- Email: [email protected]
CSF α 1-antichymotrypsin level is elevated in patients with Alzheimer-type dementia
333
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BIOLOGICAL MARKERS/SYSTEMIC MARKERS regional variation is present between frontal and temporal lobe specimens. The current neuropathologlcal dlagnosts Is based on htstopathologlcal data: vlsualization and counting of senile plaques and neuroflbrlllary tangles, which requlres expertles and ls time consuming. The biochemical ALZ-EIA, offers a rapld, easily performed and quantitative dlagnostlc test which may serve as an valuable ald to the clinics-pathological dlagnosls of Alzhelmar's Disease. We would l i k e to acknowledge The Netherlands Brain Bank for supplying human braln specimens of rapid autopsies. 1. Biochemical assay of Alzhstmer's 01sease associated protein(s) In human brain tissue: a clinical study, H.A. Ghanbarl, B.E. Mtller, H.J. Halgler, N. Arato, G. B~ssette, P. Davies, Ch.B. Nemaroff, E.K. Perry, R. Perry, R. Ravld, D.F. Swaab, W.O. Whetse11, F.P. Zemlan, subclltted 1989, 2. Regional variation of ALZ-50 lmunoreactlvtty in 6 brains by ellsa type assays, B . E . Miller, R. Ravld, D . F . Swaab, H . A . Ghanbarl, Personal communlcatlons, 1990.
330 QUANTITATIVE DETERMINATION OF THE AMYLOID A4 P ~ R PROTEIN IN CF_,REBROSPINAL FLUID *R. Prior, U. MOnning, A. Weidemann, P.Fischer, K. Blennow 1, C.G. Gott fries 1 and K. Beyreuther Center for MoleCularBiology,Universityof Heidelberg(ZMBH),ImNeuenhcimcrFek1282. D-6900 Heidelberg l, F. R. Get.tony 1Depart~nt of Psychiatwand Meurochembtry, Universityof C.Mtburg,St. J6rgen%Fl~pital, $422 03 Hisinp Backa,Sweden The amyloid A4 precursor protein (PreA4/APP) is present in cerebrospinal fluid (CSF) in a 95 kD C-terminal truncated form that lacks most of the amyloidogenic region. The quantitative evaluation of the PreA4 content of CSF may be of value in understanding the mechanisms leading to amyloid deposition and plaque formation in Alzheimer's disease brains. Using a newly developed double sandwich enzymelinked immunosorbent assay (ELISA) we detected the concentrations of total PreA4 in CSF samples from 14 patients with clinical Alzheimer's disease diagnosed according to the N I N C D S - A D R D A criteria, 5 patients with multi*infarct dementia and 10 neurologically normal age-matched control subjects. The mean concentrations of PreA4 were found to be slightly decreased in both the Alzheimer% disease and the multi-infarct dementia cases. The changes of PreA4 CSF levels were not related to alterations of the blood-brain barrier or CSF circuiation, since they were not paralleled by substantial variations of the CSF protein content. CSF levels of PreA4 may reflect mechanisms involved in both the pathogenesis and pathology of Alzheimer~ disease such as altered intracerebral or choroid plexus PreA4 expression, processing or secretion, neuronal death and reactive giiosls. Our CSF assay allows the quantitative analysis of PreA4 in different stages of sporadic and familial Alzheimer's disease, D o w n ' s syndrom and other related disorders and therefore represents a first step towards an understanding of the mechanisms that determine the CSF PreA4 content and to establish a potential diagnostic utility of quantitative CSF PreA4 evaluation.
