Recombinant vaccinia virus expression of the Alzheimer amyloid precursor (AAP)

NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS plaques in the cerebral cortex shows a correlation with the appearance and severity of dementia. Therefore, knowledge of the molecular conformation of A4 and how it aggregates to form fibrils should not only help in the understanding of neuronal death, but in the design of therapeutic "blocking" agents. A conformational model for the A4 monomer has been constructed, based on secondary structure prediction methods and sequence homology with known protein structures [Johnstone et al., BBRC 163(3):1248-55 (1989)]. The cross-beta sheet structural domain of prealbumin [Protein Data Bank, Brookhaven National Laboratory, New York, NY] was used as the structural model for the A4 tetrameric aggregate [Masters et al., EHB0 4(11):2757-63 (1983)]. The fibril model was constructed by packin@ tetrameric aggregates to form hexadecamers and higher-order aggregates. The stability of these aggregate models was assessed by performing molecular dynamics and minimization calculations. The present model agrees with low-angle X-ray diffraction data [Kirschner et al., PNAS 83:503-7 (1986)]. Predictions have been made for the binding of Congo red, Thioflavin T and alpha-l-antichymotrypsin.

311

precursor proteins (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of authentic purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis=0.2 & 0.3 nM), and less well mouse submaxillary epidermal growth factor binding protein (Kis=l & 3.5 nM), c~-chymotrypsin (Kis=3 & 6 nM), and the .¢-subunit of nerve growth factor (Kis=8 & 9 nM). Neither protein appreciably inhibited plasma & pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase or cathypsin G (Kis z 1 ~M). The remarkable similarity of the protease inhibitory properties of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein, and that the KPI domain is sufficient for the protease inhibitory properties of APP751. The magnitudes of the inhibition constants also suggests that the KPI domain-containing APPs likely function as reversible regulators of a trypsin-like enzyme in brain. The consequence of this remains to be understood, but use of BX9 as an affinity reagent may shed light on the identity of this protease(s). 242 WHAT ROLE MAY MICROGLIAL CELLS PLAY IN PLAQUE FORMATION IN ALZHEIMER'S DISEASE.

240 AMYLOIDOGENESIS IN A L Z H E I M E R ' S DISEASE (AD): M O R P H O L O G I C A S S O C I A T I O N W I T H M I C R O G L I A . *L.S. P e r l m u t t e r , E. Barron, H.C. Chui. U S C S c h o o l of M e d i c i n e , L o s Angeles, CA 90033 USA. S e v e r a l f o r m s of a m y l o i d o s e s h a v e b e e n l i n k e d t o t h e m o n o n u c l e a r p h a g o c y t i c s y s t e m (MPS). Amyloid fibrils s t r e a m i n g f r o m or into m e m b r a n o u s c y s t e n a of M P S c e l l s h a v e b e e n d e s c r i b e d in e x p e r i m e n t a l a m y l o i d o s e s (e.g., C o h e n e t al., 1960, A m J P a t h o l 37:413), h u m a n m y e l o m a s t e m - c e l l c u l t u r e (Durie e t al., 1982, N E J M 3 0 7 : 1 6 8 9 ) , a n d s c r a p i e ( W i s n i e w s k i et al., 1982, E x p e r B r a i n R e s S u p p l 5:3). A r e c e n t s t u d y of A D - a s s o c i a t e d s e n i l e p l a q u e s r e v e a l e d an i d e n t i c a l r e l a t i o n s h i p b e t w e e n m i c r o g l i a l c e l l s a n d a m y l o i d s t a r s ( W i s n i e w s k i e t al., 1989, C a n J N e u r o l Sci, 16:535). Cerebral cortical samples were obtained at autopsy f r o m 6 A D cases, f i x e d in a m i x e d a l d e h y d e s o l u t i o n , a n d p r e p a r e d for e l e c t r o n m i c r o s c o p y . A s p a r t of o n g o i n g s t u d i e s of the p a t h o g e n e s i s of t h e s e n i l e plaque, ultrathin sections were examined for the p r e s e n c e of a m y l o i d stars. Twenty-four intact stars w e r e i d e n t i f i e d . A t t h e p e r i p h e r y of t h e a m y l o i d star, cellular cytoplasm was often closely interdigitated w i t h c h a n n e l s of a m y l o i d fibrils. M o r p h o l o g i c c h a r a c t e r i s t i c s , s u c h as d e n s i t y of c y t o p l a s m a n d c e l l / n u c l e a r s h a p e w e r e u s e d t o i d e n t i f y c e l l type. The cell type surrounding amyloid stars could not always be determined, and sometimes appeared to be astroglial. In s e v e n p l a q u e s f r o m 2 cases, h o w e v e r , t h e a s s o c i a t e d c e l l s w e r e d e f i n i t i v e l y i d e n t i f i e d as m i c r o g l i a . T h e s e c e l l s h a d k i d n e y - s h a p e d nuclei, d e n s e c y t o p l a s m , d e n s e bodies, and prominent endoplasmic reticulum. Bundles of a m y l o i d f i b r i l s w e r e f o u n d i n t r a c e l l u l a r l y , u s u a l l y m e m b r a n e b o u n d in w h a t a p p e a r e d t o b e s w o l l e n s m o o t h endoplasmic reticulum. These amyloid fibrils appeared t o b e e i t h e r s t r e a m i n g o u t of o r into t h e p e r i p h e r y of t h e a m y l o i d star. The present study confirms the intimate relationship between microglia and amyloid f i b r i l s in A l z h e i m e r ' s s e n i l e p l a q u e s , a n d is c o n s i s t e n t w i t h e i t h e r s e c r e t i o n or p h a g o c y t o s i s of B amyloid by microglia. (NIH A G - 0 7 1 2 7 , A G - 0 7 6 2 4 , P S O A G 0 5 1 4 2 , S t a t e of C a l i f o r n i a H e a l t h S e r v i c e s , N a t i o n a l N e u r o l o g i c R e s e a r c h Bank)

