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AMYLOID PRECURSOR PROTEIN IN SENILE PLAQUES OF ALZHEIMER DISEASE
746 AMYLOID PRECURSOR PROTEIN IN SENILE PLAQUES OF ALZHEIMER DISEASE
62) and C-terminal (residues 638 to 658) antisera recognised the structures as the original antisera, although with less background staining. Specificity of the immunoreaction with these two antisera was further shown by blockage of senile plaque staining by adsorption with the peptide used as immunogen. Our results indicate that: (1) antibodies to APP sequences outside those contained within A4 do not recognise congophilic amyloid deposits; (2) APP deposition precedes the formation of A4 amyloid; and (3) proteolysis of APP to A4 amyloid occurs within the senile plaque. We speculate that the abnormality leading to the senile plaque is APP accumulation, which possibly defines the plaque, to
same
SIR,-Senile plaques and neurofibrillary tangles are the pathological characteristics of Alzheimer disease. Proteolysis of a larger precursor protein leads to the formation of the 42-aminoacid residue amyloid fragment (A4 or P-protein),1,2 which is a major component of the core of the plaque. cDNAs for the amyloid precursor protein (APP) have been isolated, and sequences of 695, 751, or 770 aminoacids, differing by a serine protease inhibitor insert, have been reported.3,4 However, the cellular origin and the site of proteolysis of APP in Alzheimer disease remain to be
rather than altered APP metabolism alone. Clarification of the site of APP entry and accumulation (vascular, neuronal, or endothelial) and processing, will be important in defining senile plaque pathogenesis in the aetiology of Alzheimer disease.
established. In an immunocytochemical study of brain tissue from cases of Alzheimer disease, we produced polyclonal antisera in rabbits to synthetic peptides of 11 to 42 aminoacid residues long, corresponding to twelve regions of APP and spanning residues 18 to 678 (including A4).’ Antisera were raised to keyhole limpet haemocyanin or a-thyroglobin conjugates of the peptides or, for sequences represented in A4, to the peptide alone. The peptides and conjugates were produced in our laboratory or purchased from Peninsula Laboratories (Belmont, California). The antisera specifically recognised the peptide used as immunogen in ELI SA or
Supported by grants from the National Institutes of Health (AG-07552, K04-AG00415, and AG-00795) and the American Health Assistance Foundation. Division of
Neuropathology,
GEORGE PERRY SANDRA LIPPHARDT PAUL MULVIHILL University, MADHU KANCHERLA MAGDALENA MIJARES Cleveland, Ohio 44106, PIERLUIGI GAMBETTI SATISH SHARMA USA, JAMES CORNETTE and Upjohn Company LINDA MAGGIORA BARRY GREENBERG THOMAS LOBL Kalamazoo, Michigan
Institute of Pathology, Case Western Reserve
immunodot assays. Antibodies raised to sequences containing all or part of A4 stained senile plaque cores (figure, A), irregular areas that appeared to be plaques,S and some vessels. But antibodies raised to other APP sequences recognised only a subset of this pattern, staining the periphery or irregular areas surrounding the senile plaque cores (figure, C) and similar areas displaying no core (figure, B). The staining of the periphery of plaque cores was better appreciated with Congo red (a marker for amyloid) under polarised light (figure, D). Occasional cell processes within the senile plaque were also intensely immunostained (figure, C). Although not all antibodies were effective in immunostaining, the absence of any difference in staining pattern with antibodies to APP outside A4, from aminoacid residues 18 to 678, indicates that native full-length APP is probably present in senile plaques. Immunopurified N-terminal (residues 45
Kang J, Lemaire H-G, Unterbeck A, et al The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor Nature 1987; 325: 733-36. 2 Masters CL, Simms G, Weinman NA, et al. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 1985, 82: 4245-49. 3. Ponte P, Gonzalez-DeWhitt P, Schilling J, et al A new A4 amyloid mRNA contains a domain homologous to senne protemase inhibitors Nature 1988, 331: 525-27 4. Kitagushi N, Takahashi Y, Tokushima Y, Shiojiri S, Ito H. Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity. Nature 1988; 1.
5.
331: 530-32. Giaccone G, Frangione B, Bugiani O. Cortical pre-amyloid deposition Alzheimer patients and normals. J Neuropathol Exp Neurol 1988, 47: 332.
