Parenchymal preamyloid and amyloid deposits in the brains of patients with hereditary cerebral hemorrhage with amyloidosis—Dutch type

Neuroscience Letters, 118 (1990) 223-226 Elsevier Scientific Publishers Ireland Ltd.

223

NSL 07222

Parenchymal preamyloid and amyloid deposits in the brains of patients with hereditary cerebral hemorrhage with amyloidosis Dutch type Willem Frederik Timmers a,2, Fabrizio Tagliavini a, J o o s t H a a n 2 and Bias Frangione 1 1Department of Pathology, New York University Medical Center, New York, NY 10016 (U.S.A.) and 2Department of Neurology, University Hospital, Leyden (The Netherlands) (Received 2 May 1990; Revised version received 18 June 1990; Accepted 25 June 1990)

Key words: Hereditary cerebral hermorrhage with amyloidosis-Dutch type; Amyloid fl-protein; Alzheimer's disease; Dementia; Stroke; Immunohistochemistry Hereditary cerebral hermorrhage with amyloidosis--Dutch type, one of the 'cerebral fl-amyloid diseases', like Alzheimer's disease, is characterized by extensive deposition of amyloid in small cerebral vessels. We investigated the presence of parenchymal fl-protein deposits in two Dutch patients with hereditary cerebral hemorrhage with amyloidosis. Immunostaining with anti-SP28 revealed a full spectrum of these deposits, varying from preamyloid deposits to burned-out plaques. However, their density is less than in Alzheimer's desease, and immunostainingwith Alz50 and anti-PHF did not show abnormal neurites in and around amyloid deposits in these two patients.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant disease, characterized by hemorrhagic strokes in the 5th and 6th decades of life [10, 17]. In almost 50% of the patients the first cerebral hemorrhage leads to death, whereas the remaining half suffer from recurrent strokes and often develop dementia [8, 9]. The main pathological change in the brains of HCHWA-D patients is an extensive amyloid deposition in small and medium-sized vessels of the leptomeninges and cerebral cortex. In addition, silver staining techniques (Bielschowski and methenamine-silver) have demonstrated structures that resemble senile plaques, although in a small number of cases. Distinct from Alzheimer's disease (AD) the plaque-like lesions lack a neuritic component and neurofibrillary tangles are absent [10, 15]. Amyloid fibrils from the leptomeningeal vessels of patients with HCHWA-D, contain a protein with a molecular weight of 4 kDa, whose amino acid sequence is homologous to that of vascular amyloid of AD [12, 15]. Immunohistological studies have shown that the amyloid in HCHWA-D reacted with an antiserum raised against a synthetic peptide homologous to the AD flprotein [4, 15]. Medium-sized arteries, arterioles, capillaries and venules were intensely labelled. Moreover, Correspondence: B. Frangione, Department of Pathology, New York University Medical Centre, 560 First Avenue, New York, NY 10016, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

immunostaining revealed the presence of amyloid deposits showing a homogeneous finely fibrillar structure in the neuropil [15]. As a further step in the characterization of cerebral amyloid deposits in HCHWA-D and in the definition of their relationship with those of AD, we have carried out an immunohistochemical study on the cerebral cortex of two HCHWA-D cases: A 47-year-old woman who suffered a first stroke 6 months before a second fatal stroke and who had shown a slowly progressive cognitive deterioration several months before death; and a 62-year-old man who died of his first cerebral hemorrhage. In both cases autopsy was performed within 8 h after death. Brain specimens were immediately frozen at - 7 0 ° C for biochemical analyses. Frontal, parietal and temporal blocks were fixed in 10% buffered formalin, embedded in paraplast and cut into serial sections 8/tin in thickness. The sections were either stained with Thioflavine S and Alcian blue, or incubated with one of the following antibodies: Anti-SP28, which is a rabbit polyclonal antibody to the 28 residue synthetic peptide homologous to the NH2-terminal region of the AD//-protein [3]; anti-P component (Dako, Denmark); anti-~tl-antichymotrypsin (Accurate, NY); Alz50 [18] and anti-PHF, which is a rabbit polyclonal antibody to paired helical filaments isolated from AD brains [7]. Before immunostaining with anti-SP28 (1:50) and anti-PHF (1:100) the sections were treated with 98% formic acid for 30 min. Anti-P

