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Synaptophysin and chromogranin A immunoreactivities in senile plaques of Alzheimer's disease
Brain Research, 539 (1991) 143-150 Elsevier
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Synaptophysin and chromogranin A immunoreactivities in senile plaques of Alzheimer's disease Jean-Pierre Brion 1, Anne-Marie Couck 1, Mary Bruce 2, Brian Anderton 2 and Jacqueline Flament-Durand 1 1Laboratory of Pathology and Electron Microscopy, Universit# Libre de Bruxelles, Brussels (Belgium) and 2Department of Neuroscience, Institute of Psychiatry, London (U.K.) (Accepted 2 October 1990) Key words: Synaptophysin; Chromogranin A; Senile plaque; A4 amyloid; Aizheimer's disease
Immunolabelling for synaptophysin and chromogranin A, two polypeptides associated with small clear and large dense core synaptic vesicles respectively, has been performed on tissue sections of the temporal cortex in Alzheimer's disease in combination with anti-A4 amyloid labelling. The dystrophic neurites in many senile plaques were observed to be labelled by the anti-synaptophysin or anti-chromogranin A antibodies. Some diffuse amyloid deposits, demonstrated by antibodies against synthetic amyloid A4 peptides, were associated with a punctuate increase in synaptophysin or chromogranin A immunoreactivity. The labelling of dystrophic plaque neurites may reflect the accumulation in these processes of synaptic vesicles or material derived from them. We suggest also that the punctuate increase in synaptophysin and chromogranin A immunoreactivities associated with some A4 amyloid deposits may be an early event reflecting neuronal dysfunction.
Senile plaques and neurofibrillary tangles are conspicuous neuropathological lesions observed in the brains of individuals affected with Alzheimer's disease. Neurofibrillary tangles are made of abnormal filaments (paired helical filaments, PHFs) accumulating in neurones and their processes. One major constituent protein of PHF has been shown to be the microtubule-associated protein tau 3. Senile plaques are complex structures and several types of senile plaques have been described. A distinction is made usually between primitive or neuritic plaques, classical plaques, and compact or burned-out plaques 35. The classical plaque is composed of an extracellular 'core' of amyloid material, surrounded by reactive astrocytes, microglial cells and dystrophic neurites. The dystrophic neurites in plaques contain PHF and accumulations of lysosomal bodies and other membraneous organelles. The primitive plaque shows abnormal neurites with a few whisps of amyloid material, whereas the 'burned-out' plaque is limited to a dense amyloid core, with few or no abnormal neurites which are presumed to have degenerated completely. A 42 amino acid polypeptide, called A4- or fl-amyloid, has been isolated from the plaque amyloid 22 and from the cerebrovascular amyloid 8 which accumulates in some vessel walls in Alzheimer's disease; this A4 polypeptide derives from a precursor (fl-amyloid precursor or PreA4)
which has the features of a transmembrane protein 13. More recently, 'very primitive' plaques 25 and 'preamyloid deposit '34 or 'diffuse plaque '43, the latCer being limited to a non-compact and diffuse area of immunopositive staining with A4 antibodies, have been described. The relationship between these morphologically different types of plaques and the different types of A4 amyloid deposits observed in brain 41,43 is still a matter of debate. Diffuse plaques are numerous in the younger Down's syndrome patients19; since in this disease the pathology is age-dependent and similar to that in Alzheimer's disease, diffuse plaques might represent an early stage of plaque formation and with abnorm~i| neurites appearing later. Alternatively diffuse plaques might represent only one of the different types of A4 amyloid deposits observed in the brain in Alzheimer's disease 41. Antibodies to P H F and their constituent proteins (e.g. tau) have not been reported to label neurites in diffuse plaques. However, if PHF accumulation in abnormal neurites is preceeded by other events possibly including organelle accumulations, a neuritic component to diffuse plaques may be revealed by appropriate markers. In this report, we describe the results of such a study using double immunocytochemistry with antibodies to chromogranin A, synaptophysin and A4 amyloid. Synaptophysin and chromogranin A are two markers of vesicles
Correspondence: J.P. Brion, Laboratory of Pathology and Electron Microscopy, Universit6 Libre de Bruxelles, 808, route de Lennik 1070, Brussels, Belgium. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
