- Email: [email protected]
Senile plaques: Staining for acetylcholinesterase and beta-protein. A comparative study in the entorhinal cortex
269
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE NEUROPATHOLOGY/ALZHEIMER'S DISEASE IN DEVELOPMENTAL DISABILITIES N a k a z a t o 3 , M. S h o j i a , Y. H a r i g a y az , T. K a w a r a b a y a s h i2 , K. Okamoto z , S. H i r a i z , M. T a k a t a m a4 . ICollege of M e d i c a l C a r e and T e c h n o l o g y , D e p a r t m e n t s o f Z N e u r o l o g y and a P a t h o l o g y , Gunma U n i v e r s i t y , 4 G e r i a t r i c s R e s e a r c h I n s t i t u t e and H o s p i t a l ; M a e b a s h i , Gunma 371, J a p a n . In addition to primitive, c l a s s i c , and b u r n e d - o u t p l a q u e s , we had r e p o r t e d " d i f f u s e p l a q u e s " , which were n o t d e t e c t a b l e by t h e Bodian s t a i n . H e r e , we examined the u l t r a s t r u c t u r e o f d i f f u s e p l a q u e s ( D P ) and u l t r a structural localization of ~ protein immunoreaetive s u b s t a n c e w i t h i n DP i n A l z h e i m e r b r a i n s . F i r s t , u s i n g o s m i u n - f i x e d and epon-embedded t i s s u e s we e x a m i n e d p a i r s o f r o u t i n e electron microscopic ultrathin s e c t i o n and a d j a c e n t 0 . 4 ~ m - t h i c k l i g h t m i c r o s c o p i c s e m i t h i n s e c t i o n w h i c h was t r e a t e d with f o r m i c a c i d and B p r o t e i n i m m u n o s t a i n . In the f r o n t a l c o r t e x , s e m i t h i n s e c t i o n s showed numerous DP. Many o f them had i r r e g u l a r b o r d e r s and c o n s i s t e d o f c l u s t e r o f f i n e dot s t a i n s . N e u r o n a l c e l l b o d i e s were o c c a s i o n a l l y p r e s e n t w i t h i n DP. Most o f DP u s u a l l y showed no o r v e r y s m a l l amount o f a m y l o i d f i b r i l s i n t h e narrow extraeellular spaces. Neuropil w i t h i n t h e s e a r e a s appeared morphologically almost normal. Therefore, it was i m p o s s i b l e t o d i s t i n g u i s h DP from t h e s u r r o u n d i n g neuropil without an a i d o f t h e s e m i t h i n sections stained for ~ protein. Focal d e n s e l y - s t a i n e d a r e a s showed a number o f a l t e r e d n e u r i t e s and e x t r a c e l l u ] a r amyloid f i b r i l s . Second, Vibratome sections of paraformaldehydef i x e d t i s s u e s w e r e i m m u n o s t a i n e d for" ~ p r o t e i n , and then embedded in epon. Positive immunoreacbion p r o d u c t s were s c a t t e r e d sporadically throughout the DP. Positive structures i n t h e DP w e r e I ) s m a l l bundles of amy]oid f i b r i l s , 2) amorphous m a t e r i a l s b e tween cell processes ( p r e - a m y l o i d ) , a n d 3) c e l l m e m b r a n e s of s o m e c e l l p r o c e s s e s . Our findings suggest that DP consist of small a m o u n t of a m y l o i d fibrils and pre-amyloid with minimal neuritic changes, and that pre-amyloid deposits may precede the amyloid fibril formation.
67 IMAGE ANALYSIS MICROSPECTROSCOPY OF SENILE PLAQUES I}~UNOSTAINED WITH AN ANTI BETA-PROTEIN ANTIBODY. *Miguel A. Pappol]a I, Rawhi A. Omar 2, Harry Vinters 3. Departments of Pathology, F.D.R. V.A. Medical Center, Montrose, New York I, New York Medical College, Valhalla, New York I , University ot West Virginia, Morgantown, West Virginia 2 , University of California, Los Angeles, California 3. We used image analysis microspecttoscopy (IAM) to quantitatively evaluate the genesis and distribution of amyloid deposits in senile plaques immunostained with an antibody raised against a synthetic peptide containing the beta-protein sequence. IAM is a computer assisted method which combines basic principles of ligilt densitometry with image subtraction techniques and allows selective optical isolation with simultaneous quantification of tissue components (Pappolla, 1988). In prior studies utilizing IAM we had initially observed that congophilic deposits in senile plaques have a consistent relationship to microvessels characterized by a gradient of amyloid concentration radiating outwardly from central capillaries (Pappolla, 1989). The antibody used in this study permitted detection of early primitive senile plaques at a stage not exhibiting the typical congophilia characteristic of well-developed plaques. Quantitative analysis of these presumably early amyloid deposits in serial tissue sections did not show an apparent relationship to capillaries (i.e.: no gradient was present). Instead, these deposits appeared to surround neurites which occasionally showed degenerative changes. Image analysis of more mature plaques immunostained as above revealed again the characteristic concentration gradient of amyloid previously observed using congo red staining alone. These observations indicate that involvement of the microvasculature probably takes place at a later stage in the genesis of the plaque. Moreover, it seems that amyloid precursor proteins are converted to the beta-pleated configuration once they have reached the microvasculature, where they accumulate as central congophilic cores in mature plaques.