241

THE KUNITZ PROTEASE INHIBITORY DOMAIN OF THE 13AMYLOID PRECURSOR PROTEIN IS SUFFICIENT FOR ITS PROTEASE INHIBITORY PROPERTIES. *S. Sinha1, H.F. Dovey1, P. Seubert 1, P.J. Ward 1, R.W. Blacher1, M. Blaber2, R.A. Bradshaw2, M. Arici 3, W.C. Mobley3 and I. Lieberburg 1. 1Athena Neurosciences, Inc., S. San Francisco, CA 94080, 2Department of Biological Chemistry, University of California, Irvine, CA 92714 and 3Department of Neurology & Pediatrics, University of California, SF, CA 94143. We have expressed the 57 amino-acid Kunitz protease inhibitory (KPI) domain of one of the Alzheimer's I~-amyloid

BEHROUZ N., DEFOSSEZ A.*, DELACOURTE A., MAZZUCA M. Unite INSERM 156, Laboratoire d'Histologie, Facultd de Mddecine, Place de Verdun, 59045 Lille cedex, FRANCE Numerous histochemical and immunohistochemical studies have shown the morphology and the distribution of amyloid deposits in the Alzheimer brains. Using a glycolytic pretreatment for amplification of intmunostaining performed with 6-protein amyloid A4 antisera, we detected an amyloid diffuse material that infiltrate the neuropil of the grey matter (Lab. Invest. 1989, 61, 576-583). Moreover we demonstrated the presence of pericapillary amyloid diffuse deposits in the white matter (Nature, in press). In the present study, we used simultaneously the thioflavin staining of the amyloid fibrils and the detection of the microglial cells with lectin histochemistry and Hortega's method. We observed a close contact of microglial cells with amyloid fibrils constituting senile plaques and a capillary was always present in the vicinity of senile plaques. These observations agree with the vascular origin of the amyloid protein precursor and its processing by microglial cells.

This work was supported by grants from the Caisse Rdgionale d'Assurance Maladie Nord-Picardie. 243 RECOMBINANT VACCINIA VIRUS EXPRESSION OF THE ALZHEIMER AMYLOID PRECURSOR (AAP). *B.D. Greenberg, P.A. Gonzalez-DeWhitt, D.E. Lowery and R.A. Altman. The Upjohn Company, Kalamazoo, Ml 49001 USA. We have been producing the 695-, 751- and 770-amlno acid AAP forms in several recombinant systems to enable studies of their functions, processing and intermolecular interactions, and for use as antigens to raise highly specific polyclonal and monoclonal antibodies. Using antisera raised to AAP protein generated by recombinant expression in E. coli and in insect cells, we have identified immunoreactive cellassociated glycoprotelns in a monkey fibroblast-like cell llne (CV-I) infected with recombinant vaccinia viruses containing the AAP cDNA sequences under appropriate transcriptional controls. These proteins migrate at 110-130 Kd apparent molecular weight in SDS-polyacrylamide gels following boiling in SDS-mercaptoethanol. We have additionally identified and partially purified C-termlnally truncated AAP forms from media conditioned by these infected cells. Consistent with reports of similarly truncated soluble AAP forms in human brain and