Tagliavini F,
in
HERPESVIRUSES AS CO-FACTORS IN AIDS
SIR,-Several investigators have proposed that herpesviruses as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) may be important in the development of AID S. 1-4 In-vitro data suggest that herpesviruses may be co-factors in HIV pathogenesis by transactivation of the HIV genome.5-6 Unfortunately, there are few ways to evaluate the putative role of herpesviruses as co-factors in such
AIDS and other HIV-associated diseases. Antibodies to these viruses are often present in the serum of homosexual men with HIV-associated disease but they can also be found in homosexual men without HIV infection or disease. Serological studies have usually been done on specimens obtained after AIDS has developed, and only rarely have investigators been able to study sera collected from HIV-infected individuals before disease has
developed.7,8
Staining of senile plaques by antibodies raised to sequences containing all or part of A4. Antibodies to sequences corresponding to A4 recognised core of senile plaques (A). Antisera raised to APP sequences outside A4 recognised non-congophilic plaques (B) and region surrounding core (C), which was birefringent in polarised light after Congo red staining (D). Immunostained cell processes (C, arrow) surrounded the core. Antisera were raised to peptides corresponding to APP aminoacid residues 597 to 638 in (A), 45 to 62 in (B), and 490 to 504 in (C) and (D).’ Peroxidase-antiperoxidase, x 400.
.
From our study of homosexual men in San Francisco we identified HIV-seropositive men with AIDS, AIDS-related complex and lymphadenopathy syndrome, or lymphopenia (according to defmitions current in 19859) and who had had serum samples drawn before those diseases developed. We studied sera drawn when the above syndromes developed (specimen 3), together with sera from the year before (specimen 2), and one earlier specimen (specimen 1). For comparison we identified for each individual a man who had had HIV-seropositive serum drawn in the same years but in whom disease had not developed by the time specimen 3 was drawn. As described elsewhere10 we measured antibody titres to CMV (enzyme immunoassay), to EBV (indirect immunofluorescence), and to HSV-1 and HSV-2 (enzyme-linked immunodot-blot). We found no significant differences in rises in antibody titres to CMV or EBV or seroconversions for HSV-1 or HSV-2 between the men with the HIV-associated diseases and those without (table). This study was limited to 34 pairs of men who had had several specimens drawn before the development (or not) of HIV-related
62) and C-terminal (residues 638 to 658) antisera recognised the structures as the original antisera, although with less background staining. Specificity of the immunoreaction with these two antisera was further shown by blockage of senile plaque staining by adsorption with the peptide used as immunogen. Our results indicate that: (1) antibodies to APP sequences outside those contained within A4 do not recognise congophilic amyloid deposits; (2) APP deposition precedes the formation of A4 amyloid; and (3) proteolysis of APP to A4 amyloid occurs within the senile plaque. We speculate that the abnormality leading to the senile plaque is APP accumulation, which possibly defines the plaque, to
same
SIR,-Senile plaques and neurofibrillary tangles are the pathological characteristics of Alzheimer disease. Proteolysis of a larger precursor protein leads to the formation of the 42-aminoacid residue amyloid fragment (A4 or P-protein),1,2 which is a major component of the core of the plaque. cDNAs for the amyloid precursor protein (APP) have been isolated, and sequences of 695, 751, or 770 aminoacids, differing by a serine protease inhibitor insert, have been reported.3,4 However, the cellular origin and the site of proteolysis of APP in Alzheimer disease remain to be
rather than altered APP metabolism alone. Clarification of the site of APP entry and accumulation (vascular, neuronal, or endothelial) and processing, will be important in defining senile plaque pathogenesis in the aetiology of Alzheimer disease.