144 found in neurones and in non-neuronal neuroendocrine cells5'12"4°. A labelling of dystrophic neurites with antisynaptophysin and anti-chromogranin A antibodies has been previously mentioned in some studies 2°,38 but the demonstration that synaptophysin and chromogranin A immunoreactive neurites are associated with A4 amyloid deposits has not been reported. We demonstrate here that abnormal neurites visualized with anti-synaptophysin and anti-chromogranin A antibodies are associated with A4 amyloid deposits in several type of plaques, including in diffuse plaques. The anti-synaptophysin (clone SY38) used in this study is a mouse monoclonal antibody 4° purchased from Biotest and was used diluted 1/100 in TBS (0.01 M Tris-HCI, pH 7.6, 0.15 M NaCl) containing 10% normal goat serum. The anti-chromogranin A (clone LK2H10) is a mouse monoclonal antibody TM purchased from Hybritech and used at the same dilution. The anti-amyloid A4 sera were raised against synthetic peptides corresponding to residues 1-10 (anti-A41_10) and 12-28 (anti-A412_ES) of the amyloid A4 polypeptide21'22. Synthetic peptides were coupled to keyhole limpet hemocyanin and injected in rabbits. The anti-A41_~0 and anti-A412._2s sera were tested by ELISA using the synthetic peptides and by Western blotting on human (frontal cortex) and rat total brain homogenates. The latter were prepared by homogenizing brain tissue at 0 °C (1 g/ml) in TBS, trypsin inhibitor (100 /~g/ml), and phenylmethylsulfonyl fluoride (0.5 mM). The homogenate was centrifuged for 1 h at 105 gav (4 °C) and the supernatant retained for Western blot analysis. The anti-A41_10 and anti-A4~2_2s sera were used diluted 1/250. Brain tissue samples from the temporal cortex, the hippocampus and the parahippocampal gyrus were taken at autopsy in patients with Alzheimer's disease. Tissues were fixed in 10% formalin (3 days to 3 weeks) and embedded in paraffin. The immunolabelling was performed on 7/~m tissue sections with the peroxidase-antiperoxidase (PAP) method for single labelling, using diaminobenzidine as chromogen. The PAP complex, goat anti-rabbit and goat anti-mouse antibodies were obtained from Nordic (Netherlands). In double-labelling experiments, the first labelling was performed with the PAP method and the second labelling was performed by an indirect method using a goat anti-rabbit (Nordic, diluted 1/20) conjugated to fluorosceine isothiocyanate. For detection of A4 amyloid deposits with the anti-A4 sera, slides were first treated 20 min with undiluted formic acid ~4. Some slides were counterstained with Congo red or with thioflavine S to demonstrate amyloid deposits. Controls for the immunolabelling by the anti-A4 sera were performed by using normal rabbit serum in place of the anti-A4 sera. Controls for the immunolabelling by the anti-synap-
A
B
C
D
Fig. 1. Immunoblots of total brain supernatants from rat (lanes A and B) and Alzheimer (lanes C and D) brain homogenates. Lanes A and C: anti-A412 28 serum. Lanes B and D: anti-A41_~0serum. Molecular weight markers (arrows) are myosin (200 kDa), flgalactosidase (116 kDa), phosphorylase B (97 kDa), albumin (66 kDa), and ovalbumin (43 kDa).
tophysin and the anti-chromogranin A monoclonal antibodies (IgG1) were performed by incubating sections with an isotype-matched (IgG1) monoclonal anti-keratin antibody (Immunotech, clone KL 1) at the same dilution. The anti-A41_10 and anti-A412_28 sera both react with their corresponding peptide by ELISA. By immunoblotting, the anti-A412_28 serum labels at least two bands of molecular weight between 100 and 130 kDa in rat brain and two bands of 130 kDa and 80 kDa, respectively, in human brain (Fig. 1). Proteins with similar molecular weights were identified in other studies by immunoblotting with antibodies to the N- and C-terminus of the A4 amyloid precursor 23'3°. Kunitz-type inserts and N- or O-glycosylation could explain the detection of several A4 precursors isoforms and their difference in molecular weight 37. The lower molecular weight bands might correspond to the secreted form of A4 precursor, lacking the C-terminal part of it, rather than the membranebound form. The anti-A41_~o does not label consistently any bands in these preparations. Several antisera raised to synthetic A4 peptides have been reported not to label significantly any bands by Western blotting 3°'31 which might be due to epitopes being sterically hidden in the precursor protein when bound to nitrocellulose. As previously reported 1'22'32"34'41 -43, the anti-A4 sera
