- Email: [email protected]
Alzheimer protein precursors: Tyrosine phosphorylation of the cytoplasmic domain
284
N E U R O B I O L O G Y OF A G I N G , V O L U M E l i, i990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE MECHANISMS OF NEURONAL DEGENERATION t r e a t e d PBFs w i t h o u t f u r t h e r t r e a t m e n t , i n c l u d i n g m o n o c l o n a l antibodies 423, SMI31 and SMI33, and polyclonal antibody Alz5 to the C terminus of tau. Antibodies with epitopes N terminal to the tubulln binding region did not react, including 5E2, tau-1 and polyclonal antibodies to the N-terminal inserts in human tau (BR89 and BR92) and to amino acids 16-26 (AI). This suggests these epitopes were removed by pronase. Two other tau antibodies, 795 and 751, failed to react unless the PHFs were first disrupted with 2% formic acid, 2M guanidine-HCl or 8M urea. The corresponding epitopes may be unmasked or modified to a recognizable form by these agents. Tau reactivity with antl-tau antibodies was recovered in the soluble fraction after PBFs were treated with formic acid or guanidine and spun for 3h at 185,000g. Urea supernatants did not react. On PAGE gels peptides with reactivity to antibodies 423,751 and 795 migrated separately from material with reactivity to either SM131 or Alz 5, suggesting that the corresponding epltopes are present in different peptides after pronase treatment. In conclusion, C terminal sequences of tau beyond the tubulln binding region may bind to PHF core and are recovered in separate peptides, but N terminal sequences do not appear to be associated with the PHF.
128 DIFFERENTIAL DISTRIBUTION OF LACTOFERRIN AND ALZ-5O IMMUNOREACTIVlTIES IN NEURITIC PLA~UES AND NEUROFIBRI~Y TANGLES IN ALZHEIMER'S DISEASE (AD). "A.P. Osmand, R.C. Swltzer. University of Tennessee Medical Center, Knoxville, Tennessee, 37920 USA. The selective presence of lactoferrin (LF) l~munoreactivity in the neuritic plaques (NPs) and neuroflbrlllary tangles (NFTs) in the brain tissue of AD patients has been previously described from this laboratory. Initial studies demonstrated a highly variable degree of immunoreactlvlty, largely restricted to the lesions present in medial temporal cortex, the strongest reactivity being seen in the hlppoeampal formation, subiculum, amygdala, and entorhlnal cortex. The apparent absence of LF immunoreactivty from NPs in certain regions of cortex was taken to indicate that this component was not essential to the pathogenesis of NPs or NFTs. These studies have been extended to demonstrate the invariable presence of LF immunoreactivlty in over 55 cases of AD; the use of higher affinity antlsera, more sensitive immunohlstochemlcal enzyme reagents (e.g the 'Elite' ABC reagent from Vector Laboratories) and substrates (nickel enhanced dlamlnobenzidine), has indicated a wider distribution of immunoreactivity than previously appreciated. The distribution of LF reactivity within plaques and NFTs has been further found to be distinct from Alz-50 immunoreactivity, the latter being restricted to NFTs, a small number of cortical neurons, and to neuritic processes both within NPs and throughout the involved cortical neuropil. In contrast 12 reactivity was associated with intra- and extra-cellular NFTs and showed variously both a globular and a granular appearance within NPs. This distribution could also be distinguished from amyloid E-protein immunoreactlvity and from ar~yrophilla. The uniform absence of lactoferrln from normal brain tissue suggests a unique involvement in AD; the presence of high concentrations of LF in nasal mucosa has led us to conclude that the protein may have been transported from the olfactory mucosa and, due to disruptions of normal proteolytlc mechanisms, accumulated in the cortical and subeortical lesions characteristic of this disease. Supported by grants from the NIH (NINCDS), the Alzhelmer's Research Program of the American Health Assistance Foundation, and the Robert H. and Monlca Cole Foundation.
