Mr. L. Frigerio for skillful technical assistance. This work was supported in part by the Italian Ministry of Health, Department of Social Services, and the U.S. National Institutes of Health (Grants AG05891 and AG08721 to B.F.). 1 Bugiani, O., Giaccone, G., Frangione, B., Ghetti, B. and Tagliavini, F., Alzheimer patients: preamyloid deposits are more widely distributed than senile plaques throughout the central nervous system, Neurosci. Lett., 103 (1989) 263-268. 2 Castafio, E.M., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease fl-protein, Biochem. Biophys. Res. Commun., 141 (1986) 782789. 3 Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J., Cleavage of amyloid fl peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. 4 Gardella, J.E., Ghiso, J., Gorgone, G.A., Marratta, D., Kaplan, A.P., Frangione, B. and Gorevic, P.D., Intact Alzheimer amyloid precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA, Biochem. Biophys. Res. Commun., 173 (1990) 1292-1298. 5 Giaccone, G., Tagliavini, F., Linoli, G., Bouras, C., Frigerio, L., Frangione, B. and Bugiani, O., Down patients: extracellular preamyloid deposits precede neuritic degeneration and senile plaques, Neurosci. Lett., 97 (1989) 232-238. 6 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 7 Golde, T.E., Estus, S., Younkin, L.H. and Younkin, S.G., PCR amplification of reverse transcribed RNA reveals a fourth alternatively spliced flAPP mRNA, Soc. Neurosci. Abstr., 15 (1989) 648. 8 Ikeda, S., Allsop, D. and Glenner, G.G., Morphology and distribution of plaque and related deposits in the brains of Alzheimer's disease and control cases, Lab. Invest., 60 (1989) 113-122. 9 Ishii, T., Kametani, F., Haga, S. and Sato, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease, Neuropathol. Appl. Neurobiol., 15 (1989) 135 147. 10 Joachim, C., Games, D., Morris, J., Ward, P., Frenkel, D. and Sel-
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