331 c~ ,-ANTIOh'MOTRYPSIN AND ANTITRYPSIN IN CSF OF PATIENTS WITH DAT *Y. Shinohara, M. Yamamoto, H. 0hsuga, S. 0hsuga, K. Akiyama, F. Y o s h i i , M. Tsuda ¢[), H. Kamiguchi ~1~, M. Yamamura <~>. Departments of Neurology and B i o c h e m i s t r y cz>, Tokai U n i v e r s i t y School of Medicine, Kanagawa, Japan. Since the immunohistochemical method u s i n g antibody of ~ - a n t i c h y m o t r y p s i n (ACT) demonstrated the p o s i t i v e s t a i n i n g in the s e n i l e plaques in p a t i e n t s with dementia of Alzheimer type (DAT), i t has been s u g g e s t e d t h a t ACT might play some r o l e s on the p a t h o l o g i c a l p r o c e s s e s of DAT. High c o n c e n t r a t i o n of ACT in serum has been r e p o r t e d as p o s s i b l e d i a g n o s t i c marker of DAT. The purpose of t h i s communication i s to r e p o r t the concentr a t i o n of ACT and a n t i t r y p s i n (AT) of c e r e b r o s p i n a l f l u i d (C_.SF) and serum in p a t i e n t s w i t h DAT, and to e v a l u a t e the s i g n i f i c a n c e of these p r o t e i n s . S u b j e c t s and Methods: The measurements of ~ and AT in CSF and serum were performed in 8 p a t i e n t s with DAT and 9 c o n t r o l s . D i a g n o s i s of DAT was made by the h i s t o r y , p h y s i c a l and n e u r o l o g i c a l examinations, MRI f i n d i n g s and DSM mR c r i t e r i a . S u b j e c t s who had high serum C R P ( > I . 0 0 l g / m l ) were excluded from t h i s study because the c o e x i s t e n c e of shows high concent r a t i o n s of ACT and AT. The measurements of Acr and AT in ~ and serum were made by u s i n g ELISA method and s i n g l e r a d i a l immunodiffusion method, respectively. R e s u l t s and Comments: The c o n c e n t r a t i o n s of ACT(3.G±2.0 ~ g / m l ) and A T ( 1 5 . 6 ± 7 . 8 g g/ml) in CSF of DAT p a t i e n t s were s i g n i f i c a n t l y h i g h e r than those of ACT(1.4±0.3 ~ g / m l ) and A T ( 8 . 5 ± 3 . 6 g g / m l ) in 9 c o n t r o l s ( P < 0.05 and P < 0 . 0 5 ) , r e s p e c t i v e l y . The v a l u e s of C.SF-ACT/Serum-ACT and CSFACT/CSF-AT in p a t i e n t s with DAT were a l s o s i g n i f i c a n t l y h i g h e r than those
of control ( P < 0 . 0 5 and P < 0 . 0 5 , r e s p e c t i v e l y ) . There were no s i g n i f i cant d i f f e r e n c e s of serum ACT and serum AT l e v e l s between DAT and c o n t r o l s . Our p r e s e n t study showed t h a t the c o n c e n t r a t i o n s of ACT and AT in CSF of p a t i e n t s with DAT were h i g h e r than those of c o n t r o l s in s p i t e of nons i g n i f i c a n t d i f f e r e n c e s of serum ACt and AT l e v e l s between DAT and controls. The mechanisms of i n c r e a s e of ACT and AT in CSF remains to be clarified. However, the h i g h e r value of AC[/AT in ~ of DAT might s u g g e s t t h a t ACT in CSF r e f l e c t the p a t h o l o g i c a l p r o c e s s e s of DAT.