68 NEUROFIBRILLARY TANGLES CONTAIN HEPARAN SUL FATE PROTEOGLYCANS. *G. Perry, S. Siedlal% P. Mulvihill M. Kawai, P. Cras, B. Cordell 1, J. Marian Scardina j , S.. Ledbetter :~, B.
Greenbfrg 2, P. Gambetti. Case Western Reserve University, C~veland, Ohio; "California Biotechnology, Mountain View, California; "Upjohn Company, Kalamazoo, Michigan. Recently we observed that vessels are increased in density around senile plaques (SP), suggesting that angiogenic factors play a role in SP formation. Therefore we investigated the distribution of basic fibroblast growth factor (bFGF), a well known angiogenic factor, in brain tissues from subjects with AD and controls. We found that when the antibodies were absorbed with bFGF prior to immunostaining, the antigen-antibody complex strongly bound to neurofibrillary tangles (NFT), neuropil threads, dystrophic neurites surrounding SP, amyloid component of SP as well as normal vessels. A similar pattern was obtained when tissue sections were incubated with bFGF and subsequently immunostained with the antibodies to bFGF. Ultrastructural observation showed the bFGF is bound by paired helical filaments, yet was especially pronounced in NFT containing amorphous material. Treatment of sections with a combination of heparinase and heparitinase or preincubation of bFGF with heparin, polylysine or protamine abolished binding of bFGF to tissue sections prepared from Alzheimer disease cases. Interestingly, pretreatment of the section with NaOH, a treatment known to cause B elimination of carbohydrate groups unmasked heparan sulfate proteoglycan core protein immunoreactivity in NFT. Similar NaOH treatment of enriched NFT fractions dissolved much of the protein. Analysis of the solubilized proteins immunoblotted indicated that an HSPG immunoreactive protein barely entering the separating gel was present. The present findings indicate that a specific heparan sulfate proteoglycan core protein is associated with NFT and that its presence may explain many biological and chemical properties of paired helical filaments, bFGF and antibody to bFGF were supplied by California Biotechnology. The antiserum to heparan sulfate proteoglycan core protein were supplied by Upjohn Company. Supported by N1H grants K04-AG00415, AG-007775.
69 INCREASED CHROMOGRANIN A/SYNAPTOPHYSIN RATIO I~' AI,ZH E I M E R ' S AND P I C K ' S D I S E A S E S . H. L a s s m a n n ( l ) , >~ K . A . . Jellinger (2), P.Fischer (1), R.Wei]er, H. W i n k ] o r ( 3 ) . (l) Neurological Institute, U n i v . el: V i e n n a S c h o o l of Medicine, Vienna, ( 2 ) L. B o ] t z m a n n lnst itute o! C ] J n . Neurobiologv, Vienna, (3) Department ()f P h a r m a ( ) ] o g v , Univ.of lnnsbru(k School of Med., Innsbru(k, Auslria. Chrumogranin A (Ch-A) i m m u n o r e a c t ire! m a t e r i ~] i s seen ;n n e u r i t i c plaques of Alzheimer's disease CAD) (Munoz, Neurology 38: $ 3 9 D , 1 9 8 9 ) , wbile synapto[hvsin (Syn) and s v o a p t i n I are strikingly decreased in AD ( H a m o s er e l . Neurology 3q:]55,198g', Masliah et el. J Neuropath Exp N e u r o ] 4 8 : 3 : ~ 3 , 1 9 8 9 ) . I m m u n o h i s t n c h < m i c a ] and immunoblotting studies are reported in AD, P i c k ' s disease (PD) a n d a g e d c o n t r o l s . Immunohistochen istry using po];'clonal antisera against It-A, se(17etofranin (S-If) a n d Svn s h o w e d c o n s i s t e n t st t i n i n g e l n e t r i t i e plaques f o r Ch-A ( s t a i n i n g a lar~,er number of plaques than antibodies against phosphorv]ated neurofilaments and tau protein),while diffuse ("early") plaquee were negative. [n P D , a l l Pick I)odies showed intensiw Ch-A immunoreactivitv, while n e u r i t i~ p l a q u e s and th~ P i c k bodies were n e g a t ~ve f o r S-l[. Qual i t a t i v e immuneblotting of brain tissue f r o m AD ( n - 5 ) , PD ( n = l ) a n d aged controls (n-4) with ant isera against C h - i and Syn r e v e a l e d no q u a l i t a t i v e differences between the antigens in c o n t r o l s and d i s e a s e d bt-ain. While buth AD a n d PD s h o w e d s i g n i f i c a n t l y l o w e r J e w , I s el S y n , f o u r o u t o f 5 AD c a s e s a n d t h e PD b r a i n r e v e a ] e ~ significantl~ increased !evels of (b-A. The ratio Ch-A/ Syn was i n ( t e a s e d in a l l AD b r a i n s (average %.7 f o l d ) a n d in t h e PD b r a i n (10.8 fold). The p a t h o g e n i c significance of the increased C h - A / S y n r a t io in b( t b AD 3 n d PD and its p o s s i b l e relevance as i~ d i a g n o s t c indicator