established. In an immunocytochemical study of brain tissue from cases of Alzheimer disease, we produced polyclonal antisera in rabbits to synthetic peptides of 11 to 42 aminoacid residues long, corresponding to twelve regions of APP and spanning residues 18 to 678 (including A4).’ Antisera were raised to keyhole limpet haemocyanin or a-thyroglobin conjugates of the peptides or, for sequences represented in A4, to the peptide alone. The peptides and conjugates were produced in our laboratory or purchased from Peninsula Laboratories (Belmont, California). The antisera specifically recognised the peptide used as immunogen in ELI SA or
Supported by grants from the National Institutes of Health (AG-07552, K04-AG00415, and AG-00795) and the American Health Assistance Foundation. Division of
Neuropathology,
GEORGE PERRY SANDRA LIPPHARDT PAUL MULVIHILL University, MADHU KANCHERLA MAGDALENA MIJARES Cleveland, Ohio 44106, PIERLUIGI GAMBETTI SATISH SHARMA USA, JAMES CORNETTE and Upjohn Company LINDA MAGGIORA BARRY GREENBERG THOMAS LOBL Kalamazoo, Michigan
Institute of Pathology, Case Western Reserve
immunodot assays. Antibodies raised to sequences containing all or part of A4 stained senile plaque cores (figure, A), irregular areas that appeared to be plaques,S and some vessels. But antibodies raised to other APP sequences recognised only a subset of this pattern, staining the periphery or irregular areas surrounding the senile plaque cores (figure, C) and similar areas displaying no core (figure, B). The staining of the periphery of plaque cores was better appreciated with Congo red (a marker for amyloid) under polarised light (figure, D). Occasional cell processes within the senile plaque were also intensely immunostained (figure, C). Although not all antibodies were effective in immunostaining, the absence of any difference in staining pattern with antibodies to APP outside A4, from aminoacid residues 18 to 678, indicates that native full-length APP is probably present in senile plaques. Immunopurified N-terminal (residues 45
Kang J, Lemaire H-G, Unterbeck A, et al The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor Nature 1987; 325: 733-36. 2 Masters CL, Simms G, Weinman NA, et al. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 1985, 82: 4245-49. 3. Ponte P, Gonzalez-DeWhitt P, Schilling J, et al A new A4 amyloid mRNA contains a domain homologous to senne protemase inhibitors Nature 1988, 331: 525-27 4. Kitagushi N, Takahashi Y, Tokushima Y, Shiojiri S, Ito H. Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity. Nature 1988; 1.
5.
331: 530-32. Giaccone G, Frangione B, Bugiani O. Cortical pre-amyloid deposition Alzheimer patients and normals. J Neuropathol Exp Neurol 1988, 47: 332.
Tagliavini F,
in
HERPESVIRUSES AS CO-FACTORS IN AIDS
SIR,-Several investigators have proposed that herpesviruses as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) may be important in the development of AID S. 1-4 In-vitro data suggest that herpesviruses may be co-factors in HIV pathogenesis by transactivation of the HIV genome.5-6 Unfortunately, there are few ways to evaluate the putative role of herpesviruses as co-factors in such
AIDS and other HIV-associated diseases. Antibodies to these viruses are often present in the serum of homosexual men with HIV-associated disease but they can also be found in homosexual men without HIV infection or disease. Serological studies have usually been done on specimens obtained after AIDS has developed, and only rarely have investigators been able to study sera collected from HIV-infected individuals before disease has
developed.7,8
Staining of senile plaques by antibodies raised to sequences containing all or part of A4. Antibodies to sequences corresponding to A4 recognised core of senile plaques (A). Antisera raised to APP sequences outside A4 recognised non-congophilic plaques (B) and region surrounding core (C), which was birefringent in polarised light after Congo red staining (D). Immunostained cell processes (C, arrow) surrounded the core. Antisera were raised to peptides corresponding to APP aminoacid residues 597 to 638 in (A), 45 to 62 in (B), and 490 to 504 in (C) and (D).’ Peroxidase-antiperoxidase, x 400.
.
From our study of homosexual men in San Francisco we identified HIV-seropositive men with AIDS, AIDS-related complex and lymphadenopathy syndrome, or lymphopenia (according to defmitions current in 19859) and who had had serum samples drawn before those diseases developed. We studied sera drawn when the above syndromes developed (specimen 3), together with sera from the year before (specimen 2), and one earlier specimen (specimen 1). For comparison we identified for each individual a man who had had HIV-seropositive serum drawn in the same years but in whom disease had not developed by the time specimen 3 was drawn. As described elsewhere10 we measured antibody titres to CMV (enzyme immunoassay), to EBV (indirect immunofluorescence), and to HSV-1 and HSV-2 (enzyme-linked immunodot-blot). We found no significant differences in rises in antibody titres to CMV or EBV or seroconversions for HSV-1 or HSV-2 between the men with the HIV-associated diseases and those without (table). This study was limited to 34 pairs of men who had had several specimens drawn before the development (or not) of HIV-related