Gerontology 1992;38(suppl 1): 10—14
Department of Neurology, Institute of Brain Research, University of Tokyo, and Diagnostic Research Laboratories, Fujirebio Inc., Tokyo, Japan
KeyWords Amyloid precursor protein ß-Protein Alzheimer’s disease Senile plaques
Accumulation of Amyloid Precursor Protein and 3-Protein Immunoreactivities in Axons Injured by Cerebral Infarct
Abstract To determine the distribution of amyloid precursor protein (APP), monoclonal antibodies against APP45_62 (APP1-28-9) and Pi_i7 (4A18 and 4A61) were produced. In the sections of Alzheimer’s disease (AD) brain, APP 1-28-9 was reactive with neurites around senile plaques and a few neurons but not with amyloid cores. This antibody also immunostained the axons in the ischemic lesions of brain tissues from cases with cere bral infarct. 4A18 and 4A61 were reactive with amyloid plaques but not with neurites and neurons. The latter two anti bodies also immunostained axons in ischemic lesions. These findings suggest that APP, transported by the fast axonal flows, accumulated in the injured axons in the central nervous system. The (3 immunoreactivity appearing in those axons may provide a clue to the mechanism of amyloidogenesis.
Introduction The main pathological features of Alz heimer’s disease (AD) are Alzheimer neurofi brillary tangles and senile plaques. The amy loid core in the senile plaques is composed of (3-protein. (3-Protein was discovered by Glenner and Wong [ 1] as a component of vascular amyloid. Its molecular weight is 4 kD. After
p-protein was discovered, the cDNA of its precursor protein was cloned [2], The de duced sequence of amyloid precursor protein (APP) suggested that APP has a 695 residue, a transmembrane domain with an N-terminal extracellular domain and a C-terminal cyto plasmic domain. A number of variants of APP have been cloned since then and some of them have 751 and 770 residues which have a
Nobuyuki Nukina Department of Neurology Institute of Brain Research University of Tokyo. 7-3-1 Hongo. Bunkyo-ku Tokyo 113 (Japan)
©1992 S. Karger AG. Basel 0304-324X/92/ 0387-0010$2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
N. Nukinaa I. Kanazawaa T. Mannena Y. Uchidab
Material and Methods Antibodies Peptides corresponding to APP45. « [2] and Pi_n [ 1] were synthesized and coupled with keyhole limpet hemocyanin by the maleimidobenzoyl N-hydroxysuccinimide method. BALB/c mice ( 1 mouse for APP45. 62 and 1 mouse for (h-n) were injected intraperitoneally with 100 |ig peptide with Freund’s complete adjuvant. After three booster injections of the same amount at 3-week intervals, splenocytes were fused with P3U1 myeloma cells. Hybridoma supernatants were screened for antipeptide antibodies by the enzymelinked immunosorbent assay. Strongly reactive cul tures were cloned and selected for the immunohisto chemical studies.
Immimohistochemistry We examined several brain tissue sections of pathologically diagnosed AD (8 cases) and of other neurological disease, such as Parkinson’s disease (2 cases), multiple slcerosis (1 case) and cerebral infarc tion (3 cases). Routine 10% formalin-fixed tissues were embedded in paraffin, cut into 6-mm-thick sec tions and affixed to bovine-serum-albumin-coated glass slides. The sections were deparaffinized, and endogenous peroxidase activity was inactivated by ex posure to methanol containing 0.3% (v/v) H 2O2 for 30 min. The sections were then incubated with 5 % skim milk in TBS (50 mM Tris-buffer saline, pH 7.4) for blocking. After washing with TBS, the sections were incubated for 2 h at room temperature with either of the three antibodies. The sections were washed with TBS and then immunostained using the avidin-biotin peroxidase complex kit (Vector Laboratory, Burlin game, Calif., USA). The reaction was visualized by a 10-min incubation in 40 mg 3,3'-diaminobenzidine in 100 ml of 50 m M Tris-HCl, pH 7.4, containing 0.0133% H20 2.