332 CSF ~ I-ANTICHYMOTRYPSIN LEVEL IS E L E V A T E D IN PATIENTS WITH ALZHEIMER-TYPE DEMENTIA. *E. M a t s u b a r a , M. A m a r i , M. S h o j i , Y. H a r i g a y a , H. Y a m a g u c h i , K. Okamoto, K. I s h i g u r o , H. K a n a i , T. K a w a r a b a y a s h i , S. H i r a i , M. T a k a t a m a # , K. H o s o d a $ . Department of N e u r o l ogy, G u m a u n i v e r s i t y School of Medicine, #Geriatrics Research Institute and Hospital, Maebashi, G u m a 371, Japan, $Teijin Institute for Bio-medical Research, Tokyo. In t h e f i r s t c o n f e r e n c e , we had reported that serum l-antichymotrypsin (ACT) levels were significantly elevated in t h e p a t i e n t s with Alzheimer-type dementia (ATD). Recently, we have developed radioimmunoassay (RIA) a n d e n z y m e immunoassay (EIA) of ACT. Together with serum ACT levels, we m e a s u r e d the CSF ACT level in o r d e r to c l a r i f y whether or not these parameters are useful as biochemical markers for ATD, and to determine the relationship between serum and CSF l e v e l s o f A C T in e a c h s t a g e s of ATD. The CSF subjects consisted of 12 p a t i e n t s w i t h ATD, 6 with vascular dementia, 7 with cerebrovascular disease without dementia, a n d 21 n o r m a l controls. The CSF ACT levels were measured u s i n g R I A a n d EIA. Both serum and CSF ACT levels were measured simultaneously in A T D p a t i e n t s (RIA: 9 c a s e s , EIA: 7 c a s e s ) . i. R I A : There were no age-related changes or s e x differences in n o r m a l control subjects. CSF ACT levels of ATD were significantly and specifically h i g h e r t h a n t h o s e of t h e o t h e r s u b j e c t s (p<0.05). CSF ACT levels reflected the degree of d e m e n t i a . Despite the significant elevation (p<0.05) of C S F A C T l e v e l s in A T D , s o m e c a s e s s h o w e d normal values. These cases belonged to t h e Ist s t a g e of ATD. CSF ACT levels were not correlated with serum ACT levels. 2. EIA: S i m i l a r results were obtained in t h i s study. However, the value obtained in t h i s s t u d y w a s w i d e l y r a n g e d in c o m p a r i s o n w i t h t h a t of RIA. Our findings indicate t h a t s e r u m a n d C S F A C T m a y be independently up-regulated in ATD. We c o n c l u d e d that measurement of CSF ACT levels was useful as a biochemical diagnostic marker for ATD, because CSF ACT m a y r e f l e c t t h e s t a g e o f ATD.
333 THE AMYLOID IS RELEASED
PRECURSOR BY HUMAN
PROTEIN OF ALZEEIMER'S DISEASE PLATELETS. *Ashley Bush t , Ralph
M a r t i n s ~ , Baden Hu~dDle t, Robert Moir ~, Stephanie Fuller ~, Elizabeth M i l w a r d ~ , Jon Curriet , D a v i d Ames t , A n d r e a s Weidemann i, Peter Fischer e, Gerd Multhaup ~, Konrad Beyreuther ~ and Colin Masters ~ . ~ Department of Pathology, University of Melbourne, and Mental Health Research Institute, Royal Park Hospital, Parkville, Vic., 3052, Australia. 0 The Center for Molecular Biology, University of Heidelberg, Heidelberg, Federal Republic of Germany. The neuropathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid ~A4 protein in the cerebral cortex. The mechanism of amyloidogenesis is unclear but we have previously purified and characterised BA4 as a 42 residue protein whose sequence was used to predict a precursor (APP) resembling a transmerabrane glycoprotein receptor. APP behaves as a mitogen and protease inhibitor-containing forms of A P B inhibit coagulation enzymes Xa and XIa in vitro. A hematogenous source of precursor for ~A4 deposition must be considered since most cases of AD have amyloid deposits around the vessel walls of the leptomeninges and the cerebral cortex giving rise to a congophilic angiopathy. Furthermore, there is an association between abnormal platelet merobrane fluidity and AD Western blots were carried out on human platelets, employing a monoclonal antibody raised against the full-length APP695 fusion protein. These revealed major forms of I00-Ii0 and 120130 kDa molecular weight in both cytosolic, membrane and released fractions. These species were similar in size to forms seen in brain preparations and in plasma, but in a far greater concentration. There was no difference in Western blots of