NEUROBIOLOGY OF AGING, VOLUME I i, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS

312

cerebrosplnal fluid, the soluble AAP-695 appears approximately i0 Kd smaller than the cell-assoclated form. Interestingly, soluble AAP-751 and -770 forms appear approximately 10 Kd lar~er than the corresponding cell-assoclated forms, consistent with the formation of an SDS-stable complex at their internal Kunltz protease inhibitory domains. This prlmate-based system may provide access to an endogenous protein which forms the most avid interaction with the AAP Kunitz domain yet reported. Our ongoing characterization of these proteins and protein complexes will be presented.

Our results show that cystatin C is accumulated in the amytoid deposits, either in a non-fibrillar form or as amyloid fibrils, lmmunogold labelling for cystatin C shows that the antibodies bind selectively to areas infiltrated by amyloid fibrils but to a lesser extent than the betaprotein antibodies. In cerebral biopsy cases we have observed a positive reaction for cystatin C antibodies in astrocytes. The presence of cystatin C in neurons and glial cells including astrocytes has been reported. The cystatin C produced by these cells might possibly be adsorbed to amyloid material already present in the vessel walls. 1. Eirl'kur Benedikz, Hannes BlOndal, Gu0j6n J6hannesson, Grrtar Gu~3mundsson, Gunnar Gu/~mundsson: Dementia with non-Hereditary cystatin C Angiopadiy. Alzheimer's disease and related disorders. Khalid lqbal, Henry M. Wisniewski, Bengt Winblad (eds). Alan R. Liss, New York, 1989, p.517-522.

244 Abstract withdrawn.

2. ShigeyoshiFujihara, Koichi Shimode, Morihiko Nakamura, Shotai Kobayashiand Tokugoro Tsunematsu:Cerebral amyloid angiopathy with the deposition of Cystatin C (gamma-trace)and ~protein. Alzheimer'sdisease and related disorders. Khalid Iqbal, Henry M. Wisniewski, Bengt Winblad (eds). ALanR. Liss, New York, 1989, p.939-944.

247 DIAf~K)SIS OF C ~ R A L

AMYLOID ANGIOPATHY WITH %1{E DEPOSITION OF

CYSTATIN C BY THE USE OF $1~ZYME-LINKSD ~ S O R B E I ~ T

245 THE TWO A L 2 H I ~ E R AMYLOtD ~ c%-ANTICHYMOTRYPSIN A N D ~ - ~ ProP,M A STABLE C O M P L i ~ / N V/TRO. *Hunlh~on Potter, Cmmela R. Abrahame, and David H. l)n~Icx. Depertment of Neuzobidogy, Harvard M_~_'__,~,_] School, BoAm, MA 02115. eArthritis Center, Boston Univ~ity School of Medicine, Boston, MA 02118. The ~ inhibit~ 0 h - a n f i c h y ~ (ACT) m d tlw 42 ~ : m o ~ k i

[~-prowinare intelval ~ t s

of the Ira,in amylcid depe6it~ of ~ ' s

dL_.~,_~, Down's syndrome, and nmmal ~ a o in h m m m and monkeys. ACT is also found to be asmcimed with the [i-prom/n in o,~¢ m m - ~ amylnid--a variant of hmm~m7 cm~ral hemocdu~ wRh im~Ioidosis found in Hollml& T o ~ e r these resulls i n d i c ~ th~ flm~ is a special as,~/m/c~ of ACT and the ~-Wotein, perhaps easealial to the feemalica o~"amyloid. A basis

for this amax:iation is n~ggsted by the ~ m i / ~

of the N.tea'mimd ternamino

acids of the ~-prot~m to the ~five site of a=~m Womm=. We then~an~ exa~h~d the ~ betwo~ ACT md tlg ~ - p m ~ i . vi,'o. "I~ exp=iments ~ m , ~ that the aw~iatloa t.awem ACr ~ ~ ~.Wotm i~ no¢ ~cid~md bot ~ ' i ~ s lb¢ ~d~ ~cificity m d fl~ ~xlKli~ of a Ima~v~e-inbibit~ intm-~,ctlon. The addition of synthedc N-terminal (an 1-28 and 1-12) pot,tim~s