129 A L Z - 5 0 LMMUNOREACTIVITY IN THE HIPPOCAMPAL COMPLEX OF N O R M A L E L D E R L Y A N D ALZHEIMER'S DISEASED BRAINS. *D. R. Brady and E. J. Mufson. Inst. for Biogerontology Res., P.O. Box 1278, Sun City, AZ 85372. AIz-50, a putative marker for neurodegeneration, topographically labels neurons, neurites and neurofibrillary tangles (NFT) throughout the hippocampal complex (HPC) in Alzheimer's disease (AD). Prior studies have focused mainly on the location of Alz-50 in restricted portions of the HPC, with virtually no detailed investigations on this antigenic response over the full extent of this complex in aged controls and AD. The present investigation details the topographic and intracellular distribution of Alz-50 in the HPC from postmortem tissue (4.8 +_ 0.4 h) of 19 AD (79.9 +_ 1.7 years) and 13 control (74.9 + 3.2 years)
brains. In 10 of the controls, virtually all Alz-50 immunoreactivity was restricted to the HPC, revealing Golgi-like staining of numerous neurons in C A I , CA2 and occasionally, granule cells of the dentate gyrus. In these aged controls with abundant Alz-50 neuronal staining, visible neuropil labeling was observed in the subiculum, strata moleculare and oriens of CAI and CA2, and layer 4 of the entorhinal cortex, Some Alz-50 containing profiles also double-labeled with thioflavin S, indicating NFTs. In the other 3 controls, only a few AIz-50 labeled cells were seen without concomitant neuropil staining in the HPC. In contrast, profuse Alz-5O staining of neurons and neuropil was seen in AD brains, providing clear differentiation of HPC architectonic subfields. Generally, Alz-50 positive neurons appeared dystrophic with dense aggregates of label restricted to its cell body and proximal dendrites. While the appearance of the immunoreactivity was consistent across subfields, the intracellular distribution ranged from a Golgi-like appearance in CA4 to a thin rim of cytoplasmic labeling around the nuclear outline in the subiculum. Although heavier, The pattern of neuropil labeling seen in AD was similar to controls. Ultrastructurally, Alz-50 labeled paired helical filaments, straight filaments and an amorphous substance in the soma. In the CAI stratum moleculare, bundles of labeled filaments were observed in distal dendritic, myelinated axonal and presynaptic compartments. These results demonstrate a focal population of hippocampal neurons that express Alz-50 antigen in the normal elderly and AD brain. Although some of these labeled neurons coincidently contain NFTs, the results cannot rule out the expression of Alz-50 as a consequence of normal neuronal degeneration in the aged brain. Furthermore, the inverse relationship between decreased dendritic arbors and increased neuropil staining in AD brains may reflect a discontinuity between the neuron and its distal dendrites and axon. (Support: ADCRC #82-0686, NIH #26146 and AHAF). 130
ALZHEIMER PROTEIN PRECUR~RS: PHOSPIIORYLATION
OF THE
TYROSINE CYTOPLASMIC DOMAIN
*Dirk Bunk¢, lRobert M. Kypta, Ursula M0nning, l S a r a h A. Courmeidg¢, 2Colin L. Masters and Konrad T. Beyreuther Center for Molecular Biology, University of Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-6900 Heidelberg 1, FRG lEuropean Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRO 2Department of Pathology, University of Melbourne, Parkville, Victoria 3052. Australia. Depositions of amyloid A4 protein are one characteristic feature of Alzheimer's Disease. This protein is derived from t r a n s m v m b r a n e p r e c u r s o r proteins w h i c h c a r r y a c y t o p l a s m i c domain. We started an a n a l y s i s o f the p o s t t r a n s l a t i o n a l modifications of this e n d o d o m a i n with a synthetic c y t o p l a s m i c domain in order to improve our understanding of the physiological functions of these precursors. Using the protein tyrosinc kinase pp60V'src we find the synthetic peptide to be s p e c i f i c a l l y p h o s p h o r y l a t c d on t y r o s i n ¢ 682 ( n u m b e r i n g a c c o r d i n g to A P P 6 9 5 ) . The Km-valu¢ of this reaction is o f the same order of magnitude as that of the major phosphory!ation site of pp60 v - s r c , which is p h o s p h o r y l a t e d on t y r o s i n ¢ u n d e r p h y s i o l o g i c a l conditions. Tyrosine 682 is flanked by a total of eight amino acids which are homologous to the putative cytoplasmic domain of the D r o s o p h i l a ventral nervous system c o n d e n s a t i o n defective (end) gene transcript. The end geoc region was shown to be essential for the c o n d e n s a t i o n of the nervous system of the fruit fly. According to our results, the Alzheimer precursor proteins contain an evolutionary conserved functional accepter site for p h o s p h o r y l a t i o n through protein tyrosin¢ kinases. Since tyroaine phosphorylation in c n d o d o m a i n s is a s i g n a l w h i c h m a y be involved in s i g n a l t r a n s d u c t i o n and r e c e p t o r r e c y c l i n g , our findings s u g g e s t t h a t the a m y t o i d p r e c u r s o r p r o t e i n s of A l z h e i m e r ' s Disease may participate in signal transduction or may u n d e r g o an e n d o g e n o u s enzymatic regulation.