Results Monoclonal antibodies against APP 45_62 (APP 1-28-9) and (3i_i7 (4A18 and 4A61) were obtained by the above procedures. Monoclonal antibody APP 1-28-9 against APP45-62 strongly stained neurites around se nile plaques (fig. la). Amyloid cores and dif fuse plaques were not recognized with this antibody. Although neurites around senile plaques were strongly stained, neuropil threads were not recognized. APP 1-28-9 stained the cytoplasm of some neurons in AD sections (fig. lb) as well as the sections of other diseases, such as multiple sclerosis. APP 1-28-9 sometimes reacted with tangles (fig. lc) and Pick bodies. It also reacted with some reactive astrocytes. The axons in cere bral infarct were strongly immunolabeled by APP 1-28-9(fig. Id). Monoclonal antibodies 4A18 and 4A61 against (3i_n recognized senile plaques includ ing diffuse ones (fig. 2a). However, they did
11
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protease inhibitor domain homologous to the Kunitz type protease inhibitors [3-5], Using antibodies against APP, APP has been also detected in brain membrane fraction as 110 to 130 kD proteins [6], Since (3-protein is derived from a much larger precursor, the proteolytic processing which produces this fragment must exist. However, this processing has not yet been clarified. Several studies using antibodies against APP showed that APP is expressed in neurons and neurites around senile plaques, suggesting that (3-protein might be processed in those structures. The distribution of APP is particularly of interest because the origins of (3-protein are still controversial, and the processing of APP to (3-protein has not yet been determined. Because the immunoreactivity of antibodies against synthetic peptides decreases in the routine formalin fixation, using these anti bodies it has been difficult to conduct the immunochemical studies on the formalinfixed tissue from many pathological speci mens, including the tissue from AD at differ ent stages. In order to make it possible to determine the APP expression in routine for malin-fixed tissue, we raised monoclonal anti bodies against synthetic peptides.
12
Nukina/Kanazawa/Mannen/Uchida
Accumulation of APP and P-Protein Immunoreactivities
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
Fig. 1. Immunostainings of the monoclonal antibody APPl-28-9 which is an antibody against APP45. 62. a Neurites around senile plaques are immunostained by the antibody. X 200. b A few neurons have granular immunoreactivities in their perikarya. X 300. c Tangle bearing neurons were sometimes immunostained (arrows). X 200. d Axons in ischemic lesions were strongly immunolabeled. X 100.
not react with neurites around senile plaques or neuronal cell bodies. They did not stain normal axons, but showed strong immunostaining of axons in ischemic lesions of cere bral infarct (fig. 2b). These two monoclonal antibodies showed identical staining pat terns.
Discussion Our findings confirm the results of pre vious reports using polyclonal antibodies against APP synthetic peptides and P-protein. Polyclonal antibodies against APP45_62 have been reported to stain neurites [7, 8]. Though one report showed the staining of amyloid with the antibodies to APP45_62 [9], the amy loid cores were not stained in our study. Although it was reported that APP immuno-
reactivities were observed in neuronal cell bodies [8], APP 1-29-8 could not stain neu ronal cell bodies in our study. A few neurons were immunostained with the antibody APP 1-29-8, suggesting that this immunoreactivity is associated with a pathological reac tion of neurons rather than the normal distri bution of APP [ 10]. It is interesting that anti bodies against APP 45_62 and Pi_n stained ax ons in ischemic lesions. APP was demon strated to be transported by means of the fast axonal transport in the peripheral nervous system [11]. Accumulation of APP in axons in ischemic lesions can be explained by the disturbance of the fast axonal flow in these lesions. It was also suggested that APP is axonally trans ported from the perikarya to neuritic endings. Increased APP immunoreactivities in neu rites and axons in the ischemic lesions sup
13
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Fig. 2. Immunostainings of the monoclonal antibody 4A61 which is an antibody against Pi-17, a Amyloid plaques were immunostained by 4A61, but neurites were not. X 100. b Axons in ischemic lesion were immunostained by 4A61. X 100.