for
these
diseases
are
cril ica]]~,
discussed.
70 Senile plaques: Staining for acetylchollnesterase and betaprotein. A comparative study In the entorhlnal cortex. by &Ulrich, *WMeier-Ruge, A.Probst, F:.Meier and S.Ipsen, Neuropathology Division and Division of Gerontological Brain Research, Institute of Pathology, CH-4003 Basel, Switzerland. In 20 cases dying older than 70 years coming to autopsy within less than 12 hours after death, tissue-blocks from the hippocampus with
270
NEUROBIOLOGY OF' AGING, VOLUME ii, 199() ABSI'RACTS Of~ SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE NEUROPATHOLOGY/ALZHEIMER'S DISEASE IN DEVELOPMENTAL DISABI[.ITIES
adjacent entorhinal cortex and neocortex and from the neostriatum were stained for acetylcholinesterase (ACHE). Adjacent hippocampal tissues from the five brains with the most numerous senile plaques as shown in AChE-tissue blocks were embedded in paraffin and sections were stained with a polyclonal antibody against synthetic beta-protein 1-42 (Masters et al.,) after treatment with 90% formic acid. The morphological aspects of plaques as stained for beta-protein and AChE were compared. In further selected cases with many plaques adjacent cryostat sections were stained for beta-protein and AChE respectively. The same plaques and plaque-like structures including the amorphous, diffuse and subpial beta-protein deposits not demonstrable with conventional histological techniques were observed. These findings suggest, that high AChE activity is intimately associated with the process of beta-protein formation and accumulation in plaques and that this association already occurs in a very early stage of plaque formation. Plaque-bound AChE activity could possibly not be observed in the beta-protein deposits of the striatum, possibly because the reaction was masked by high background AChE activity of the striatum. 71 NORMAL CONSTITUTIVE PROCESSING OF THE ALZHEIMER AMYLOID PRECURSOR PROTEIN PROTEOLYZES THE BETA AMYLOID PEPTIDE *F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culweli, T. Oltersdorf, D. McClure ¶ and P.J. Ward. Athena Neurosciences Inc., 800F Gateway Blvd. South San Francisco, CA 94080 and Lilly Research Labs ¶ , Indianapolis, Indiana 46285. The beta-amyloid peptide [13-AP] is a small fragment of the much larger and broadly distributed amyloid precursor protein [APP]; the relative absence of 13-AP deposition in normal brain suggests that altered processing of APP occurs in AD and may represent a key pathogenic event in the disease. Constitutive processing of the APP results in the secretion of a large, soluble, N-terminal APP fragment and the generation of a membrane-bound 9 kD C-terminal APP fragment. We have purified both APP fragments from human embryonic kidney 293 cells transfected with cDNAs encoding the 695 and 751 amino acid forms of the APP; direct protein microsequencing, mass spectrometry and amino acid analyses of these N- and C-terminal APP fragments have shown that both APP695 and APP751 are p r o t e o l y t i c a l l y ~:l~vg~ within the g-AP s e o u e n ~ to generate these fragments. Thus, the formation an~l ~12O~iti0i3 of the B-AP is erecluded bv normal constitutivQ orocessino in 293 cells. These observations suggest the p o s s i b i l i t y that ~-AP deposition and the pathological formation of amyloid-bearing senile plaques in Alzheimer's disease may result from a deficit in this processing event. 72 AMYLOID P COMPONINT I N NllUitOirZillP.XLL~¥ TANGLES,
CORTICflkL PLI&QUIB ~HD CBRIBROSPIHI&L FLUID IN ~L~I~XMER'8 DISRKSB AND RBLKTBD D/BORDERS. *R. N. Kalaria. Departments of Neurology and Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, U S A Serum amyloid P identical to amyloid P component, (AP) is an ul-glycoprotein shown to be consistently present in all types of amyloid deposits except cerebral lesions of neurofibrillary tangles and