224 component and anti-al-antichymotrypsin antisera were used at a dilution of 1:100, whereas Alz50 was used at 1:5 dilution. The immunodetection was carried out using an avidin-biotin-peroxidase system (Amersham, IL). Peroxidase activity was evidenced with 3,3'-diaminobenzidine and 0.015% H202. Negative control sections were incubated with normal rabbit serum (1:50) or with Tris buffer as the first step reagent. A quantitative evaluation of amyloid deposits in the cortical neuropil was made on sections stained with Thioflavine S and examined with a Zeiss photomicroscope equipped for epifluorescence (excitation filter 410--490 nm, barrier filter 520 nm). A ribbon of frontal, parietal and temporal cortices, subdivided into adjacent square fields, was scanned from the pial surface to the border between the 6th layer and the white matter at a magnification of 250 x. The plaquelike lesions exhibiting yellow fluorescence were counted, and their mean number per mm 2 of cortical surface was calculated. Fluorescence microscopic examination of the slides stained with Thioflavine S revealed that the leptomeningeal and cortical vessels of both patients were heavily

laden with amyloid. On some occasions the amyloid was restricted to the vessel wall (Fig. 1A), whereas in other instances spreading into the adjacent brain parenchyma was present (Fig. 1B). In the neuropil of both patients plaque-like lesions of different morphology were detected: amyloid deposits with a finely fibrillar structure (Fig. 1C); amyloid deposits with a dense central core and a peripheral ring of loosely arranged bundles (Fig. 1D); and isolated amyloid cores. Although frequently clustered around vessels, some amyloid plaques did not have an apparent relation with the vascular system. Quantification of the amyloid deposits revealed that the plaquelike lesions had a density of approximately 10 per mm 2 of cortical surface, and were mainly distributed in the upper and middle layers of the cortex. Only 5% of the plaque-like lesions showed a central core, and less than 1% were isolated cores. Amyloid laden vessels and the majority of the plaque-like lesions, particularly the amyloid cores, were deeply stained by Alcian blue. Immunostaining with anti-SP28 showed a strong specific reaction in a number of meningeal and cortical vessels. In the neuropil this antiserum labeled two principal

Fig. 1. ThiottavineS stainingof the cerebral cortex of a patient with HCHWA-Dshowingamyloiddepositionrestricted to the vesselwall (A) and spreading into the adjacent brain parenchyma(B). In the neuropil amyloiddeposits with an even density (C) and deposits with a dense central core (D) can be seen.

225

lesions in both patients: (I) poorly defined, finely granular, irregularly shaped deposits with a great variety in size; these deposits were not evidenced by Thioflavine S in adjacent sections, and corresponded to the 'preamyloid deposits' found in AD patients, adult Down patients and aged non-demented individuals [2, 14] (Fig. 2A,B). (II) Deposits constituted of material with the tinctorial properties of mature amyloid, i.e. yellow fluorescence following Thioflavine S treatment. These amyloid deposits were morphologically heterogeneous. As observed after staining with Thioflavine S, most of them exhibited a homogeneous finely fibrillar structure (Fig. 2C). Some of them appeared as strongly immunoreactive cores, with a peripheral ring of patchy immunoreactivity (Fig. 2D,E). Few of them were strongly reactive compact deposits resembling 'burned-out plaques' (Fig. 2F). Both anti-~l-antichymotrypsin and anti-P component intensely labelled the amyloid deposits in meningeal and cortical vessels. Moreover, the majority of amyloid deposits in the neuropil were also decorated by these antisera, although the immunoreactivity was generally weaker than that seen in the vessels. On the other hand, only a few preamyloid deposits were labeled by anti-0e1-