port this theory. However, our findings of no (3 immunoreactivity in neurites are puzzling. Possible explanations are the following: (1) (3 immunoreactivity in axons shows the accu mulation of normal APP, and APP accumu lated in neurites is perhaps modified by still undetermined processings and loses its (3 im munoreactivity, and (2) APP accumulated in axons might be different from the APP found in neurites. For example if APP accumulated in axons is proteolyzed in situ, (3 immunoreac tivity could appear. It has also been reported that some antibodies against (3-protein recog nize neuronal cell bodies [12], our antibodies
could not. The APP in the injured axons might be different from the APP in the neu ronal perikarya. Although we cannot explain the discrepancy in the immunoreactivities of APP and ß in neurites and axons, the appear ance of ß immunoreactivity other than in senile plaque may provide a clue to the mech anism of amyloidogenesis.
Acknowledgements This work was kindly supported in part by an award from the Sandoz Foundation for Gerontological Research.
References
14
5 Ranzi RE, McClatchey Al, Lamperti ED, Villa-Komaroff L, Gusella J, Neve RL: Protease inhibitor do main encoded by an amyloid pro tein precursor mRNA associated with Alzheimer’s disease. Nature 1988;331:528-530. 6 Selkoe DJ, Podlisny MB, Joachim CL, et al: (3-Amyloid precursor pro tein of Alzheimer disease occurs as 110- to 135-kilodalton membraneassociated proteins in neural and nonneural tissues. Proc Natl Acad Sei USA 1988;85:7341-7345. 7 Palmert MR, Podlisny MB, Witker DS, et al: Antisera to an amino-ter minal peptide detect the amyloid protein precursor of Alzheimer’s disease and recognize senile plaques. Biochem Biophys Res Commun 1988;156:432-437. 8 Cole GM, Masliah E, Shelton ER, Chan HW. Terry RD, Saitoh T: Ac cumulation of amyloid precursor fragment in Alzheimer plaques. Neurobiol Aging 1991;12:85-91.
Nukina/Kanazawa/Mannen/Uchida
9 Perry G, Lipphardt S, Kanchaerla M, et al: Amyloid precursor protein in senile plaques of Alzheimer dis ease. Lancet 1988;ii:746. 10 Joachim C, Games D, Morris J. Ward P, Frenkel D. Selkoe D: Anti bodies to non-beta regions of the beta-amyloid precursor protein de tect a subset of senile plaques. Am J Pathol 1991;138:373-384. 11 Koo EH, Sisodia SS, Archer DR. et al: Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport. Proc Natl Acad Sci USA 1990:87:15611565. 12 Stern RA, Otvos U , Trojanowski JQ, Lee VY: Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alz heimer’s disease beta-protein recog nize amyloid and diverse glial and neuronal cell types in the central nervous system. Am J Pathol 1989: 134:973-978.
Accumulation of APP and ß-Protein Immunoreactivities
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
1 Glenner GG, Wong CW: Alzheim er’s disease: Initial report of the pu rification and characterization of a novel cerebrovascular amyloid pro tein. Biochem Biophys Res Commun 1984;120:885-890. 2 Kang J, Lemaire HG, Unterbeck A, et al: The precursor of Alzheimer’s disease amyloid A4 protein resem bles a cell-surface receptor. Nature 1987;325:733-736. 3 Kitaguchi N, Takahashi Y. Toku shima Y. Shiojiri S, Ito H: Novel precursor of Alzheimer’s disease amyloid protein shows protease in hibitory activity. Nature 1988:331: 530-532. 4 Ponte P, Gonzalez-DeWhitt P, Schilling J, et al: A new A4 amyloid mRNA contains a domain homolo gous to serine protease inhibitors. Nature 1988;331:525-527.