131
THE NEURONALANTIGENA68 RECOGNIZEDBY ALZ-50 IN AD BRAINS IS AN ABNORMALLYPHOSPHORYLATEDTAU PROTEIN. S.Flament (1), *L.Buee (1-2), A.Delacourte ( i ) . (I) Lab.INSERM of Neurosciences, Faculte de Medecine de L i l l e , 59045 FRANCE. (2) The Mount Sinai Medical Center, Department!of Geriatrics and Adult Development, New York, NY, 10029-6574 USA.
N E U R O B I O L O G Y OF A G I N G , V O L U M E l i, i990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE MECHANISMS OF NEURONAL DEGENERATION t r e a t e d PBFs w i t h o u t f u r t h e r t r e a t m e n t , i n c l u d i n g m o n o c l o n a l antibodies 423, SMI31 and SMI33, and polyclonal antibody Alz5 to the C terminus of tau. Antibodies with epitopes N terminal to the tubulln binding region did not react, including 5E2, tau-1 and polyclonal antibodies to the N-terminal inserts in human tau (BR89 and BR92) and to amino acids 16-26 (AI). This suggests these epitopes were removed by pronase. Two other tau antibodies, 795 and 751, failed to react unless the PHFs were first disrupted with 2% formic acid, 2M guanidine-HCl or 8M urea. The corresponding epitopes may be unmasked or modified to a recognizable form by these agents. Tau reactivity with antl-tau antibodies was recovered in the soluble fraction after PBFs were treated with formic acid or guanidine and spun for 3h at 185,000g. Urea supernatants did not react. On PAGE gels peptides with reactivity to antibodies 423,751 and 795 migrated separately from material with reactivity to either SM131 or Alz 5, suggesting that the corresponding epltopes are present in different peptides after pronase treatment. In conclusion, C terminal sequences of tau beyond the tubulln binding region may bind to PHF core and are recovered in separate peptides, but N terminal sequences do not appear to be associated with the PHF.
128 DIFFERENTIAL DISTRIBUTION OF LACTOFERRIN AND ALZ-5O IMMUNOREACTIVlTIES IN NEURITIC PLA~UES AND NEUROFIBRI~Y TANGLES IN ALZHEIMER'S DISEASE (AD). "A.P. Osmand, R.C. Swltzer. University of Tennessee Medical Center, Knoxville, Tennessee, 37920 USA. The selective presence of lactoferrin (LF) l~munoreactivity in the neuritic plaques (NPs) and neuroflbrlllary tangles (NFTs) in the brain tissue of AD patients has been previously described from this laboratory. Initial studies demonstrated a highly variable degree of immunoreactlvlty, largely restricted to the lesions present in medial temporal cortex, the strongest reactivity being seen in the hlppoeampal formation, subiculum, amygdala, and entorhlnal cortex. The apparent absence of LF immunoreactivty from NPs in certain regions of cortex was taken to indicate that this component was not essential to the pathogenesis of NPs or NFTs. These studies have been extended to demonstrate the invariable presence of LF immunoreactivlty in over 55 cases of AD; the use of higher affinity antlsera, more sensitive immunohlstochemlcal enzyme reagents (e.g the 'Elite' ABC reagent from Vector Laboratories) and substrates (nickel enhanced dlamlnobenzidine), has indicated a wider distribution of immunoreactivity than previously appreciated. The distribution of LF reactivity within plaques and NFTs has been further found to be distinct from Alz-50 immunoreactivity, the latter being restricted to NFTs, a small number of cortical neurons, and to neuritic processes both within NPs and throughout the involved cortical neuropil. In contrast 12 reactivity was associated with intra- and extra-cellular NFTs and showed variously both a globular and a granular appearance within NPs. This distribution could also be distinguished from amyloid E-protein immunoreactlvity and from ar~yrophilla. The uniform absence of lactoferrln from normal brain tissue suggests a unique involvement in AD; the presence of high concentrations of LF in nasal mucosa has led us to conclude that the protein may have been transported from the olfactory mucosa and, due to disruptions of normal proteolytlc mechanisms, accumulated in the cortical and subeortical lesions characteristic of this disease. Supported by grants from the NIH (NINCDS), the Alzhelmer's Research Program of the American Health Assistance Foundation, and the Robert H. and Monlca Cole Foundation.