145 detect numerous A4 amyloid deposits in tissue sections after formic acid treatment, i.e. plaque amyioid in neuritic, classical, burned-out plaques and more diffuse and less intensively labelled A4-immunoreactive areas, some of the latter having the appearance of the described diffuse plaques. The amyloid material deposited in vessels walls was strongly labelled. Sub-pial amyloid deposits were also observed to be labelled. The antiA4t2_28 labelled, however, more deposits than the antiA41_10 as previously found in cases of Down's syndrome 32. Sections incubated with normal rabbit serum did not show any labelling. The anti-synaptophysin labels all the thickness of the gray matter and some fibers in the white matter. The labelling in the gray matter has a punctuate aspect and could sometimes be seen concentrated at the periphery of neuronal pericarya and proximal dendrites, the latter labelling corresponding probably to synaptic contacts. The anti-chromogranin A antibody labelled less intensively the neuropil and showed a granular labelling of some neuronal bodies. In areas rich in neurofibrillary tangles, the anti-synaptophysin labelling was weaker. This was especially prominent in star cell dusters of layer II and in layer IV of the presubiculum. The anti-synaptophysin and the anti-chromogranin antibodies demonstrated the abnormal neurites in many senile plaques. This labelling was more prominent in abnormal neurites in neuritic plaques and in thick, dilated neurites surrounding an amyloid core in classical plaques (Figs. 2A, 3A,C and 4A). This labelling of neurites was sometimes discrete, limited to one or two small neurites (Fig. 3G). Another plaque-like structure showing a synaptophysin or a chromogranin A immuno-
reactivity is shown in Figs. 2B and 5A. In these focal areas the anti-synaptophysin (Figs. 2B,C and 3E) and anti-chromogranin A (Fig. 5A,B) labelling appears as a more or less homogeneous distribution of punctuate staining, often giving the impression of a focal increase in the neuropil labelling. Thioflavine S counterstaining and anti-A412_28 labelling after anti-synaptophysin or antichromogranin A labelling showed that these granular areas of synaptophysin or chromogranin A immunoreactivity were associated with diffuse A4 amyloid immunoreactivity. These areas were less stained (thioflavine S) or labelled (anti-A412_28) than the amyloid deposits of neuritic, classical or burned-out plaques. The double immunolabelling combining the anti-synaptophysin or the anti-chromogranin A and the antiA4~2_28 showed that practically all plaques with synaptophysin or chromogranin A immunoreactive neurites were also anti-A4 positive (Figs. 3D,F and 5D), but not all A4 amyloid deposits were associated with increases in synaptophysin or chromogranin immunoreactivities. A strong labelling of plaque neurites with the antisynaptophysin and the anti-chromogranin A antibodies was obtained only in tissue samples with a short fixation time (less than 24 h). No labelling was observed with the monoclonal anti-keratin antibody. Synaptophysin is a 38 kDa polypeptide which appears to be an integral membrane protein found in small vesicles with clear centers, i.e. in vesicles containing classical neurotransmitters in neurones ~2,4°. The function of synaptophysin is still little understood but it has been observed that synaptic-like vesicles appear in fibroblasts transfected with a synaptophysin gene17; this might indicate that this protein plays an important role in the
Fig. 2. Immunolabellingon paraffin sections of Alzheimer brain tissue (temporal cortex) with the anti-synaptophysinantibodies, showing the different types of immunoreactivityin senile plaques. A: Abnormal, dilated neurites are strongly labelled in this plaque. The amyloid core is unlabelled. B and C: The labelling in these two plaques appears as homogeneous and punctuate, looking as a focal increase in the neuropil immunoreactivity. The labelling in C surrounds an unlabelled core. Bar = 10 am.
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Fig. 3. Immunolabelling on paraffin sections of Alzheimer brain tissue (temporal cortex). A,B: abnormal, dilated neurites in this plaque are labelled by the anti-synaptophysin antibodies (arrows in A) and surround an amyloid core demonstrated by its birefringence after Congo red counterstaining (B). C,D: double immunolabelling. The synaptophysin-immunoreactiveneurites in this plaque (C) surround a compact amyloid core labelled by the anti-A4~2_2aserum (D). E,F: immunolabelling with the anti-synaptophysin (E) followed by thioflavine S counterstaining (F). One senile plaque (arrow) is associated with a punctuate increase in synaptophysin immunoreactivity and discrete diffuse positive amyloid immunoreaetivity. The adjacent plaque (arrowhead) show a discrete synaptophysin immunoreactivity and a compact amyloid core. G,H: double immunolabelling. The plaque amyloid labelled by the anti-A412_2sserum (H) is associated with only a few small synaptophysin-immunoreactive neurites (arrow in G). Bar = 10/~m.
