- Email: [email protected]
Expression of Alzheimer's disease amyloid precursor protein during post-natal development of rat brain
304
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS
Young, S. Aubln, S.K. Ludwln and *R. Kisilevsky. Department of Pathology, Queen's Unlverslty, Kingston, Ontario, Canada, K7L 3N6. Highly sulphated glycoaamlnoglycans (GAGs) have been identified as part of all amylold deposits examined to date. Where it has been possible to identify the proteoglyean, these efforts have shown it to be the heparan sulphate proteoglycan (HSPG). In amylold associated with Inflammation the HSPG is deposited eolncldentally with the amylold peptlde regardless of the induction procedure used or tissue site of deposition. In addition, sulphate ions, probably as part of the HSPG, have been shown to play a role in maintaining amylold fibril structure. Previous light and EM studies have shown the GAG to be part of the plaque amylold fibril, and also present in plaque-llke lesions which do not contain an amylold core. Immunohlstochemleal techniques have shown the HSPG to be part of plaque amylold in sltu. In the present study we isolated neuritic plaques using the procedures of Selkoe et al, followed by further purification in sucrose gradients. The isolated plaques continue to stain positively with Congo red and the sulphated alclan blue techniques. Solubillzatlon of these plaques in guanldlne thlocyanate was followed by their dialysis and lyophillzatlon. Samples were subjected to electrophoresls in SDS polyacrylamlde gels followed by Western blotting with polyelonal antls1~ra prepared against the purified basement membrane HSPG of EHS tumors. The results revealed that basement membrane HSPG is part of the Alzhelmer'a plaque. Pre-treatment of these samples with nitrous acid, followed by eleetrophoreals and Western blotting, reduced the molecular weight of the antigen derived from these plaques, providing further evidence that the antigen is a form of HSPG. HSPG association with two fundamentally different forms of amylold and the fact that basement membrane HSPG is an extremely large molecule (greater than 400 KDa) and has never been shown to be present in the circulation, suggests that the HSPG molecule is synthesized locally and plays a role in the development of the characteristic amylold lesions seen in the brains of Alzhelmer's and Down's syndrome patients. Supported by MRC Grants MT-3153 and MA-I0477, and the Upj ohn Company.
211 EARLY ACCL~KK~TION OF HEP~RAIN SULFATE IN N~JRONS AND IN THE BEI"AKt{~/LOID PROTEIN CONTAINING LESIONS OF ALZHEIP~R'S D I S K ~ AND DONN'S ~ . "A.D. Snow, H. Mar, D. Nochlin, R.T. Seki~nJchi, K. Kirnsta, Y. Koike, T.N. Wight. University of washir~on, Seattle, WA 98195 and Aich/ Medical University, Aich/, Japan. A mcnoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha l-ap4 glucuronic acid-co~tain/ng determinant in heparan sulfate (HS) chains of a baselrellt-mmmbrane derived heparan sulfate proteoglycan (HSPG), identified and immunolocalized HS specifically to the alwloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle bearing neuror~, in the brains of Alzheimer's and Down's syndrome (DS) patiants. Ultrastructural immunohistochemistry demmlstrated that HS within neurons of Alzheirner's disease (AD) brain was localized to lipofuscin granules, an aging p i ~ t previously shown to also contain beta-m~yloid protein (BAP) (Bancher et al, 1988). Heparan sulfate was also localized to neurite-containing, non-fibrillar "primitive" plaques which also dml~nstrated positive BAP ir0munoreactivity in both AD and DS brain. Antibodies to im~inin, fibronectin and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurcrm of the h i ~ and am~jdala, as early as 1 day after birth. Yotmg age-matched controls did not demonstrate similar positive HS immm%oreactivity in neurons whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest depositiom of BAP was first observed as "~morphous" or "diffuse" cortical deposits in DS brain in patients aged 18 and 24, prior to the accumulation of fibrillar am~loid (o~served in DS patients 35 years and older). These cortical deposits also contained positive-HS immmloreaetivity in, lying that HS accumulation in conjmaction with the BAP is an eat:Iv event that may ultimately contribute to the early age-related a ~ l a t i o n (ie. as early as 35 years of age in DS) of NPs, NFTs and/or CA. Furthermore, the co-localization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may he important in lesion development in these two diseases.