antichymotrypsin and none contained amyloid P-component. Immunostaining with Alz50 and anti-PHF of sections adjacent to those treated with Thioflavine S, or incubated with anti-SP28 did not give any indication for the presence of neurofibrillary tangles or dystrophic neurites in and around the amyloid deposits in the neuropil. The major neuropathological feature of HCHWA-D patients is the amyloid deposition in the walls of cerebral blood vessels. In addition, we showed the presence of preamyloid and amyloid deposits in the neuropil. The occurence of these deposits in the brain of the 47-yearold patient makes it highly unlikely that the deposits are related to aging of the brain. The morphology of the preamyloid and amyloid deposits after anti-SP28 immunostaining as well as their intracortical distribution appeared to be similar to that observed in AD and Down patients, but the density of the amyloid deposits was less. In both patients preamyloid deposits prevailed over amyloid deposits, and relatively few amyloid deposits showed a central core or resembled burned-out plaques. In the brains of young Down patients preamyloid deposits also prevail over

Fig. 2. Anti-SP28 immunostaining of the cerebral cortex of a patient with HCHWA-D. The 'preamyloid' deposits (A,B), labelled by anti-SP28, did not react with Thioflavine S or Alcian blue in adjacent sections. Other deposits, decorated by Thioflavine S or Alcian blue in adjacent sections, were arranged in radial bands (C) or showed a homogeneous finely fibrillar structure (D). The strongly immunoreactive cores either had a peripheral ring in which the immunoreactive material appeared patchy (E), or were seen as homogeneously stained 'burned out' plaques (F).

226 senile plaques, whereas the o p p o s i t e occurs in aged D o w n patients [6]. This suggests t h a t p r e a m y l o i d d e p o sits precede a m y l o i d d e p o s i t i o n [6]. In H C H W A - D patients, Alz50 a n d a n t i - P H F d i d n o t show a b n o r m a l neurites in a n d a r o u n d a m y l o i d deposits. In a d d i t i o n to fl-protein i m m u n o r e a c t i v i t y , P - c o m p o nent a n d 0 t l - a n t i c h y m o t r y p s i n were detected. P - c o m p o nent is a c o m m o n c o m p o n e n t o f virtually all types o f a m y l o i d o s i s [5, 13], b u t ~ l - a n t i c h y m o t r y p s i n seems to have a unique a s s o c i a t i o n with fl-protein [1, 11]. The labelling o f p r e a m y l o i d a n d a m y l o i d deposits in H C H W A - D with a n t i - ~ t l - a n t i c h y m o t r y p s i n a n d a n t i - P c o m p o n e n t is similar to t h a t d e s c r i b e d in A D [16]. O u r findings suggest t h a t in H C H W A - D the process o f a m y l o i d f o r m a t i o n a n d d e p o s i t i o n in the n e u r o p i l is less extensive t h a n in A D , b u t seems to be qualitatively similar to t h a t o f A D . The prevalence o f v a s c u l a r a n d p a r e n c h y m a l changes with an absence o f a b n o r m a l i t i e s o f n e u r o n a l cell bodies suggest t h a t in H C H W A - D the vascular system is the site o f origin o f f l - p r o t e i n for p r e a m y l o i d a n d s u b s e q u e n t a m y l o i d d e p o s i t i o n in the b r a i n parenchyma. W e are grateful to Dr. P. D a v i e s for p r o v i d i n g Alz50 a n t i b o d y . This w o r k was s u p p o r t e d by N . I . H . G r a n t s A G 05891 a n d A R 01431. W . F . T . a n d J.H. are m e m b e r s o f the A m y l o i d R e s e a r c h G r o u p , Leyden. F . T . is on sabbatical leave f r o m I s t i t u t o N e u r o l o g i c o C a r l o Besta, M i l a n o , Italy.

1 Abraham, C.R., Selkoe, D.J. and Potter, H., Immunochemical identification of the serine protease inhibitor ~t1-antichymotrypsin in the brain amyloid deposits of Alzlaeimer's disease, Cell, 52 (1988) 487-501. 2 Bugiani, O., Giaccone, G., Frangione, B. Ghetti, B. and Tagiiavini, F., Alzheimer patients: preamyloid deposits are more widely distributed than senile plaques throughout the central nervous system, Neurosci. Lett., 103 (1989) 263-268. 3 Castafio, E.M., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease fl-protein, Biochem. Biophys. Res. Commun., 141 (1986) 782789. 4 Coria, F., Castafio, E.M. and Frangione B., Brain amyloid in normal aging and cerebral amyloid angiopathy is antigenic,ally related to Alzheimer's disease ,B-protein, Am. J. Pathol., 129 (1987) 422428.