senile plaques. We used immunohistochemical methods to demonstrate AP immunoreactivity in both tangles and plaques, as well as vessels, in lightly fixed cryostat tissue sections of neocortex and hippocampus from subjects with Alzheimer's disease (AD) and other related neurodegenerative diseases. Heavy deposition of immunoperoxidase reaction product was evident in all the classical amyloidotic lesions of all cortical areas of AD cases examined. Senile plaques of Cruetzfeld-Jakob disease and Down's syndrome, Pick bodies of a Pick's case and tangles and vessels in Parkinson's disease were also stained. The distribution and intensity of immunostaining were similar to that of thioflavin S staining in serial sections. However, in some AD cases plaques were stained by AP that were not apparent by thioflavin S. Serial sections stained with antiserum to amyloid A or albumin, preimmune rabbit serum or preabsorbed AP serum showed no evidence for reactivity. AP immunoreactivity is also localised in amyloid deposits containing heparan sulphate proteoglycans and calcium. Intense AP reactivity in the 26K dalton band was also evident in immunoblots of cerebrospinal fluid from AD subjects. These observations provide evidence for extravasation of the protein across the bloodbrain barrier (BBB) in disease and may suggest impairment of the BBB. However, expression of AP by reactive cells within or those infiltrating into brain through the BBB cannot be ruled out. Supported by an ADRDA investigator-initiated award.
73 M O R P H O L O G I C A L / N E U R O C H E M I C A L C O R R E L A T E S OF DEPRESSION AND PSYCHOSIS IN PRIMARY DEMENTIA. G.S. Zubenko, J. Moossy, *J. Rosen, A.J. Martinez, G.R. Rag, D. Claassen, U. Kopp. University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 USA Major depression and psychosis are important sources of comorbidity in patients with Alzbeimer's disease. Clinically-significant major depression emerged in 14 of 37 patients with primary dementia. Major depression in this context was associated with increased degeneration in the locus ceruleus (LC) and the substantia nigra (SN). Indices of neurodegeneration included neuronal loss, extraneuronal pigment, astrocytosis, and cells containing neurofibrillary tangles (NFT) or Lewy bodies (LB). Models that incorporated data from both of these nuclei more accurately predicted the emergence of major depression than those that included data from either nucleus alone, suggesting an interactive role of these aminergic systems in the pathogenesis of depression. As expected from the morphological studies, major depression was associated with significant reductions in the levels of norepinephrine (NE), particularly in neocortex. Dopamine (DA) levels were not decreased in seven cortical and subccrtical areas, even though the degrees of neurodegeneration in the LC and SN associated with depression were similar. The source of the apparent differential sensitivity of LC neurons to degenerative changes is uncertain. Major depression was associated with a consistent reduct.ion in serotonin (5HT) levels in all regions examined, although the reduction did not reach statistical significance in any single region. Choline acetyltransferase (CHAT), while reduced compared to control levels, was relatively preserved in subcortical regions of patients with major depression and suggests that a minimum threshold of cholinergic function may be required for the expression of major depression. Psychosis, defmed as the presence of delusions or hallucinations, emerged in 13 of 27 patients with histologically-confirmed Alzheimer's disease prior to death. In contrast to major depression, psychosis was associated with an increased density of senile plaques and neurofibrillary tangles in the prosubiculum and frontal cortex, respectively. Moreover, psychosis was not associated with consistent changes in the levels of arcane neurotransmitters or ChAT activity in the cortical and subcortical regions examined. In summary, major depression and psychosis are debilitating behavioral syndromes that commonly occur in patients with primary dementia. However, they appear to emerge independently and to have different anatomical, morphological, and neurochemical substrates. 74
DEGENERATION OF CORTICOCORTICAL GLUTAMATERGIC PATHWAYS AND ITS TREATMENT D.M. Bowen, Department of Neurochemistry, Institute for Neurology, London, U.K.