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BIOLOGICAL MARKERS/SYSTEMIC MARKERS regional variation is present between frontal and temporal lobe specimens. The current neuropathologlcal dlagnosts Is based on htstopathologlcal data: vlsualization and counting of senile plaques and neuroflbrlllary tangles, which requlres expertles and ls time consuming. The biochemical ALZ-EIA, offers a rapld, easily performed and quantitative dlagnostlc test which may serve as an valuable ald to the clinics-pathological dlagnosls of Alzhelmar's Disease. We would l i k e to acknowledge The Netherlands Brain Bank for supplying human braln specimens of rapid autopsies. 1. Biochemical assay of Alzhstmer's 01sease associated protein(s) In human brain tissue: a clinical study, H.A. Ghanbarl, B.E. Mtller, H.J. Halgler, N. Arato, G. B~ssette, P. Davies, Ch.B. Nemaroff, E.K. Perry, R. Perry, R. Ravld, D.F. Swaab, W.O. Whetse11, F.P. Zemlan, subclltted 1989, 2. Regional variation of ALZ-50 lmunoreactlvtty in 6 brains by ellsa type assays, B . E . Miller, R. Ravld, D . F . Swaab, H . A . Ghanbarl, Personal communlcatlons, 1990.
330 QUANTITATIVE DETERMINATION OF THE AMYLOID A4 P ~ R PROTEIN IN CF_,REBROSPINAL FLUID *R. Prior, U. MOnning, A. Weidemann, P.Fischer, K. Blennow 1, C.G. Gott fries 1 and K. Beyreuther Center for MoleCularBiology,Universityof Heidelberg(ZMBH),ImNeuenhcimcrFek1282. D-6900 Heidelberg l, F. R. Get.tony 1Depart~nt of Psychiatwand Meurochembtry, Universityof C.Mtburg,St. J6rgen%Fl~pital, $422 03 Hisinp Backa,Sweden The amyloid A4 precursor protein (PreA4/APP) is present in cerebrospinal fluid (CSF) in a 95 kD C-terminal truncated form that lacks most of the amyloidogenic region. The quantitative evaluation of the PreA4 content of CSF may be of value in understanding the mechanisms leading to amyloid deposition and plaque formation in Alzheimer's disease brains. Using a newly developed double sandwich enzymelinked immunosorbent assay (ELISA) we detected the concentrations of total PreA4 in CSF samples from 14 patients with clinical Alzheimer's disease diagnosed according to the N I N C D S - A D R D A criteria, 5 patients with multi*infarct dementia and 10 neurologically normal age-matched control subjects. The mean concentrations of PreA4 were found to be slightly decreased in both the Alzheimer% disease and the multi-infarct dementia cases. The changes of PreA4 CSF levels were not related to alterations of the blood-brain barrier or CSF circuiation, since they were not paralleled by substantial variations of the CSF protein content. CSF levels of PreA4 may reflect mechanisms involved in both the pathogenesis and pathology of Alzheimer~ disease such as altered intracerebral or choroid plexus PreA4 expression, processing or secretion, neuronal death and reactive giiosls. Our CSF assay allows the quantitative analysis of PreA4 in different stages of sporadic and familial Alzheimer's disease, D o w n ' s syndrom and other related disorders and therefore represents a first step towards an understanding of the mechanisms that determine the CSF PreA4 content and to establish a potential diagnostic utility of quantitative CSF PreA4 evaluation.