5 Coria, F., Castafio, E.M., Prelli, F., Larrondo-Lillo, M., Van Duinen, S., Shelanksi, M.L. and Frangione B., Isolation and characterization of amyloid P-component from Alzheimer's disease and other types of cerebral amyloidosis, Lab. Invest., 58 (1988) 454-458. 6 Giaccone, G., Tagiiavini, F., Linoli, G., Bouras, C., Frigerio, L., Frangione, B. and Bugiani O., Down patients: extracellular preamyloid deposits precede neuritic degeneration and senile plaques, Neurosci. Lett., 97 (1989) 232-238. 7 Gorevic, P.D., Goni, F., Pons-Estel, B., Alvarez, F., Peress, N.S. and Frangione, B., Isolation and partial characterization of neurofibrillary tangles and amyloid plaque core in Alzheimer's disease: immunohistochemical studies, J. Neuropathol. Exp. Neurol., 45 (1986) 647-664. 8 Haan, J., Roos, R.A.C., Brirt, P.E., Herpers, M.J.H.M., Luyendijk, W. and Bots, G.T.A.M., Hereditary cerebral hemorrhage with amyloidosis - Dutch type, Clin. Neurol. Neurosurg., 91 (1989) 285-290. 9 Haan, J., Lanser, J.B.K., Zijderveld, I., Van der Does, I.G.F. and Roos, R.A.C., Dementia in hereditary cerebral hemorrhage with amyloidosis - Dutch type, Arch. Neurol., in press. 10 Luyendijk, W., Bots, G.T.A.M., Vegter-van der Vlis, M., Went, L.N. and Frangione, B., Hereditary cerebral hemorrhage caused by cortical amyloid angiopathy, J. Neurol. Sci. 85 (1988) 267-280. 11 Picken, M.M., Larrondo-Lillo, M., Coria, F., Gallo, G., Shelanski, M.L. and Frangione, B., Distribution of the protease inhibitor etlantichymotrypsin in cerebral and systemic amyloid, J. Neuropathol. Exp. Neurol., 49 (1990) 4148. 12 Prelli, F., Castafio, E.M., Van Duinen, S., Bots, G.T.A.M., Luyendijk, W. and Frangione, B., Different processing of Alzheimer's p-protein precursor in the vessel wall of patients with hereditary cerebral hemorrhage with amyloidosis - Dutch type, Biochem. Biophys. Res. Commun., 151 (1988) 1150-1155. 13 Skinner, M., Cohen, A.S., Shirahama, T. and Cathcart, E.S., Pcomponent (pentagonal unit) of amyloid: isolation, characterization and sequence analysis, J. Lab. Clin. Med:, 84 (1974) 604. 14 Tagiiavini, F., Giaccone, G., Frangione, B. and Bugiani, O. Preamyloid deposits in the cerebral cortex of patients with Alzheimer's disease and nondemented individuals. Neurosci. Lett., 93 (1988) 191-196. 15 Van Duinen, S., Castafio, E.M., Prelli, F., Bots, G.T.A.M., Luyendijk, W. and Frangione B., Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5991-5994. 16 Verga, L., Frangione, B., Tagliavini, F., Giaecone, G., Migheli, A. and Bugiani, O., Alzheimer patients and Down patients: cerebral preamyloid deposits differ ultrastructurally and histochemically from the amyloid of senile plaques, Neurosci. Lett., 105 (1989) 294299. 17 Wattendorff, A.R., Bots, G.T.A.M., Went, L.N. and Endtz, L.J., Familial cerebral amyloid angiopathy presenting as recurrent cerebral haemorrhage, J. Neurol. Sci., 55 (1982) 121-135. 18 Wolozin, B.L., Pruchnicki, A., Dickson, D.W. and Davies, P., A neuronal antigen in the brains of Alzheimer patients, Science 232 (1986) 6484550.