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE NEUROPATHOLOGY/ALZHEIMER'S DISEASE IN DEVELOPMENTAL DISABILITIES N a k a z a t o 3 , M. S h o j i a , Y. H a r i g a y az , T. K a w a r a b a y a s h i2 , K. Okamoto z , S. H i r a i z , M. T a k a t a m a4 . ICollege of M e d i c a l C a r e and T e c h n o l o g y , D e p a r t m e n t s o f Z N e u r o l o g y and a P a t h o l o g y , Gunma U n i v e r s i t y , 4 G e r i a t r i c s R e s e a r c h I n s t i t u t e and H o s p i t a l ; M a e b a s h i , Gunma 371, J a p a n . In addition to primitive, c l a s s i c , and b u r n e d - o u t p l a q u e s , we had r e p o r t e d " d i f f u s e p l a q u e s " , which were n o t d e t e c t a b l e by t h e Bodian s t a i n . H e r e , we examined the u l t r a s t r u c t u r e o f d i f f u s e p l a q u e s ( D P ) and u l t r a structural localization of ~ protein immunoreaetive s u b s t a n c e w i t h i n DP i n A l z h e i m e r b r a i n s . F i r s t , u s i n g o s m i u n - f i x e d and epon-embedded t i s s u e s we e x a m i n e d p a i r s o f r o u t i n e electron microscopic ultrathin s e c t i o n and a d j a c e n t 0 . 4 ~ m - t h i c k l i g h t m i c r o s c o p i c s e m i t h i n s e c t i o n w h i c h was t r e a t e d with f o r m i c a c i d and B p r o t e i n i m m u n o s t a i n . In the f r o n t a l c o r t e x , s e m i t h i n s e c t i o n s showed numerous DP. Many o f them had i r r e g u l a r b o r d e r s and c o n s i s t e d o f c l u s t e r o f f i n e dot s t a i n s . N e u r o n a l c e l l b o d i e s were o c c a s i o n a l l y p r e s e n t w i t h i n DP. Most o f DP u s u a l l y showed no o r v e r y s m a l l amount o f a m y l o i d f i b r i l s i n t h e narrow extraeellular spaces. Neuropil w i t h i n t h e s e a r e a s appeared morphologically almost normal. Therefore, it was i m p o s s i b l e t o d i s t i n g u i s h DP from t h e s u r r o u n d i n g neuropil without an a i d o f t h e s e m i t h i n sections stained for ~ protein. Focal d e n s e l y - s t a i n e d a r e a s showed a number o f a l t e r e d n e u r i t e s and e x t r a c e l l u ] a r amyloid f i b r i l s . Second, Vibratome sections of paraformaldehydef i x e d t i s s u e s w e r e i m m u n o s t a i n e d for" ~ p r o t e i n , and then embedded in epon. Positive immunoreacbion p r o d u c t s were s c a t t e r e d sporadically throughout the DP. Positive structures i n t h e DP w e r e I ) s m a l l bundles of amy]oid f i b r i l s , 2) amorphous m a t e r i a l s b e tween cell processes ( p r e - a m y l o i d ) , a n d 3) c e l l m e m b r a n e s of s o m e c e l l p r o c e s s e s . Our findings suggest that DP consist of small a m o u n t of a m y l o i d fibrils and pre-amyloid with minimal neuritic changes, and that pre-amyloid deposits may precede the amyloid fibril formation.
67 IMAGE ANALYSIS MICROSPECTROSCOPY OF SENILE PLAQUES I}~UNOSTAINED WITH AN ANTI BETA-PROTEIN ANTIBODY. *Miguel A. Pappol]a I, Rawhi A. Omar 2, Harry Vinters 3. Departments of Pathology, F.D.R. V.A. Medical Center, Montrose, New York I, New York Medical College, Valhalla, New York I , University ot West Virginia, Morgantown, West Virginia 2 , University of California, Los Angeles, California 3. We used image analysis microspecttoscopy (IAM) to quantitatively evaluate the genesis and distribution of amyloid deposits in senile plaques immunostained with an antibody raised against a synthetic peptide containing the beta-protein sequence. IAM is a computer assisted method which combines basic principles of ligilt densitometry with image subtraction techniques and allows selective optical isolation with simultaneous quantification of tissue components (Pappolla, 1988). In prior studies utilizing IAM we had initially observed that congophilic deposits in senile plaques have a consistent relationship to microvessels characterized by a gradient of amyloid concentration radiating outwardly from central capillaries (Pappolla, 1989). The antibody used in this study permitted detection of early primitive senile plaques at a stage not exhibiting the typical congophilia characteristic of well-developed plaques. Quantitative analysis of these presumably early amyloid deposits in serial tissue sections did not show an apparent relationship to capillaries (i.e.: no gradient was present). Instead, these deposits appeared to surround neurites which occasionally showed degenerative changes. Image analysis of more mature plaques immunostained as above revealed again the characteristic concentration gradient of amyloid previously observed using congo red staining alone. These observations indicate that involvement of the microvasculature probably takes place at a later stage in the genesis of the plaque. Moreover, it seems that amyloid precursor proteins are converted to the beta-pleated configuration once they have reached the microvasculature, where they accumulate as central congophilic cores in mature plaques.