of rig ~ t e i n p~events ACT from inhibitln8 ehymotrypain. F ~ , when ACT and ~ ~-p,Uein p a p e ~ m mi~ed pri~ to poty-,=ylamide gel etect~,lRggesis, a new ~ v e

band is genev~ed cetttaining a

small fi'actlen d thc p s ~ s u SDS-sudsU~ complexes. The use of crosslinking agents stabilizes an even 8ream" amount of the paplide oato ACT,

while the p~e-additlen of equimolar amoents of chymou.ypsin to ACT ix~vents the compkx formation with []~ol=in. These results suggest a physiological function of the ~-pmtein as a ~eS~lat~ of pmmae activity. They alto seW.St a model foe the Alzlgim~-li~ amyloid filmae~, in which the ~-protein forms a core filament to which ACT binds on the out~ surface.

246 BETA-PROTEIN AND CYSTATIN C IMMUNOREACTIVITY IN AMYLOID ANGIOPATHY. E. Benedikz (1), H. Bl~ndal (t). G. Gu•mundsson (2). 1) Deparanents of Anatomy and Patigdogy, University of Iceland. 2) Department of Neurology, University Hospital, Landspftalinn. Recently two groups independently reported (1,2) the coexistence of immunc~eactlvity to the proteinase inhibitor cystatin C and beta-protein in amyloid deposits found in the cerebeal arteries o f some elderly patients with c e r e ~ amyloid angiopathy. In both studies all patients except one had suffered fioin a cerebral ~ a g e . A variant of ¢yststin C is deposited as amyloid, mainly in the cerebral arteries, in patients with H e r e d i ~ Cystatin C Amyloidosis CrICCA). These pafien~ suffer from hemon'hages, usuaUy mutfiplc, at a young age, In the cun~nt study we investigate further cases previously reported using immunohistochemistry and immunogold labeling for electron microscopy.

ASSAY (ELISA)

IN CER~BRDSPINAL FLUID. ~ Shimode, K. Fujihara, S. Nakamura, M. Kobayashi, S. Tsunematsu, T. Third Division of Internal Medicine, Shimane Medical University, Isumo 693 ,Japan. We have recently reported the Japanese cases of cerebral amy[old angiopathy (CAA) causing cerebral hemorrhage, whose amyloid showed the antigenicity of cystatin C as reported in hereditary cerebral hemorrhage with amyloidosis (HCHWA)in Iceland. I,ower level of cystatin C in the cerebrospinal fluid (CSF) is known to be one of useful diagnostic markers of HCHWA m Iceland. We tried to establish the assay system of CSF level of cystatin C for the diagnosis of CAA with the deposition of cystatin C. Monoclonal mouse anti-cystatin C and polyclonal rabbit anti-cystatin C antibody were kindly supplied by Dr. Grubb. From 25 cases of cerebral hemorrhage and 68 reference cases with other neurological diseases, CSF we[~ collected and immediately kept frozen with additional serine proteinase inhibitor, benzamidlnium chloride. We used the sandwich enzyme immunoassay. "['he standard curve of quantitative determination of cyatatm C was obtained. There was no cross-reactivity of protein AA, A[,. or trypsin inhibitor. In comparison with reference patients and 13 patients with cerebral hemorrhage, 12 patients with cerebral hemorrhage showed low level of cystatin C and clinical manifestations suggestive {~t' CAA. One of the 12 patients was confirmed CAA with the deposition of cystatin C by immunohistochemical method. Our study showed the feasibility of the use of the ELISA for the diagnosis of cerebral amylnid angiopathy with the deposition of cystatin C.

248 AMYLOID PROTEIN DEPOSITS IN THE HUMAN CEREBRAL CORTEX: COMPARATIVE IMMUNOHISTOCHEMICAL STUDIES IN ALZHEIMER'S DISEASE AND CONTROL CASES, BEHROUZ N. *(I). PARENT M. (l), MANN D.M.A. (2), D ~ S E Z

A. (t),

DELACOURTE A. (1), MAZZUCA M. (1) (1) Unit&INSERM t56, Laboratoire

d'Histologie, ADERMA, Facult&de Md~lecineLille (France): (2) Laboratory of Neuropathology, University of Manchester (UX). Two polyclonal antibodies were raised against synthetic papfides corresponding to residues 1-10 and 15-28 of file Alzheimer amyloid beta p r o ~

(BPA4). After periodic acid pretreatmcnt, an immunoperoxidase study investigated the localization of BPA4 deposits in the cerebral cortex of