212 EXPRESSION OF ALZHE1MER'S DISEASEAMYLOIDPRECURSOR PROTEIN DURINGPOST-NATAL DEVELOPMENTOF RAT BRAIN. *E. Milward 1, R. Martins 1, B. Rumble 1, R. Moir 1, A. Weidemann 2, K. Beyreuther 2, C. Masters 1. Department of Pathology, University of Melbourne and Mental Health Institute of Victoria, Royal Park Hospital, Parkville, Vie., 3052, Australia 1 and Center for Molecular Biology, University of Heidelberg, Heidelberg, FRG 2. The major protein component ([3A4) of the amyloid plaques that characterize Alzheimer's disease comes from a larger amyloid precursor protein (APP). Levels of APP during post-natal development of rat brain were measured by separate radioimmunoassays using antibodies specifically directed against either the amino- or the carboxyl-terminal of APP. Both assays revealed an overall increase in APP levels of 1.5 to 2-fold between birth and weaning. Adult levels were invariably lower than levels at weaning although the size of the decrease varied considerably between experiments. A minor, transient, perinatal increase in the APP level sometimes occurred. Western analysis of membrane-associated forms revealed three distinct complexes. A 100-110kD complex was detected at essentially constant levels at all ages examined during post-natal development and in the adult. In contrast, a 65-75 kD complex occurred at levels that were relatively low in the neonate but increased markedly during the second week. Although high levels were maintained until at least post-natal day 24, the complex was not detected in the young adult. A 50-60 kD complex was barely detectable during the first post-natal week but increased substantially after the second post-natal week. This complex was also maintained until at least post-natal day 24 but was absent in the adult. Northern analysis revealed a major 3.4-3.6 kb APP mRNA species and a minor t.8 kb form. No differences in their respective patterns of expression were apparent. Total APP mRNA, standardized relative to levels of glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) mRNA, increased by up to 2-fold between birth and weaning. A transient, perinatal increase in APP mRNA levels sometimes occurred. Adult levels of APP mRNA were similar to, or slightly higher than, levels at weaning. In summary, data suggest that expression of APP can be regulated at a post-transcriptional level. Furthermore, expression of different forms of APP may be differentially regulated, both during development and in the adult.
213 INDIANA lgll~RRn OF G_RlllCrM&~.ffrl~uJguglg-~J~Lttqti~ Dli~..~Z: ISOL&TION OF LOW MOI.ZCL."LAR Wra~tft" PRO'r~IN I ~ O M AMYI.,OIDIq.,AQUE CORI~. * F. Tagliavinil~, F. Prellil, J. Ghisol, O. Bugiani% M.R, Farlow3, B. Ghetti3, B. ~al3gionel. 1 New York University Medical Center. New York. NY, USA;
2 Istituto Neurelcgico Carlo Bet~, Milano, Italy; 3 Indiana University School of Medicine, Indianapolis, IN, USA. The neuropathologic hallmark of Gerstmann-Str~mssierScheinker (GSS) disease is the deposition in cerebral and cerebellar gray structures of amyloid fibrils immlmoreactive to antisera s r l r m t the Prion Protein 27-30 (PiP 27-30), a major structural component of scrapie-associated fibrils. Our aim was to purify a n d immunochemical!y characterize proteins e x t r a , ted from amyloid plaque cores isolated from cerebral cortex a n d cerebellum
of two patients from the Indiana kindred of OSS disease. Amyloidrich preparations fractienated by gel filtration yielded two major peaks (i.e. the void volume and a peak of 10-12 kDa) and five minor peaks. These fractions were su~ected to SDS-PAGE and i m m n n o b l o t analysis u s i n g three po]ye.lonal a n t i s e r a raised againat P r P 2%30 purified from brains of scrapie-infected animals, a n a n t i s e r u m to a synthetic poptide corresponding to residues 89-108 of eDNA deduced h a m s t e r PrP, a n a n t i s e r u m to amyloid P component a n d n o r m a l rablmt sera, Innnunoblot analysis of these fractiens d e m o n s t r a t e d t h a t all the anti-PrP a n t i s e r a strongly labeled the 10-12 k D a band, the m a j o r constituent of the amytoid extract, a n d leas intensely the 27 a n d 20 kDa bands. The anti-P .c~ml]ponent a n t i s e r u m d ve~.y stained a b a n d of 28 kDa. O u r d a t a cate t h a t GSS amyloid fibrils contain two main proteins: P component, which is common to all types Of mnyloid, a n d a 10-12
kDa peptide reacting with anti-PrP antisera. Although the amino termlrm| sequence of the 10-12 kDa subunit is heterogeneous, the presence of Gly-Gln-Pro suggests that the amyloid fibril is a degradation product of the Prion Protein, most probably starting at the repetitive sequence region.