331 c~ ,-ANTIOh'MOTRYPSIN AND ANTITRYPSIN IN CSF OF PATIENTS WITH DAT *Y. Shinohara, M. Yamamoto, H. 0hsuga, S. 0hsuga, K. Akiyama, F. Y o s h i i , M. Tsuda ¢[), H. Kamiguchi ~1~, M. Yamamura <~>. Departments of Neurology and B i o c h e m i s t r y cz>, Tokai U n i v e r s i t y School of Medicine, Kanagawa, Japan. Since the immunohistochemical method u s i n g antibody of ~ - a n t i c h y m o t r y p s i n (ACT) demonstrated the p o s i t i v e s t a i n i n g in the s e n i l e plaques in p a t i e n t s with dementia of Alzheimer type (DAT), i t has been s u g g e s t e d t h a t ACT might play some r o l e s on the p a t h o l o g i c a l p r o c e s s e s of DAT. High c o n c e n t r a t i o n of ACT in serum has been r e p o r t e d as p o s s i b l e d i a g n o s t i c marker of DAT. The purpose of t h i s communication i s to r e p o r t the concentr a t i o n of ACT and a n t i t r y p s i n (AT) of c e r e b r o s p i n a l f l u i d (C_.SF) and serum in p a t i e n t s w i t h DAT, and to e v a l u a t e the s i g n i f i c a n c e of these p r o t e i n s . S u b j e c t s and Methods: The measurements of ~ and AT in CSF and serum were performed in 8 p a t i e n t s with DAT and 9 c o n t r o l s . D i a g n o s i s of DAT was made by the h i s t o r y , p h y s i c a l and n e u r o l o g i c a l examinations, MRI f i n d i n g s and DSM mR c r i t e r i a . S u b j e c t s who had high serum C R P ( > I . 0 0 l g / m l ) were excluded from t h i s study because the c o e x i s t e n c e of shows high concent r a t i o n s of ACT and AT. The measurements of Acr and AT in ~ and serum were made by u s i n g ELISA method and s i n g l e r a d i a l immunodiffusion method, respectively. R e s u l t s and Comments: The c o n c e n t r a t i o n s of ACT(3.G±2.0 ~ g / m l ) and A T ( 1 5 . 6 ± 7 . 8 g g/ml) in CSF of DAT p a t i e n t s were s i g n i f i c a n t l y h i g h e r than those of ACT(1.4±0.3 ~ g / m l ) and A T ( 8 . 5 ± 3 . 6 g g / m l ) in 9 c o n t r o l s ( P < 0.05 and P < 0 . 0 5 ) , r e s p e c t i v e l y . The v a l u e s of C.SF-ACT/Serum-ACT and CSFACT/CSF-AT in p a t i e n t s with DAT were a l s o s i g n i f i c a n t l y h i g h e r than those
of control ( P < 0 . 0 5 and P < 0 . 0 5 , r e s p e c t i v e l y ) . There were no s i g n i f i cant d i f f e r e n c e s of serum ACT and serum AT l e v e l s between DAT and c o n t r o l s . Our p r e s e n t study showed t h a t the c o n c e n t r a t i o n s of ACT and AT in CSF of p a t i e n t s with DAT were h i g h e r than those of c o n t r o l s in s p i t e of nons i g n i f i c a n t d i f f e r e n c e s of serum ACt and AT l e v e l s between DAT and controls. The mechanisms of i n c r e a s e of ACT and AT in CSF remains to be clarified. However, the h i g h e r value of AC[/AT in ~ of DAT might s u g g e s t t h a t ACT in CSF r e f l e c t the p a t h o l o g i c a l p r o c e s s e s of DAT.