formation of the synaptic vesicle membranes. Chromogranin A belongs to a family of secretory proteins (chromogranins/secretogranins) which have been found in large dense core vesicles present in neurones and in non-neuronal neuroendoerine cells5. In neurones, these large dense core vesicles contain neuropeptides. The function of chromogranins is not yet well determined; these proteins might be processed to smaller peptides which could exert extraceUular biological activities. They might also play a role in the packaging and/or processing of neuropeptides 27. In Alzheimer's disease brain tissue we observed that in areas rich in neurofibrillary tangles (star cell clusters in layer II and layer IV of presubiculum) there is a decrease in synaptophysin immunoreactivity as compared to the neigbouring neuropil. This probably reflects the loss of neurones in these areas and of the synaptic arborization of tangle-beating neurones 29. A decrease in the synaptophysin immunoreactivity in the neuropil in Alzheimer's
disease, as compared to normal controls, has been previously reported 11,2°. We suggest that the labelling of neurites in primitive and classical plaques reflects the accumulation in these processes of synaptic vesicles or material derived from them. Previous ultrastructural observations of these plaques has shown that abnormal, dilated neurites in plaques contain PHF, accumulations of multilamellar bodies, multivesicular bodies, lysosomial bodies and degraded mitochondria 4'6'9,35. Some of these membranous organelles may contain material derived from synaptic vesicles, e.g. multivesicular bodies may represent a form of autophagy of vesicles. Since synaptic vesicles, like other membranous organelles, move along microtubules (fast neuroplasmic transport), their accumulation in abnormal plaque neurites might witness for axoplasmic flow disturbances in these neurites, as previously suggested 4'6"1°'26'35. Synaptic contacts on abnormal neurites in senile
Fig. 4. Immunolabelling on paraffin sections of Alzheimer brain tissue with the anti-chromogranin A antibodies. A: the dystrophic neurites in this neuritic plaque are labelled. B,C: the labelled neurites in this plaque (arrows in B) surround an amyloid core shown in C after counterstaining with thioflavine S. Bar = 10 ~m.
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Fig. 5. Immunolabellingon paraffin sections of Alzheimer brain tissue. A,B: immunolabelling with the anti-chromogranin A antibodies (A) followed by thioflavine S counterstaining (B). The amyloid deposit shown in B is associated with a punctuate increase in chromogranin A immunoreactivity. C,D: double immunolabelling with the anti-chromogranin A (C) and the the anti-A412 2~serum (D). The A4 amyloiddeposit is surrounded by a punctuate increase in chromogranin A immunoreactivity. Bar = 10/~m.
plaques have been described in electron microscopy9 and may contribute to the synaptophysin and chromogranin labelling of plaque neurites. Many presynaptic boutons were however reported to contain less synaptic vesicles than normal presynaptic boutons; this suggests that the increase in synaptophysin and chromogranin labelling in plaque neurites is rather related to the accumulation of material in these dystrophic neurites. The finding of polypeptides associated with synaptic vesicles in some plaque neurites suggests also that these neurites are axonal rather than dendritic terminals. The neuritic processes in plaques have been previously visualized by their content in specific neurotransmitters
or in enzymes involved in the metabolism of these neurotransmitters. The identified neurotransmitters in plaque neurites corresponded to several neuropeptides 33 as well as classical neurotransmitters 2'~5. This would be consistent with our results showing the presence of both types of vesicle-associated proteins in plaque neurites. In addition we observed also a homogeneous distribution of punctuate synaptophysin and chromogranin A immunoreactivities in focal areas associated with diffuse A4 amyloid deposits. These structures probably correspond to the previously described diffuse plaques 43 or preamyloid deposits 34. The A4 immunoreactive structures in these diffuse plaques are made up of extracellular
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a b n o r m a l dystrophic neurites are sometimes seen in electron microscopy in diffuse plaques 45. The synaptophysin and chromogranin immunoreactivity in these early plaques might reflect early events occuring at the level of neurites, e.g. a b n o r m a l synaptogenesis or sprouting. Several studies have suggested that a b n o r m a l neurites in senile plaques exhibit sprouting p h e n o m e n a TM. T h e latter p h e n o m e n a might be related to the p r e s u m p tive trophic and proliferative abilities of A 4 amyloid p o l y p e p t i d e and its precursor 28'39. Alternatively, these
of dilated, dystrophic plaque neurites. T h e accumulation of several n e u r o p e p t i d e s in plaque neurites has been suggested to p r e c e d e the accumulation of P H F in the same neurites 16 and our observations are consistent with such an interpretation. O u r results indicate thus that synaptophysin and chromogranin A , two m a r k e r s of synaptic vesicles, can be used to follow and identify p l a q u e neurites in Alzheimer's disease. We suggest also that changes in the synaptic network might be an early change of neurites found in contact with amyloid deposits.
p u n c t u a t e increases in synaptophysin and chromogranin A immunoreactivities might be a consequence of early disturbances of neuroplasmic transport of synaptic vesicles. The increases in synaptophysin and chromogranin A immunoreactivities could thus precede the d e v e l o p m e n t
This study was supported by grants from the Belgian ER.EC. and ER.S.M. (Nos. 2.4523.87, 9.8009.889, 9.4501.85), NATO, MRC and the WeUcome Trust. We thank the Brain Bank of the Institute of Psychiatry (Department of Neuropathology) for furnishing some tissue samples.