68 NEUROFIBRILLARY TANGLES CONTAIN HEPARAN SUL FATE PROTEOGLYCANS. *G. Perry, S. Siedlal% P. Mulvihill M. Kawai, P. Cras, B. Cordell 1, J. Marian Scardina j , S.. Ledbetter :~, B.
Greenbfrg 2, P. Gambetti. Case Western Reserve University, C~veland, Ohio; "California Biotechnology, Mountain View, California; "Upjohn Company, Kalamazoo, Michigan. Recently we observed that vessels are increased in density around senile plaques (SP), suggesting that angiogenic factors play a role in SP formation. Therefore we investigated the distribution of basic fibroblast growth factor (bFGF), a well known angiogenic factor, in brain tissues from subjects with AD and controls. We found that when the antibodies were absorbed with bFGF prior to immunostaining, the antigen-antibody complex strongly bound to neurofibrillary tangles (NFT), neuropil threads, dystrophic neurites surrounding SP, amyloid component of SP as well as normal vessels. A similar pattern was obtained when tissue sections were incubated with bFGF and subsequently immunostained with the antibodies to bFGF. Ultrastructural observation showed the bFGF is bound by paired helical filaments, yet was especially pronounced in NFT containing amorphous material. Treatment of sections with a combination of heparinase and heparitinase or preincubation of bFGF with heparin, polylysine or protamine abolished binding of bFGF to tissue sections prepared from Alzheimer disease cases. Interestingly, pretreatment of the section with NaOH, a treatment known to cause B elimination of carbohydrate groups unmasked heparan sulfate proteoglycan core protein immunoreactivity in NFT. Similar NaOH treatment of enriched NFT fractions dissolved much of the protein. Analysis of the solubilized proteins immunoblotted indicated that an HSPG immunoreactive protein barely entering the separating gel was present. The present findings indicate that a specific heparan sulfate proteoglycan core protein is associated with NFT and that its presence may explain many biological and chemical properties of paired helical filaments, bFGF and antibody to bFGF were supplied by California Biotechnology. The antiserum to heparan sulfate proteoglycan core protein were supplied by Upjohn Company. Supported by N1H grants K04-AG00415, AG-007775.
69 INCREASED CHROMOGRANIN A/SYNAPTOPHYSIN RATIO I~' AI,ZH E I M E R ' S AND P I C K ' S D I S E A S E S . H. L a s s m a n n ( l ) , >~ K . A . . Jellinger (2), P.Fischer (1), R.Wei]er, H. W i n k ] o r ( 3 ) . (l) Neurological Institute, U n i v . el: V i e n n a S c h o o l of Medicine, Vienna, ( 2 ) L. B o ] t z m a n n lnst itute o! C ] J n . Neurobiologv, Vienna, (3) Department ()f P h a r m a ( ) ] o g v , Univ.of lnnsbru(k School of Med., Innsbru(k, Auslria. Chrumogranin A (Ch-A) i m m u n o r e a c t ire! m a t e r i ~] i s seen ;n n e u r i t i c plaques of Alzheimer's disease CAD) (Munoz, Neurology 38: $ 3 9 D , 1 9 8 9 ) , wbile synapto[hvsin (Syn) and s v o a p t i n I are strikingly decreased in AD ( H a m o s er e l . Neurology 3q:]55,198g', Masliah et el. J Neuropath Exp N e u r o ] 4 8 : 3 : ~ 3 , 1 9 8 9 ) . I m m u n o h i s t n c h < m i c a ] and immunoblotting studies are reported in AD, P i c k ' s disease (PD) a n d a g e d c o n t r o l s . Immunohistochen istry using po];'clonal antisera against It-A, se(17etofranin (S-If) a n d Svn s h o w e d c o n s i s t e n t st t i n i n g e l n e t r i t i e plaques f o r Ch-A ( s t a i n i n g a lar~,er number of plaques than antibodies against phosphorv]ated neurofilaments and tau protein),while diffuse ("early") plaquee were negative. [n P D , a l l Pick I)odies showed intensiw Ch-A immunoreactivitv, while n e u r i t i~ p l a q u e s and th~ P i c k bodies were n e g a t ~ve f o r S-l[. Qual i t a t i v e immuneblotting of brain tissue f r o m AD ( n - 5 ) , PD ( n = l ) a n d aged controls (n-4) with ant isera against C h - i and Syn r e v e a l e d no q u a l i t a t i v e differences between the antigens in c o n t r o l s and d i s e a s e d bt-ain. While buth AD a n d PD s h o w e d s i g n i f i c a n t l y l o w e r J e w , I s el S y n , f o u r o u t o f 5 AD c a s e s a n d t h e PD b r a i n r e v e a ] e ~ significantl~ increased !evels of (b-A. The ratio Ch-A/ Syn was i n ( t e a s e d in a l l AD b r a i n s (average %.7 f o l d ) a n d in t h e PD b r a i n (10.8 fold). The p a t h o g e n i c significance of the increased C h - A / S y n r a t io in b( t b AD 3 n d PD and its p o s s i b l e relevance as i~ d i a g n o s t c indicator