Department of Neurology, Institute of Brain Research, University of Tokyo, and Diagnostic Research Laboratories, Fujirebio Inc., Tokyo, Japan
KeyWords Amyloid precursor protein ß-Protein Alzheimer’s disease Senile plaques
Accumulation of Amyloid Precursor Protein and 3-Protein Immunoreactivities in Axons Injured by Cerebral Infarct
Abstract To determine the distribution of amyloid precursor protein (APP), monoclonal antibodies against APP45_62 (APP1-28-9) and Pi_i7 (4A18 and 4A61) were produced. In the sections of Alzheimer’s disease (AD) brain, APP 1-28-9 was reactive with neurites around senile plaques and a few neurons but not with amyloid cores. This antibody also immunostained the axons in the ischemic lesions of brain tissues from cases with cere bral infarct. 4A18 and 4A61 were reactive with amyloid plaques but not with neurites and neurons. The latter two anti bodies also immunostained axons in ischemic lesions. These findings suggest that APP, transported by the fast axonal flows, accumulated in the injured axons in the central nervous system. The (3 immunoreactivity appearing in those axons may provide a clue to the mechanism of amyloidogenesis.
Introduction The main pathological features of Alz heimer’s disease (AD) are Alzheimer neurofi brillary tangles and senile plaques. The amy loid core in the senile plaques is composed of (3-protein. (3-Protein was discovered by Glenner and Wong [ 1] as a component of vascular amyloid. Its molecular weight is 4 kD. After
p-protein was discovered, the cDNA of its precursor protein was cloned [2], The de duced sequence of amyloid precursor protein (APP) suggested that APP has a 695 residue, a transmembrane domain with an N-terminal extracellular domain and a C-terminal cyto plasmic domain. A number of variants of APP have been cloned since then and some of them have 751 and 770 residues which have a
Nobuyuki Nukina Department of Neurology Institute of Brain Research University of Tokyo. 7-3-1 Hongo. Bunkyo-ku Tokyo 113 (Japan)
©1992 S. Karger AG. Basel 0304-324X/92/ 0387-0010$2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
N. Nukinaa I. Kanazawaa T. Mannena Y. Uchidab
Material and Methods Antibodies Peptides corresponding to APP45. « [2] and Pi_n [ 1] were synthesized and coupled with keyhole limpet hemocyanin by the maleimidobenzoyl N-hydroxysuccinimide method. BALB/c mice ( 1 mouse for APP45. 62 and 1 mouse for (h-n) were injected intraperitoneally with 100 |ig peptide with Freund’s complete adjuvant. After three booster injections of the same amount at 3-week intervals, splenocytes were fused with P3U1 myeloma cells. Hybridoma supernatants were screened for antipeptide antibodies by the enzymelinked immunosorbent assay. Strongly reactive cul tures were cloned and selected for the immunohisto chemical studies.
Immimohistochemistry We examined several brain tissue sections of pathologically diagnosed AD (8 cases) and of other neurological disease, such as Parkinson’s disease (2 cases), multiple slcerosis (1 case) and cerebral infarc tion (3 cases). Routine 10% formalin-fixed tissues were embedded in paraffin, cut into 6-mm-thick sec tions and affixed to bovine-serum-albumin-coated glass slides. The sections were deparaffinized, and endogenous peroxidase activity was inactivated by ex posure to methanol containing 0.3% (v/v) H 2O2 for 30 min. The sections were then incubated with 5 % skim milk in TBS (50 mM Tris-buffer saline, pH 7.4) for blocking. After washing with TBS, the sections were incubated for 2 h at room temperature with either of the three antibodies. The sections were washed with TBS and then immunostained using the avidin-biotin peroxidase complex kit (Vector Laboratory, Burlin game, Calif., USA). The reaction was visualized by a 10-min incubation in 40 mg 3,3'-diaminobenzidine in 100 ml of 50 m M Tris-HCl, pH 7.4, containing 0.0133% H20 2.