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Synaptophysin and chromogranin A immunoreactivities in senile plaques of Alzheimer's disease Jean-Pierre Brion 1, Anne-Marie Couck 1, Mary Bruce 2, Brian Anderton 2 and Jacqueline Flament-Durand 1 1Laboratory of Pathology and Electron Microscopy, Universit# Libre de Bruxelles, Brussels (Belgium) and 2Department of Neuroscience, Institute of Psychiatry, London (U.K.) (Accepted 2 October 1990) Key words: Synaptophysin; Chromogranin A; Senile plaque; A4 amyloid; Aizheimer's disease
Immunolabelling for synaptophysin and chromogranin A, two polypeptides associated with small clear and large dense core synaptic vesicles respectively, has been performed on tissue sections of the temporal cortex in Alzheimer's disease in combination with anti-A4 amyloid labelling. The dystrophic neurites in many senile plaques were observed to be labelled by the anti-synaptophysin or anti-chromogranin A antibodies. Some diffuse amyloid deposits, demonstrated by antibodies against synthetic amyloid A4 peptides, were associated with a punctuate increase in synaptophysin or chromogranin A immunoreactivity. The labelling of dystrophic plaque neurites may reflect the accumulation in these processes of synaptic vesicles or material derived from them. We suggest also that the punctuate increase in synaptophysin and chromogranin A immunoreactivities associated with some A4 amyloid deposits may be an early event reflecting neuronal dysfunction.
Senile plaques and neurofibrillary tangles are conspicuous neuropathological lesions observed in the brains of individuals affected with Alzheimer's disease. Neurofibrillary tangles are made of abnormal filaments (paired helical filaments, PHFs) accumulating in neurones and their processes. One major constituent protein of PHF has been shown to be the microtubule-associated protein tau 3. Senile plaques are complex structures and several types of senile plaques have been described. A distinction is made usually between primitive or neuritic plaques, classical plaques, and compact or burned-out plaques 35. The classical plaque is composed of an extracellular 'core' of amyloid material, surrounded by reactive astrocytes, microglial cells and dystrophic neurites. The dystrophic neurites in plaques contain PHF and accumulations of lysosomal bodies and other membraneous organelles. The primitive plaque shows abnormal neurites with a few whisps of amyloid material, whereas the 'burned-out' plaque is limited to a dense amyloid core, with few or no abnormal neurites which are presumed to have degenerated completely. A 42 amino acid polypeptide, called A4- or fl-amyloid, has been isolated from the plaque amyloid 22 and from the cerebrovascular amyloid 8 which accumulates in some vessel walls in Alzheimer's disease; this A4 polypeptide derives from a precursor (fl-amyloid precursor or PreA4)
which has the features of a transmembrane protein 13. More recently, 'very primitive' plaques 25 and 'preamyloid deposit '34 or 'diffuse plaque '43, the latCer being limited to a non-compact and diffuse area of immunopositive staining with A4 antibodies, have been described. The relationship between these morphologically different types of plaques and the different types of A4 amyloid deposits observed in brain 41,43 is still a matter of debate. Diffuse plaques are numerous in the younger Down's syndrome patients19; since in this disease the pathology is age-dependent and similar to that in Alzheimer's disease, diffuse plaques might represent an early stage of plaque formation and with abnorm~i| neurites appearing later. Alternatively diffuse plaques might represent only one of the different types of A4 amyloid deposits observed in the brain in Alzheimer's disease 41. Antibodies to P H F and their constituent proteins (e.g. tau) have not been reported to label neurites in diffuse plaques. However, if PHF accumulation in abnormal neurites is preceeded by other events possibly including organelle accumulations, a neuritic component to diffuse plaques may be revealed by appropriate markers. In this report, we describe the results of such a study using double immunocytochemistry with antibodies to chromogranin A, synaptophysin and A4 amyloid. Synaptophysin and chromogranin A are two markers of vesicles
Correspondence: J.P. Brion, Laboratory of Pathology and Electron Microscopy, Universit6 Libre de Bruxelles, 808, route de Lennik 1070, Brussels, Belgium. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