332 CSF ~ I-ANTICHYMOTRYPSIN LEVEL IS E L E V A T E D IN PATIENTS WITH ALZHEIMER-TYPE DEMENTIA. *E. M a t s u b a r a , M. A m a r i , M. S h o j i , Y. H a r i g a y a , H. Y a m a g u c h i , K. Okamoto, K. I s h i g u r o , H. K a n a i , T. K a w a r a b a y a s h i , S. H i r a i , M. T a k a t a m a # , K. H o s o d a $ . Department of N e u r o l ogy, G u m a u n i v e r s i t y School of Medicine, #Geriatrics Research Institute and Hospital, Maebashi, G u m a 371, Japan, $Teijin Institute for Bio-medical Research, Tokyo. In t h e f i r s t c o n f e r e n c e , we had reported that serum l-antichymotrypsin (ACT) levels were significantly elevated in t h e p a t i e n t s with Alzheimer-type dementia (ATD). Recently, we have developed radioimmunoassay (RIA) a n d e n z y m e immunoassay (EIA) of ACT. Together with serum ACT levels, we m e a s u r e d the CSF ACT level in o r d e r to c l a r i f y whether or not these parameters are useful as biochemical markers for ATD, and to determine the relationship between serum and CSF l e v e l s o f A C T in e a c h s t a g e s of ATD. The CSF subjects consisted of 12 p a t i e n t s w i t h ATD, 6 with vascular dementia, 7 with cerebrovascular disease without dementia, a n d 21 n o r m a l controls. The CSF ACT levels were measured u s i n g R I A a n d EIA. Both serum and CSF ACT levels were measured simultaneously in A T D p a t i e n t s (RIA: 9 c a s e s , EIA: 7 c a s e s ) . i. R I A : There were no age-related changes or s e x differences in n o r m a l control subjects. CSF ACT levels of ATD were significantly and specifically h i g h e r t h a n t h o s e of t h e o t h e r s u b j e c t s (p<0.05). CSF ACT levels reflected the degree of d e m e n t i a . Despite the significant elevation (p<0.05) of C S F A C T l e v e l s in A T D , s o m e c a s e s s h o w e d normal values. These cases belonged to t h e Ist s t a g e of ATD. CSF ACT levels were not correlated with serum ACT levels. 2. EIA: S i m i l a r results were obtained in t h i s study. However, the value obtained in t h i s s t u d y w a s w i d e l y r a n g e d in c o m p a r i s o n w i t h t h a t of RIA. Our findings indicate t h a t s e r u m a n d C S F A C T m a y be independently up-regulated in ATD. We c o n c l u d e d that measurement of CSF ACT levels was useful as a biochemical diagnostic marker for ATD, because CSF ACT m a y r e f l e c t t h e s t a g e o f ATD.
333 THE AMYLOID IS RELEASED
PRECURSOR BY HUMAN
PROTEIN OF ALZEEIMER'S DISEASE PLATELETS. *Ashley Bush t , Ralph
M a r t i n s ~ , Baden Hu~dDle t, Robert Moir ~, Stephanie Fuller ~, Elizabeth M i l w a r d ~ , Jon Curriet , D a v i d Ames t , A n d r e a s Weidemann i, Peter Fischer e, Gerd Multhaup ~, Konrad Beyreuther ~ and Colin Masters ~ . ~ Department of Pathology, University of Melbourne, and Mental Health Research Institute, Royal Park Hospital, Parkville, Vic., 3052, Australia. 0 The Center for Molecular Biology, University of Heidelberg, Heidelberg, Federal Republic of Germany. The neuropathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid ~A4 protein in the cerebral cortex. The mechanism of amyloidogenesis is unclear but we have previously purified and characterised BA4 as a 42 residue protein whose sequence was used to predict a precursor (APP) resembling a transmerabrane glycoprotein receptor. APP behaves as a mitogen and protease inhibitor-containing forms of A P B inhibit coagulation enzymes Xa and XIa in vitro. A hematogenous source of precursor for ~A4 deposition must be considered since most cases of AD have amyloid deposits around the vessel walls of the leptomeninges and the cerebral cortex giving rise to a congophilic angiopathy. Furthermore, there is an association between abnormal platelet merobrane fluidity and AD Western blots were carried out on human platelets, employing a monoclonal antibody raised against the full-length APP695 fusion protein. These revealed major forms of I00-Ii0 and 120130 kDa molecular weight in both cytosolic, membrane and released fractions. These species were similar in size to forms seen in brain preparations and in plasma, but in a far greater concentration. There was no difference in Western blots of