Virchows Archiv B Cell Pathol (1992) 61:331-335

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l,~tm~ Mot~t~ l~oto~v 9 Springer-Verlag 1992

The distribution of i-antichymotrypsin and amyloid production in the brain in Alzheimer's disease Taihei Miyakawa, Shoiehi Katsuragi, Kensho Yamashita, Kunio Araki, Tetsuo Hashimura, Takemi Kimura, and Kiyoshi Ohuchi Department of Neuropsychiatry, Kumamoto University Medical School, Honjo Machi, Kumamoto, Japan Received April 9 //Accepted July 24, 1991

Summary. In this immunohistopathological study 0qantichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer's disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-cq-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, 0q-antichymotrypsin seems to play a role in the production o f amyloid fibrils in Alzheimer's disease.

Key words: Preamyloids - Amyloid Fibrils - ctl-antichymotrypsin

Introduction Senile plaques (SP) and neurofibrillary tangles (NFT) in the brain are very important features of Alzheimer's disease. Amyloid fibrils are generally considered to be essential components of the SP, but recent histochemical and immunohistochemical studies have concerned the type of plaque that cannot be detected with Congo red or conventional silver stains. These structures have been variously termed plaque A (Probst et al. 1987), diffuse plaque (Yamaguchi et al. 1989), pre-amyloid deposits (Tagliavini et al. 1988), type 3 plaque (Ikeda et al. 1989) or pre-plaque (Mann et al. 1989) and do not contain degenerate neurites or amyloid fibrils. These preamyloid structures are considered to be an initial stage before amyloid fibril formation in the development o f the SP. However, where and how amyloid fibrils are formed in the brain is not yet certain.

Offprint requests to: T. Miyakawa

Recently, Abraham et al. (1988) reported that ~ 1-antichymotrypsin might be an important factor in the production o f amyloid fibrils. In the present ultrastructural study, we report the distribution o f 0q-antichymotrypsin and discuss it possible role in inhibiting serine protease in relation to amyloid fibril formation in Alzheimer's disease.

Materials and methods The brains from five patients with Alzheimer's disease and three unaffected control patients aged 55-60 years were studied. Parts of the cerebral cortex from the temporal and occipital lobes were removed immediately after death and immersed in 4% formalin for 1 week. Sections (100 lim thick) were cut from each block using a vibratome. An antibody (rabbit immunoglobulin) to human ctlantichymotrypsin (DAKO Corp. Denmark) was then applied as follows. The sections were hydrated and incubated in a blocking solution of normal rabbit serum in 10 mM Tris buffer containing 0.15 M NaC1 (pH 7.6) for 20 min, then incubated with the primary antibody (diluted to 500:1) overnight at 4~ C. The antibody was detected using an avidin-biotin complex (Vector Lab., U.S.A.). Two negative control studies were also performed as follows. Control sections from the brains of three normal, undemented patients were treated with the same procedures described above. In addition, the incubation with the primary antibody was omitted in processing some of the sections of the brains from patients with Alzheimer's disease. All sections were immersed in 2.5% osmium tetroxide, dehydrated in graded alcohol and embedded in epon. Ultra-thin sections were cut on a Porter-Blum ultratome, stained with uranyl acetate and lead acetate, and observed with JEOL 2000 EX electron microscopy. In addition, methenamine silver-stained sections (Ikeda et al. 1989) was also used and observed by light and electron microscopy.

Results Light microscopical findings By light microscopy, diffuse plaques were clearly stained with methenamine silver and anti-0q-antichymotrypsin. In the silver methenamine preparations, many diffuse

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Fig. I a. Light micrograph of the brain in Alzheimer's disease. Diffuse plaques, a typical senile plaque (TSP) and capillary walls are darkly stained. Methenamine silver stain. (BV) blood vessel, x 440. b Light micrograph of the brain in Alzheimer's disease. Diffuse plaques are darkly stained with anti-~l-antichymotrypsin. Not only diffuse plaques but also the cytoplasm of cells (arrows)are darkly stained. (Bit) blood vessel, x 440

Fig. 2. Deposits (arrow)of anti-~l-antichymotrypsin are observed in the cytoplasm of a capillary endothelial cell. (L) lumen of capillary. x 14000

and typical plaques were observed (Fig. I a). In serial sections stained with anti-~l-antichymotrypsin, not only the diffuse and typical senile plaques were positive, but also the cytoplasm of several cell types, including glial and neuronal cells (Fig. 1 b).