144 found in neurones and in non-neuronal neuroendocrine cells5'12"4°. A labelling of dystrophic neurites with antisynaptophysin and anti-chromogranin A antibodies has been previously mentioned in some studies 2°,38 but the demonstration that synaptophysin and chromogranin A immunoreactive neurites are associated with A4 amyloid deposits has not been reported. We demonstrate here that abnormal neurites visualized with anti-synaptophysin and anti-chromogranin A antibodies are associated with A4 amyloid deposits in several type of plaques, including in diffuse plaques. The anti-synaptophysin (clone SY38) used in this study is a mouse monoclonal antibody 4° purchased from Biotest and was used diluted 1/100 in TBS (0.01 M Tris-HCI, pH 7.6, 0.15 M NaCl) containing 10% normal goat serum. The anti-chromogranin A (clone LK2H10) is a mouse monoclonal antibody TM purchased from Hybritech and used at the same dilution. The anti-amyloid A4 sera were raised against synthetic peptides corresponding to residues 1-10 (anti-A41_10) and 12-28 (anti-A412_ES) of the amyloid A4 polypeptide21'22. Synthetic peptides were coupled to keyhole limpet hemocyanin and injected in rabbits. The anti-A41_~0 and anti-A412._2s sera were tested by ELISA using the synthetic peptides and by Western blotting on human (frontal cortex) and rat total brain homogenates. The latter were prepared by homogenizing brain tissue at 0 °C (1 g/ml) in TBS, trypsin inhibitor (100 /~g/ml), and phenylmethylsulfonyl fluoride (0.5 mM). The homogenate was centrifuged for 1 h at 105 gav (4 °C) and the supernatant retained for Western blot analysis. The anti-A41_10 and anti-A4~2_2s sera were used diluted 1/250. Brain tissue samples from the temporal cortex, the hippocampus and the parahippocampal gyrus were taken at autopsy in patients with Alzheimer's disease. Tissues were fixed in 10% formalin (3 days to 3 weeks) and embedded in paraffin. The immunolabelling was performed on 7/~m tissue sections with the peroxidase-antiperoxidase (PAP) method for single labelling, using diaminobenzidine as chromogen. The PAP complex, goat anti-rabbit and goat anti-mouse antibodies were obtained from Nordic (Netherlands). In double-labelling experiments, the first labelling was performed with the PAP method and the second labelling was performed by an indirect method using a goat anti-rabbit (Nordic, diluted 1/20) conjugated to fluorosceine isothiocyanate. For detection of A4 amyloid deposits with the anti-A4 sera, slides were first treated 20 min with undiluted formic acid ~4. Some slides were counterstained with Congo red or with thioflavine S to demonstrate amyloid deposits. Controls for the immunolabelling by the anti-A4 sera were performed by using normal rabbit serum in place of the anti-A4 sera. Controls for the immunolabelling by the anti-synap-
A
B
C
D
Fig. 1. Immunoblots of total brain supernatants from rat (lanes A and B) and Alzheimer (lanes C and D) brain homogenates. Lanes A and C: anti-A412 28 serum. Lanes B and D: anti-A41_~0serum. Molecular weight markers (arrows) are myosin (200 kDa), flgalactosidase (116 kDa), phosphorylase B (97 kDa), albumin (66 kDa), and ovalbumin (43 kDa).
tophysin and the anti-chromogranin A monoclonal antibodies (IgG1) were performed by incubating sections with an isotype-matched (IgG1) monoclonal anti-keratin antibody (Immunotech, clone KL 1) at the same dilution. The anti-A41_10 and anti-A412_28 sera both react with their corresponding peptide by ELISA. By immunoblotting, the anti-A412_28 serum labels at least two bands of molecular weight between 100 and 130 kDa in rat brain and two bands of 130 kDa and 80 kDa, respectively, in human brain (Fig. 1). Proteins with similar molecular weights were identified in other studies by immunoblotting with antibodies to the N- and C-terminus of the A4 amyloid precursor 23'3°. Kunitz-type inserts and N- or O-glycosylation could explain the detection of several A4 precursors isoforms and their difference in molecular weight 37. The lower molecular weight bands might correspond to the secreted form of A4 precursor, lacking the C-terminal part of it, rather than the membranebound form. The anti-A41_~o does not label consistently any bands in these preparations. Several antisera raised to synthetic A4 peptides have been reported not to label significantly any bands by Western blotting 3°'31 which might be due to epitopes being sterically hidden in the precursor protein when bound to nitrocellulose. As previously reported 1'22'32"34'41 -43, the anti-A4 sera