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS
Young, S. Aubln, S.K. Ludwln and *R. Kisilevsky. Department of Pathology, Queen's Unlverslty, Kingston, Ontario, Canada, K7L 3N6. Highly sulphated glycoaamlnoglycans (GAGs) have been identified as part of all amylold deposits examined to date. Where it has been possible to identify the proteoglyean, these efforts have shown it to be the heparan sulphate proteoglycan (HSPG). In amylold associated with Inflammation the HSPG is deposited eolncldentally with the amylold peptlde regardless of the induction procedure used or tissue site of deposition. In addition, sulphate ions, probably as part of the HSPG, have been shown to play a role in maintaining amylold fibril structure. Previous light and EM studies have shown the GAG to be part of the plaque amylold fibril, and also present in plaque-llke lesions which do not contain an amylold core. Immunohlstochemleal techniques have shown the HSPG to be part of plaque amylold in sltu. In the present study we isolated neuritic plaques using the procedures of Selkoe et al, followed by further purification in sucrose gradients. The isolated plaques continue to stain positively with Congo red and the sulphated alclan blue techniques. Solubillzatlon of these plaques in guanldlne thlocyanate was followed by their dialysis and lyophillzatlon. Samples were subjected to electrophoresls in SDS polyacrylamlde gels followed by Western blotting with polyelonal antls1~ra prepared against the purified basement membrane HSPG of EHS tumors. The results revealed that basement membrane HSPG is part of the Alzhelmer'a plaque. Pre-treatment of these samples with nitrous acid, followed by eleetrophoreals and Western blotting, reduced the molecular weight of the antigen derived from these plaques, providing further evidence that the antigen is a form of HSPG. HSPG association with two fundamentally different forms of amylold and the fact that basement membrane HSPG is an extremely large molecule (greater than 400 KDa) and has never been shown to be present in the circulation, suggests that the HSPG molecule is synthesized locally and plays a role in the development of the characteristic amylold lesions seen in the brains of Alzhelmer's and Down's syndrome patients. Supported by MRC Grants MT-3153 and MA-I0477, and the Upj ohn Company.
211 EARLY ACCL~KK~TION OF HEP~RAIN SULFATE IN N~JRONS AND IN THE BEI"AKt{~/LOID PROTEIN CONTAINING LESIONS OF ALZHEIP~R'S D I S K ~ AND DONN'S ~ . "A.D. Snow, H. Mar, D. Nochlin, R.T. Seki~nJchi, K. Kirnsta, Y. Koike, T.N. Wight. University of washir~on, Seattle, WA 98195 and Aich/ Medical University, Aich/, Japan. A mcnoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha l-ap4 glucuronic acid-co~tain/ng determinant in heparan sulfate (HS) chains of a baselrellt-mmmbrane derived heparan sulfate proteoglycan (HSPG), identified and immunolocalized HS specifically to the alwloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle bearing neuror~, in the brains of Alzheimer's and Down's syndrome (DS) patiants. Ultrastructural immunohistochemistry demmlstrated that HS within neurons of Alzheirner's disease (AD) brain was localized to lipofuscin granules, an aging p i ~ t previously shown to also contain beta-m~yloid protein (BAP) (Bancher et al, 1988). Heparan sulfate was also localized to neurite-containing, non-fibrillar "primitive" plaques which also dml~nstrated positive BAP ir0munoreactivity in both AD and DS brain. Antibodies to im~inin, fibronectin and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurcrm of the h i ~ and am~jdala, as early as 1 day after birth. Yotmg age-matched controls did not demonstrate similar positive HS immm%oreactivity in neurons whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest depositiom of BAP was first observed as "~morphous" or "diffuse" cortical deposits in DS brain in patients aged 18 and 24, prior to the accumulation of fibrillar am~loid (o~served in DS patients 35 years and older). These cortical deposits also contained positive-HS immmloreaetivity in, lying that HS accumulation in conjmaction with the BAP is an eat:Iv event that may ultimately contribute to the early age-related a ~ l a t i o n (ie. as early as 35 years of age in DS) of NPs, NFTs and/or CA. Furthermore, the co-localization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may he important in lesion development in these two diseases.