Results Monoclonal antibodies against APP 45_62 (APP 1-28-9) and (3i_i7 (4A18 and 4A61) were obtained by the above procedures. Monoclonal antibody APP 1-28-9 against APP45-62 strongly stained neurites around se nile plaques (fig. la). Amyloid cores and dif fuse plaques were not recognized with this antibody. Although neurites around senile plaques were strongly stained, neuropil threads were not recognized. APP 1-28-9 stained the cytoplasm of some neurons in AD sections (fig. lb) as well as the sections of other diseases, such as multiple sclerosis. APP 1-28-9 sometimes reacted with tangles (fig. lc) and Pick bodies. It also reacted with some reactive astrocytes. The axons in cere bral infarct were strongly immunolabeled by APP 1-28-9(fig. Id). Monoclonal antibodies 4A18 and 4A61 against (3i_n recognized senile plaques includ ing diffuse ones (fig. 2a). However, they did
11
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
protease inhibitor domain homologous to the Kunitz type protease inhibitors [3-5], Using antibodies against APP, APP has been also detected in brain membrane fraction as 110 to 130 kD proteins [6], Since (3-protein is derived from a much larger precursor, the proteolytic processing which produces this fragment must exist. However, this processing has not yet been clarified. Several studies using antibodies against APP showed that APP is expressed in neurons and neurites around senile plaques, suggesting that (3-protein might be processed in those structures. The distribution of APP is particularly of interest because the origins of (3-protein are still controversial, and the processing of APP to (3-protein has not yet been determined. Because the immunoreactivity of antibodies against synthetic peptides decreases in the routine formalin fixation, using these anti bodies it has been difficult to conduct the immunochemical studies on the formalinfixed tissue from many pathological speci mens, including the tissue from AD at differ ent stages. In order to make it possible to determine the APP expression in routine for malin-fixed tissue, we raised monoclonal anti bodies against synthetic peptides.
12
Nukina/Kanazawa/Mannen/Uchida
Accumulation of APP and P-Protein Immunoreactivities
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
Fig. 1. Immunostainings of the monoclonal antibody APPl-28-9 which is an antibody against APP45. 62. a Neurites around senile plaques are immunostained by the antibody. X 200. b A few neurons have granular immunoreactivities in their perikarya. X 300. c Tangle bearing neurons were sometimes immunostained (arrows). X 200. d Axons in ischemic lesions were strongly immunolabeled. X 100.
not react with neurites around senile plaques or neuronal cell bodies. They did not stain normal axons, but showed strong immunostaining of axons in ischemic lesions of cere bral infarct (fig. 2b). These two monoclonal antibodies showed identical staining pat terns.
Discussion Our findings confirm the results of pre vious reports using polyclonal antibodies against APP synthetic peptides and P-protein. Polyclonal antibodies against APP45_62 have been reported to stain neurites [7, 8]. Though one report showed the staining of amyloid with the antibodies to APP45_62 [9], the amy loid cores were not stained in our study. Although it was reported that APP immuno-
reactivities were observed in neuronal cell bodies [8], APP 1-29-8 could not stain neu ronal cell bodies in our study. A few neurons were immunostained with the antibody APP 1-29-8, suggesting that this immunoreactivity is associated with a pathological reac tion of neurons rather than the normal distri bution of APP [ 10]. It is interesting that anti bodies against APP 45_62 and Pi_n stained ax ons in ischemic lesions. APP was demon strated to be transported by means of the fast axonal transport in the peripheral nervous system [11]. Accumulation of APP in axons in ischemic lesions can be explained by the disturbance of the fast axonal flow in these lesions. It was also suggested that APP is axonally trans ported from the perikarya to neuritic endings. Increased APP immunoreactivities in neu rites and axons in the ischemic lesions sup
13
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
Fig. 2. Immunostainings of the monoclonal antibody 4A61 which is an antibody against Pi-17, a Amyloid plaques were immunostained by 4A61, but neurites were not. X 100. b Axons in ischemic lesion were immunostained by 4A61. X 100.