Electron microscopicalfindings

Fig. 3. In the cytoplasm of an astroglial cell, deposits darkly stained with anti-~l-antichymotrypsin are observed close to rough endoplasmic reticulum and membranous structures. (GF) glial fibrils. x 19000

By electron microscopy, the cytoplasm o f endothelial cells and vascular feet contained numerous deposits which reacted with anti-~l-antichymotrypsin (Fig. 2). In

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Fig. 4. Amyloid fibrils (A) darkly stained with anti-~l-antichymotrypsin and in direct contact with the membranes (arrows) of astroglial vascular feet (VF). (BM) basement membrane. (EC) endothelial cell of a capillary, x 49000

Fig. 5. Amyloid fibrils (A) are darkly stained with anti-cq-antichymotrypsin and the fibrils directly attach to fibrous structures connecting to the membranes of the astroglial cell process (arrow). x 57000

the cytoplasm of the astroglial cells, abundant reaction product to anti-~l-antichymotrypsin was found (Fig. 3). Amyloid fibrils were darkly stained with anti-~q-antichymotrypsin, and were frequently detected in direct contact with fibrous structures connected to the membranes of the astrocytes of vascular feet (Fig. 4) and astroglial processes (Fig. 5). In preparations stained with silver methenamine, deposits were mainly arrayed along the membranes (Fig. 6).

blood-brain barrier. On the other hand, a nerve cell (Masters etal. 1985; Allsop etal. 1986; Powers etal. 1988) or microglial cell origin (Probst etal. 1987; Wisniewski etal. 1973, 1989; Yamaguchi etal. 1988; Haga et al. 1989) for amyloid fibrils in senile plaques have also been proposed. Yamaguchi et al. (1990) demonstrated the ultrastructural localization of /%protein in diffuse plaques, especially small ones, by a sensitive method of immunoperoxidase labeling using a vibratome. They described that immunoreactivity on the membrane is usually located around amorphous and/or fibrillar stained structures, and was occasionally continuous with them. The/~-prorein immunoreactivity on cell membranes was seen consistently in all the diffuse plaques which lacked capillaries blood vessels within them. The capillaries outside the areas staining positively with the/~-protein showed no apparent immunostaining in their walls. Masters et al. (1985) suggested that extracellular amyloid may arise from APP in altered neurons, based on the degree of N-terminal heterogeneity compared with the protein component of Alzheimer's disease amyloid [NFT, amyloid plaque core and congophilic angiopathy]. In addition, Kang et al. (1987) isolated what appeared to be a cell membrane protein as the putative precursor. However, these represent different kinds of membranes such as those of nerve cells, glial cells and endothelial cells in the brain. As described above, in

Discussion Recent immunohistochemical studies have demonstrated 9 l-antichymotrypsin in the amyloid of all senile plaques (Abraham et al. 1988, Shoji et al. 1988) and Abraham et al. (1988) speculated that the overproduction of ~lantichymotrypsin inhibits the protease dissolving amyloid precursor protein (APP) and induces amyloid formation in the brain in Alzheimer's disease. On the other hand, (Matsubara et al. 1988) detected a high levels of ~l-antichymotrypsin in serum of patients with Alzheimer's disease. Miyakawa et al. (1974, 1979, 1982) insisted that all senile plaques contained at least some amyloid fibrils, which seemed to be produced at the basement membranes of capillary endothelial cells. These findings suggest that amyloid formation has a close relation to the

334 small blood vessels occurring at a relatively young age. Joachim et al. (1989) reported amyloid deposits in tissues other than the brain in Alzheimer's disease. On the other hand, in the brains of non-demented individuals, preamyloid deposits were observed (Tagliavini et al. 1988). This means that fl-protein may be derived from a common circulating precursor. However, there is as yet no definite evidence of where and how the amyloid is produced from preamyloid precursor.

Acknowledgement. We wish to express our gratitude to Dr. H. Yamaguchi (College of Medical Care and Technology, Gunma University) for his kindness in giving us some useful materials used in the present study.

References

Fig. 6. Silver granules reacted with preamyloid arrayed along the membranes. Methenamine silver stain, x 23 000

the present study cq-antichymotrypsin was demonstrated in amyloid fibrils, in endothelial cells and in astrocytes. On the other hand, APP was mainly arrayed along membranes in the brains of both Alzheimer and control patients, whereas, ~l-antichymotrypsin could hardly be detected in the controls. From this it might be concluded that APP may not be an abnormal protein, but that in Alzheimer's disease cq-antichymotrypsin could inhibit the proteases which normally break down APP in the brain. In the present study, we detected amyloid fibrils in contact with structures stained with anti-~l-antichymotrypsin which were also in direct contact with the surface membranes o f astrocytes. Although cq-antichymotrypsin itself may not play the major role in amyloid production, these ultrastructural findings suggest the possibility that amyloid fibrils may be formed from APP derived from the surface membranes but not broken down in the normal way by proteases, because of the inhibitory effects of ~l-antichymotrypsin at several areas such as the basement membranes of capillaries and the extracellular spaces facing the membranes of astrocytes and their vascular feet. Glenner and Wong (1984) reported that in a large proportion o f patients with Alzheimer's presenile dementia, the major causative mechanism was an alteration of the blood-brain barrier resulting from the deposition o f Congo red-positive material in the walls of