212 EXPRESSION OF ALZHE1MER'S DISEASEAMYLOIDPRECURSOR PROTEIN DURINGPOST-NATAL DEVELOPMENTOF RAT BRAIN. *E. Milward 1, R. Martins 1, B. Rumble 1, R. Moir 1, A. Weidemann 2, K. Beyreuther 2, C. Masters 1. Department of Pathology, University of Melbourne and Mental Health Institute of Victoria, Royal Park Hospital, Parkville, Vie., 3052, Australia 1 and Center for Molecular Biology, University of Heidelberg, Heidelberg, FRG 2. The major protein component ([3A4) of the amyloid plaques that characterize Alzheimer's disease comes from a larger amyloid precursor protein (APP). Levels of APP during post-natal development of rat brain were measured by separate radioimmunoassays using antibodies specifically directed against either the amino- or the carboxyl-terminal of APP. Both assays revealed an overall increase in APP levels of 1.5 to 2-fold between birth and weaning. Adult levels were invariably lower than levels at weaning although the size of the decrease varied considerably between experiments. A minor, transient, perinatal increase in the APP level sometimes occurred. Western analysis of membrane-associated forms revealed three distinct complexes. A 100-110kD complex was detected at essentially constant levels at all ages examined during post-natal development and in the adult. In contrast, a 65-75 kD complex occurred at levels that were relatively low in the neonate but increased markedly during the second week. Although high levels were maintained until at least post-natal day 24, the complex was not detected in the young adult. A 50-60 kD complex was barely detectable during the first post-natal week but increased substantially after the second post-natal week. This complex was also maintained until at least post-natal day 24 but was absent in the adult. Northern analysis revealed a major 3.4-3.6 kb APP mRNA species and a minor t.8 kb form. No differences in their respective patterns of expression were apparent. Total APP mRNA, standardized relative to levels of glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) mRNA, increased by up to 2-fold between birth and weaning. A transient, perinatal increase in APP mRNA levels sometimes occurred. Adult levels of APP mRNA were similar to, or slightly higher than, levels at weaning. In summary, data suggest that expression of APP can be regulated at a post-transcriptional level. Furthermore, expression of different forms of APP may be differentially regulated, both during development and in the adult.
213 INDIANA lgll~RRn OF G_RlllCrM&~.ffrl~uJguglg-~J~Lttqti~ Dli~..~Z: ISOL&TION OF LOW MOI.ZCL."LAR Wra~tft" PRO'r~IN I ~ O M AMYI.,OIDIq.,AQUE CORI~. * F. Tagliavinil~, F. Prellil, J. Ghisol, O. Bugiani% M.R, Farlow3, B. Ghetti3, B. ~al3gionel. 1 New York University Medical Center. New York. NY, USA;
2 Istituto Neurelcgico Carlo Bet~, Milano, Italy; 3 Indiana University School of Medicine, Indianapolis, IN, USA. The neuropathologic hallmark of Gerstmann-Str~mssierScheinker (GSS) disease is the deposition in cerebral and cerebellar gray structures of amyloid fibrils immlmoreactive to antisera s r l r m t the Prion Protein 27-30 (PiP 27-30), a major structural component of scrapie-associated fibrils. Our aim was to purify a n d immunochemical!y characterize proteins e x t r a , ted from amyloid plaque cores isolated from cerebral cortex a n d cerebellum
of two patients from the Indiana kindred of OSS disease. Amyloidrich preparations fractienated by gel filtration yielded two major peaks (i.e. the void volume and a peak of 10-12 kDa) and five minor peaks. These fractions were su~ected to SDS-PAGE and i m m n n o b l o t analysis u s i n g three po]ye.lonal a n t i s e r a raised againat P r P 2%30 purified from brains of scrapie-infected animals, a n a n t i s e r u m to a synthetic poptide corresponding to residues 89-108 of eDNA deduced h a m s t e r PrP, a n a n t i s e r u m to amyloid P component a n d n o r m a l rablmt sera, Innnunoblot analysis of these fractiens d e m o n s t r a t e d t h a t all the anti-PrP a n t i s e r a strongly labeled the 10-12 k D a band, the m a j o r constituent of the amytoid extract, a n d leas intensely the 27 a n d 20 kDa bands. The anti-P .c~ml]ponent a n t i s e r u m d ve~.y stained a b a n d of 28 kDa. O u r d a t a cate t h a t GSS amyloid fibrils contain two main proteins: P component, which is common to all types Of mnyloid, a n d a 10-12
kDa peptide reacting with anti-PrP antisera. Although the amino termlrm| sequence of the 10-12 kDa subunit is heterogeneous, the presence of Gly-Gln-Pro suggests that the amyloid fibril is a degradation product of the Prion Protein, most probably starting at the repetitive sequence region.