145 detect numerous A4 amyloid deposits in tissue sections after formic acid treatment, i.e. plaque amyioid in neuritic, classical, burned-out plaques and more diffuse and less intensively labelled A4-immunoreactive areas, some of the latter having the appearance of the described diffuse plaques. The amyloid material deposited in vessels walls was strongly labelled. Sub-pial amyloid deposits were also observed to be labelled. The antiA4t2_28 labelled, however, more deposits than the antiA41_10 as previously found in cases of Down's syndrome 32. Sections incubated with normal rabbit serum did not show any labelling. The anti-synaptophysin labels all the thickness of the gray matter and some fibers in the white matter. The labelling in the gray matter has a punctuate aspect and could sometimes be seen concentrated at the periphery of neuronal pericarya and proximal dendrites, the latter labelling corresponding probably to synaptic contacts. The anti-chromogranin A antibody labelled less intensively the neuropil and showed a granular labelling of some neuronal bodies. In areas rich in neurofibrillary tangles, the anti-synaptophysin labelling was weaker. This was especially prominent in star cell dusters of layer II and in layer IV of the presubiculum. The anti-synaptophysin and the anti-chromogranin antibodies demonstrated the abnormal neurites in many senile plaques. This labelling was more prominent in abnormal neurites in neuritic plaques and in thick, dilated neurites surrounding an amyloid core in classical plaques (Figs. 2A, 3A,C and 4A). This labelling of neurites was sometimes discrete, limited to one or two small neurites (Fig. 3G). Another plaque-like structure showing a synaptophysin or a chromogranin A immuno-
reactivity is shown in Figs. 2B and 5A. In these focal areas the anti-synaptophysin (Figs. 2B,C and 3E) and anti-chromogranin A (Fig. 5A,B) labelling appears as a more or less homogeneous distribution of punctuate staining, often giving the impression of a focal increase in the neuropil labelling. Thioflavine S counterstaining and anti-A412_28 labelling after anti-synaptophysin or antichromogranin A labelling showed that these granular areas of synaptophysin or chromogranin A immunoreactivity were associated with diffuse A4 amyloid immunoreactivity. These areas were less stained (thioflavine S) or labelled (anti-A412_28) than the amyloid deposits of neuritic, classical or burned-out plaques. The double immunolabelling combining the anti-synaptophysin or the anti-chromogranin A and the antiA4~2_28 showed that practically all plaques with synaptophysin or chromogranin A immunoreactive neurites were also anti-A4 positive (Figs. 3D,F and 5D), but not all A4 amyloid deposits were associated with increases in synaptophysin or chromogranin immunoreactivities. A strong labelling of plaque neurites with the antisynaptophysin and the anti-chromogranin A antibodies was obtained only in tissue samples with a short fixation time (less than 24 h). No labelling was observed with the monoclonal anti-keratin antibody. Synaptophysin is a 38 kDa polypeptide which appears to be an integral membrane protein found in small vesicles with clear centers, i.e. in vesicles containing classical neurotransmitters in neurones ~2,4°. The function of synaptophysin is still little understood but it has been observed that synaptic-like vesicles appear in fibroblasts transfected with a synaptophysin gene17; this might indicate that this protein plays an important role in the
Fig. 2. Immunolabellingon paraffin sections of Alzheimer brain tissue (temporal cortex) with the anti-synaptophysinantibodies, showing the different types of immunoreactivityin senile plaques. A: Abnormal, dilated neurites are strongly labelled in this plaque. The amyloid core is unlabelled. B and C: The labelling in these two plaques appears as homogeneous and punctuate, looking as a focal increase in the neuropil immunoreactivity. The labelling in C surrounds an unlabelled core. Bar = 10 am.
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Fig. 3. Immunolabelling on paraffin sections of Alzheimer brain tissue (temporal cortex). A,B: abnormal, dilated neurites in this plaque are labelled by the anti-synaptophysin antibodies (arrows in A) and surround an amyloid core demonstrated by its birefringence after Congo red counterstaining (B). C,D: double immunolabelling. The synaptophysin-immunoreactiveneurites in this plaque (C) surround a compact amyloid core labelled by the anti-A4~2_2aserum (D). E,F: immunolabelling with the anti-synaptophysin (E) followed by thioflavine S counterstaining (F). One senile plaque (arrow) is associated with a punctuate increase in synaptophysin immunoreactivity and discrete diffuse positive amyloid immunoreaetivity. The adjacent plaque (arrowhead) show a discrete synaptophysin immunoreactivity and a compact amyloid core. G,H: double immunolabelling. The plaque amyloid labelled by the anti-A412_2sserum (H) is associated with only a few small synaptophysin-immunoreactive neurites (arrow in G). Bar = 10/~m.