for
these
diseases
are
cril ica]]~,
discussed.
70 Senile plaques: Staining for acetylchollnesterase and betaprotein. A comparative study In the entorhlnal cortex. by &Ulrich, *WMeier-Ruge, A.Probst, F:.Meier and S.Ipsen, Neuropathology Division and Division of Gerontological Brain Research, Institute of Pathology, CH-4003 Basel, Switzerland. In 20 cases dying older than 70 years coming to autopsy within less than 12 hours after death, tissue-blocks from the hippocampus with
270
NEUROBIOLOGY OF' AGING, VOLUME ii, 199() ABSI'RACTS Of~ SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE NEUROPATHOLOGY/ALZHEIMER'S DISEASE IN DEVELOPMENTAL DISABI[.ITIES
adjacent entorhinal cortex and neocortex and from the neostriatum were stained for acetylcholinesterase (ACHE). Adjacent hippocampal tissues from the five brains with the most numerous senile plaques as shown in AChE-tissue blocks were embedded in paraffin and sections were stained with a polyclonal antibody against synthetic beta-protein 1-42 (Masters et al.,) after treatment with 90% formic acid. The morphological aspects of plaques as stained for beta-protein and AChE were compared. In further selected cases with many plaques adjacent cryostat sections were stained for beta-protein and AChE respectively. The same plaques and plaque-like structures including the amorphous, diffuse and subpial beta-protein deposits not demonstrable with conventional histological techniques were observed. These findings suggest, that high AChE activity is intimately associated with the process of beta-protein formation and accumulation in plaques and that this association already occurs in a very early stage of plaque formation. Plaque-bound AChE activity could possibly not be observed in the beta-protein deposits of the striatum, possibly because the reaction was masked by high background AChE activity of the striatum. 71 NORMAL CONSTITUTIVE PROCESSING OF THE ALZHEIMER AMYLOID PRECURSOR PROTEIN PROTEOLYZES THE BETA AMYLOID PEPTIDE *F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culweli, T. Oltersdorf, D. McClure ¶ and P.J. Ward. Athena Neurosciences Inc., 800F Gateway Blvd. South San Francisco, CA 94080 and Lilly Research Labs ¶ , Indianapolis, Indiana 46285. The beta-amyloid peptide [13-AP] is a small fragment of the much larger and broadly distributed amyloid precursor protein [APP]; the relative absence of 13-AP deposition in normal brain suggests that altered processing of APP occurs in AD and may represent a key pathogenic event in the disease. Constitutive processing of the APP results in the secretion of a large, soluble, N-terminal APP fragment and the generation of a membrane-bound 9 kD C-terminal APP fragment. We have purified both APP fragments from human embryonic kidney 293 cells transfected with cDNAs encoding the 695 and 751 amino acid forms of the APP; direct protein microsequencing, mass spectrometry and amino acid analyses of these N- and C-terminal APP fragments have shown that both APP695 and APP751 are p r o t e o l y t i c a l l y ~:l~vg~ within the g-AP s e o u e n ~ to generate these fragments. Thus, the formation an~l ~12O~iti0i3 of the B-AP is erecluded bv normal constitutivQ orocessino in 293 cells. These observations suggest the p o s s i b i l i t y that ~-AP deposition and the pathological formation of amyloid-bearing senile plaques in Alzheimer's disease may result from a deficit in this processing event. 72 AMYLOID P COMPONINT I N NllUitOirZillP.XLL~¥ TANGLES,
CORTICflkL PLI&QUIB ~HD CBRIBROSPIHI&L FLUID IN ~L~I~XMER'8 DISRKSB AND RBLKTBD D/BORDERS. *R. N. Kalaria. Departments of Neurology and Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, U S A Serum amyloid P identical to amyloid P component, (AP) is an ul-glycoprotein shown to be consistently present in all types of amyloid deposits except cerebral lesions of neurofibrillary tangles and