Abraham CR, Selkoe D J, Potter HC (1988) Immunochemical identification of the serine protease inhibitor ~q-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell 52: 487-501 Allsop D, Landon M, Kidd M, Lowe JS, Reynolds GP, Gardner A (1986) Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer's disease. Neurosci Lett 68: 252-256 Glenner GG, Wong CW (1984) Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Comm 120:885890 Haga S, Akai K, Ishii T (1989) Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain. An immunohistochemical study using a novel monoclonal antibody. Acta Neuropathol (Berl) 77: 569-575 Ikeda S, Allsop D, Glenner GG (1989) The morphology and distribution of plaque and related deposits in the brain of Alzheimer's disease and control cases: An immunohistochemical study using amyloid ]~-proteinantibody. Lab Invest 60:113-122 Joachim CL, Mori H, Selkoe DJ (1989) Amyloid fl-protein deposition in tissues other than brain in Alzheimer's disease. Nature 341 : 226-230 Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Muller-Hill B (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325:733-736 Mann DMA, Brown A, Prinja D, Daries CA, Landon M, Masters CL, Beyreuther K (1989) An analysis of the morphology of senile plaques in Down's syndrome patients of different ages using immunocytochemical and lectin histochemical methods. Neuropathol Appl Neurobiol 15: 317-329 Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K (1985) Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque and blood vessels. EMBO J 4:2747-2763 Matsubara E, Amari M, Shoji M, Harigaya Y, Yamaguchi H, Okamoto K, Hirai S (1988) Significance of measurement of serum ~q-antichymotrypsin in senile-dementia of Alzheimer type. Igaku no Ayumi (in Japanese) 145:907-908 Miyakawa T, Sumiyoshi S, Murayama E, Deshimaru M (1974) Ultrastructure of capillary plaque-like degeneration in senile dementia. Acta Neuropathol (Berl) 29:229-236 Miyakawa T, Uehara Y (1979) Observation of amyloid angiopathy and senile plaque under a scanning electron microscope. Acta Neuropathol (Berl) 48 : 153-156

335 Miyakawa T, Shimoji A, Kuramoto R, Higuchi Y (1982) The relationship between senile plaques and cerebral blood vessels in Alzheimer's disease and senile dementia. Morphological mechanism of senile plaques production. Virchows Arch [B] 40:121129 Powers JM, Skeen J (1988) Ultrastructural heterogeneity in cerebral amyloid of Alzheimer's disease. Acta Neuropathol (Berl) 76:613-623 Probst A, Brunnschweiler H, Lantenschlager C, Ulrich J (1987) A special type of senile plaque, possibly an initial stage. Acta Neuropathol (Bed) 74:133-141 Shoji M, Harigaya K, Ishiguro K, Yamaguchi H, Hirai S (1988) al-antichymotrypsin in the brains of senile dementia. Igaku no Ayumi (in Japanese) 145:907-908 Tagliavini F, Giaccone G, Frangione B, Bugiani O (1988) Preamyloid deposits in the cerebral cortex of patients with Alzheimer's

disease and nondemented individuals. Neurosci Lett 93:191196 Wisniewski HM, Terry RD (1973) Reexamination of the pathogenesis of the senile plaque. Progr Neuropathol 11 : 1-26 Wisniewski HM, Weigiel J, Wang KC, Kujawa M, Lach B (1989) Ultrastructural studies of the cells forming amyloid fibers in classical plaques. Can J Neurol Sci [Suppl] 16:535-542 Yamaguchi H, Hirai S, Morimatsu M, Shoji M, Harigaya Y (1988) Diffuse type of senile plaques in the brains of Alzheimer-type dementia. Acta Neuropathol (Berl) 77:113-119 Yamaguchi H, Nakazato Y, Hirai S, Shoji M, Harigaya Y (1989) Electron micrographs of diffuse plaques. Am J Pathol 135: 593597 Yamaguchi H, Nakazato Y, Hirai S, Shoji M (1990) Immunoelectron microscopic localization of amyloid fl protein in the diffuse plaques of Alzheimer-type dementia. Brain Res 508 : 320-324

The distribution of alpha 1-antichymotrypsin and amyloid production in the brain in Alzheimer's disease.

In this immunohistopathological study alpha 1-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endoth...
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