port this theory. However, our findings of no (3 immunoreactivity in neurites are puzzling. Possible explanations are the following: (1) (3 immunoreactivity in axons shows the accu mulation of normal APP, and APP accumu lated in neurites is perhaps modified by still undetermined processings and loses its (3 im munoreactivity, and (2) APP accumulated in axons might be different from the APP found in neurites. For example if APP accumulated in axons is proteolyzed in situ, (3 immunoreac tivity could appear. It has also been reported that some antibodies against (3-protein recog nize neuronal cell bodies [12], our antibodies
could not. The APP in the injured axons might be different from the APP in the neu ronal perikarya. Although we cannot explain the discrepancy in the immunoreactivities of APP and ß in neurites and axons, the appear ance of ß immunoreactivity other than in senile plaque may provide a clue to the mech anism of amyloidogenesis.
Acknowledgements This work was kindly supported in part by an award from the Sandoz Foundation for Gerontological Research.
References
14
5 Ranzi RE, McClatchey Al, Lamperti ED, Villa-Komaroff L, Gusella J, Neve RL: Protease inhibitor do main encoded by an amyloid pro tein precursor mRNA associated with Alzheimer’s disease. Nature 1988;331:528-530. 6 Selkoe DJ, Podlisny MB, Joachim CL, et al: (3-Amyloid precursor pro tein of Alzheimer disease occurs as 110- to 135-kilodalton membraneassociated proteins in neural and nonneural tissues. Proc Natl Acad Sei USA 1988;85:7341-7345. 7 Palmert MR, Podlisny MB, Witker DS, et al: Antisera to an amino-ter minal peptide detect the amyloid protein precursor of Alzheimer’s disease and recognize senile plaques. Biochem Biophys Res Commun 1988;156:432-437. 8 Cole GM, Masliah E, Shelton ER, Chan HW. Terry RD, Saitoh T: Ac cumulation of amyloid precursor fragment in Alzheimer plaques. Neurobiol Aging 1991;12:85-91.
Nukina/Kanazawa/Mannen/Uchida
9 Perry G, Lipphardt S, Kanchaerla M, et al: Amyloid precursor protein in senile plaques of Alzheimer dis ease. Lancet 1988;ii:746. 10 Joachim C, Games D, Morris J. Ward P, Frenkel D. Selkoe D: Anti bodies to non-beta regions of the beta-amyloid precursor protein de tect a subset of senile plaques. Am J Pathol 1991;138:373-384. 11 Koo EH, Sisodia SS, Archer DR. et al: Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport. Proc Natl Acad Sci USA 1990:87:15611565. 12 Stern RA, Otvos U , Trojanowski JQ, Lee VY: Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alz heimer’s disease beta-protein recog nize amyloid and diverse glial and neuronal cell types in the central nervous system. Am J Pathol 1989: 134:973-978.
Accumulation of APP and ß-Protein Immunoreactivities
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/27/2018 10:24:44 AM
1 Glenner GG, Wong CW: Alzheim er’s disease: Initial report of the pu rification and characterization of a novel cerebrovascular amyloid pro tein. Biochem Biophys Res Commun 1984;120:885-890. 2 Kang J, Lemaire HG, Unterbeck A, et al: The precursor of Alzheimer’s disease amyloid A4 protein resem bles a cell-surface receptor. Nature 1987;325:733-736. 3 Kitaguchi N, Takahashi Y. Toku shima Y. Shiojiri S, Ito H: Novel precursor of Alzheimer’s disease amyloid protein shows protease in hibitory activity. Nature 1988:331: 530-532. 4 Ponte P, Gonzalez-DeWhitt P, Schilling J, et al: A new A4 amyloid mRNA contains a domain homolo gous to serine protease inhibitors. Nature 1988;331:525-527.