senile plaques. We used immunohistochemical methods to demonstrate AP immunoreactivity in both tangles and plaques, as well as vessels, in lightly fixed cryostat tissue sections of neocortex and hippocampus from subjects with Alzheimer's disease (AD) and other related neurodegenerative diseases. Heavy deposition of immunoperoxidase reaction product was evident in all the classical amyloidotic lesions of all cortical areas of AD cases examined. Senile plaques of Cruetzfeld-Jakob disease and Down's syndrome, Pick bodies of a Pick's case and tangles and vessels in Parkinson's disease were also stained. The distribution and intensity of immunostaining were similar to that of thioflavin S staining in serial sections. However, in some AD cases plaques were stained by AP that were not apparent by thioflavin S. Serial sections stained with antiserum to amyloid A or albumin, preimmune rabbit serum or preabsorbed AP serum showed no evidence for reactivity. AP immunoreactivity is also localised in amyloid deposits containing heparan sulphate proteoglycans and calcium. Intense AP reactivity in the 26K dalton band was also evident in immunoblots of cerebrospinal fluid from AD subjects. These observations provide evidence for extravasation of the protein across the bloodbrain barrier (BBB) in disease and may suggest impairment of the BBB. However, expression of AP by reactive cells within or those infiltrating into brain through the BBB cannot be ruled out. Supported by an ADRDA investigator-initiated award.
73 M O R P H O L O G I C A L / N E U R O C H E M I C A L C O R R E L A T E S OF DEPRESSION AND PSYCHOSIS IN PRIMARY DEMENTIA. G.S. Zubenko, J. Moossy, *J. Rosen, A.J. Martinez, G.R. Rag, D. Claassen, U. Kopp. University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 USA Major depression and psychosis are important sources of comorbidity in patients with Alzbeimer's disease. Clinically-significant major depression emerged in 14 of 37 patients with primary dementia. Major depression in this context was associated with increased degeneration in the locus ceruleus (LC) and the substantia nigra (SN). Indices of neurodegeneration included neuronal loss, extraneuronal pigment, astrocytosis, and cells containing neurofibrillary tangles (NFT) or Lewy bodies (LB). Models that incorporated data from both of these nuclei more accurately predicted the emergence of major depression than those that included data from either nucleus alone, suggesting an interactive role of these aminergic systems in the pathogenesis of depression. As expected from the morphological studies, major depression was associated with significant reductions in the levels of norepinephrine (NE), particularly in neocortex. Dopamine (DA) levels were not decreased in seven cortical and subccrtical areas, even though the degrees of neurodegeneration in the LC and SN associated with depression were similar. The source of the apparent differential sensitivity of LC neurons to degenerative changes is uncertain. Major depression was associated with a consistent reduct.ion in serotonin (5HT) levels in all regions examined, although the reduction did not reach statistical significance in any single region. Choline acetyltransferase (CHAT), while reduced compared to control levels, was relatively preserved in subcortical regions of patients with major depression and suggests that a minimum threshold of cholinergic function may be required for the expression of major depression. Psychosis, defmed as the presence of delusions or hallucinations, emerged in 13 of 27 patients with histologically-confirmed Alzheimer's disease prior to death. In contrast to major depression, psychosis was associated with an increased density of senile plaques and neurofibrillary tangles in the prosubiculum and frontal cortex, respectively. Moreover, psychosis was not associated with consistent changes in the levels of arcane neurotransmitters or ChAT activity in the cortical and subcortical regions examined. In summary, major depression and psychosis are debilitating behavioral syndromes that commonly occur in patients with primary dementia. However, they appear to emerge independently and to have different anatomical, morphological, and neurochemical substrates. 74
DEGENERATION OF CORTICOCORTICAL GLUTAMATERGIC PATHWAYS AND ITS TREATMENT D.M. Bowen, Department of Neurochemistry, Institute for Neurology, London, U.K.