formation of the synaptic vesicle membranes. Chromogranin A belongs to a family of secretory proteins (chromogranins/secretogranins) which have been found in large dense core vesicles present in neurones and in non-neuronal neuroendoerine cells5. In neurones, these large dense core vesicles contain neuropeptides. The function of chromogranins is not yet well determined; these proteins might be processed to smaller peptides which could exert extraceUular biological activities. They might also play a role in the packaging and/or processing of neuropeptides 27. In Alzheimer's disease brain tissue we observed that in areas rich in neurofibrillary tangles (star cell clusters in layer II and layer IV of presubiculum) there is a decrease in synaptophysin immunoreactivity as compared to the neigbouring neuropil. This probably reflects the loss of neurones in these areas and of the synaptic arborization of tangle-beating neurones 29. A decrease in the synaptophysin immunoreactivity in the neuropil in Alzheimer's
disease, as compared to normal controls, has been previously reported 11,2°. We suggest that the labelling of neurites in primitive and classical plaques reflects the accumulation in these processes of synaptic vesicles or material derived from them. Previous ultrastructural observations of these plaques has shown that abnormal, dilated neurites in plaques contain PHF, accumulations of multilamellar bodies, multivesicular bodies, lysosomial bodies and degraded mitochondria 4'6'9,35. Some of these membranous organelles may contain material derived from synaptic vesicles, e.g. multivesicular bodies may represent a form of autophagy of vesicles. Since synaptic vesicles, like other membranous organelles, move along microtubules (fast neuroplasmic transport), their accumulation in abnormal plaque neurites might witness for axoplasmic flow disturbances in these neurites, as previously suggested 4'6"1°'26'35. Synaptic contacts on abnormal neurites in senile
Fig. 4. Immunolabelling on paraffin sections of Alzheimer brain tissue with the anti-chromogranin A antibodies. A: the dystrophic neurites in this neuritic plaque are labelled. B,C: the labelled neurites in this plaque (arrows in B) surround an amyloid core shown in C after counterstaining with thioflavine S. Bar = 10 ~m.
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Fig. 5. Immunolabellingon paraffin sections of Alzheimer brain tissue. A,B: immunolabelling with the anti-chromogranin A antibodies (A) followed by thioflavine S counterstaining (B). The amyloid deposit shown in B is associated with a punctuate increase in chromogranin A immunoreactivity. C,D: double immunolabelling with the anti-chromogranin A (C) and the the anti-A412 2~serum (D). The A4 amyloiddeposit is surrounded by a punctuate increase in chromogranin A immunoreactivity. Bar = 10/~m.
plaques have been described in electron microscopy9 and may contribute to the synaptophysin and chromogranin labelling of plaque neurites. Many presynaptic boutons were however reported to contain less synaptic vesicles than normal presynaptic boutons; this suggests that the increase in synaptophysin and chromogranin labelling in plaque neurites is rather related to the accumulation of material in these dystrophic neurites. The finding of polypeptides associated with synaptic vesicles in some plaque neurites suggests also that these neurites are axonal rather than dendritic terminals. The neuritic processes in plaques have been previously visualized by their content in specific neurotransmitters
or in enzymes involved in the metabolism of these neurotransmitters. The identified neurotransmitters in plaque neurites corresponded to several neuropeptides 33 as well as classical neurotransmitters 2'~5. This would be consistent with our results showing the presence of both types of vesicle-associated proteins in plaque neurites. In addition we observed also a homogeneous distribution of punctuate synaptophysin and chromogranin A immunoreactivities in focal areas associated with diffuse A4 amyloid deposits. These structures probably correspond to the previously described diffuse plaques 43 or preamyloid deposits 34. The A4 immunoreactive structures in these diffuse plaques are made up of extracellular
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a b n o r m a l dystrophic neurites are sometimes seen in electron microscopy in diffuse plaques 45. The synaptophysin and chromogranin immunoreactivity in these early plaques might reflect early events occuring at the level of neurites, e.g. a b n o r m a l synaptogenesis or sprouting. Several studies have suggested that a b n o r m a l neurites in senile plaques exhibit sprouting p h e n o m e n a TM. T h e latter p h e n o m e n a might be related to the p r e s u m p tive trophic and proliferative abilities of A 4 amyloid p o l y p e p t i d e and its precursor 28'39. Alternatively, these
of dilated, dystrophic plaque neurites. T h e accumulation of several n e u r o p e p t i d e s in plaque neurites has been suggested to p r e c e d e the accumulation of P H F in the same neurites 16 and our observations are consistent with such an interpretation. O u r results indicate thus that synaptophysin and chromogranin A , two m a r k e r s of synaptic vesicles, can be used to follow and identify p l a q u e neurites in Alzheimer's disease. We suggest also that changes in the synaptic network might be an early change of neurites found in contact with amyloid deposits.
p u n c t u a t e increases in synaptophysin and chromogranin A immunoreactivities might be a consequence of early disturbances of neuroplasmic transport of synaptic vesicles. The increases in synaptophysin and chromogranin A immunoreactivities could thus precede the d e v e l o p m e n t
This study was supported by grants from the Belgian ER.EC. and ER.S.M. (Nos. 2.4523.87, 9.8009.889, 9.4501.85), NATO, MRC and the WeUcome Trust. We thank the Brain Bank of the Institute of Psychiatry (Department of Neuropathology) for furnishing some tissue samples.
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