129 A L Z - 5 0 LMMUNOREACTIVITY IN THE HIPPOCAMPAL COMPLEX OF N O R M A L E L D E R L Y A N D ALZHEIMER'S DISEASED BRAINS. *D. R. Brady and E. J. Mufson. Inst. for Biogerontology Res., P.O. Box 1278, Sun City, AZ 85372. AIz-50, a putative marker for neurodegeneration, topographically labels neurons, neurites and neurofibrillary tangles (NFT) throughout the hippocampal complex (HPC) in Alzheimer's disease (AD). Prior studies have focused mainly on the location of Alz-50 in restricted portions of the HPC, with virtually no detailed investigations on this antigenic response over the full extent of this complex in aged controls and AD. The present investigation details the topographic and intracellular distribution of Alz-50 in the HPC from postmortem tissue (4.8 +_ 0.4 h) of 19 AD (79.9 +_ 1.7 years) and 13 control (74.9 + 3.2 years)
brains. In 10 of the controls, virtually all Alz-50 immunoreactivity was restricted to the HPC, revealing Golgi-like staining of numerous neurons in C A I , CA2 and occasionally, granule cells of the dentate gyrus. In these aged controls with abundant Alz-50 neuronal staining, visible neuropil labeling was observed in the subiculum, strata moleculare and oriens of CAI and CA2, and layer 4 of the entorhinal cortex, Some Alz-50 containing profiles also double-labeled with thioflavin S, indicating NFTs. In the other 3 controls, only a few AIz-50 labeled cells were seen without concomitant neuropil staining in the HPC. In contrast, profuse Alz-5O staining of neurons and neuropil was seen in AD brains, providing clear differentiation of HPC architectonic subfields. Generally, Alz-50 positive neurons appeared dystrophic with dense aggregates of label restricted to its cell body and proximal dendrites. While the appearance of the immunoreactivity was consistent across subfields, the intracellular distribution ranged from a Golgi-like appearance in CA4 to a thin rim of cytoplasmic labeling around the nuclear outline in the subiculum. Although heavier, The pattern of neuropil labeling seen in AD was similar to controls. Ultrastructurally, Alz-50 labeled paired helical filaments, straight filaments and an amorphous substance in the soma. In the CAI stratum moleculare, bundles of labeled filaments were observed in distal dendritic, myelinated axonal and presynaptic compartments. These results demonstrate a focal population of hippocampal neurons that express Alz-50 antigen in the normal elderly and AD brain. Although some of these labeled neurons coincidently contain NFTs, the results cannot rule out the expression of Alz-50 as a consequence of normal neuronal degeneration in the aged brain. Furthermore, the inverse relationship between decreased dendritic arbors and increased neuropil staining in AD brains may reflect a discontinuity between the neuron and its distal dendrites and axon. (Support: ADCRC #82-0686, NIH #26146 and AHAF). 130
ALZHEIMER PROTEIN PRECUR~RS: PHOSPIIORYLATION
OF THE
TYROSINE CYTOPLASMIC DOMAIN
*Dirk Bunk¢, lRobert M. Kypta, Ursula M0nning, l S a r a h A. Courmeidg¢, 2Colin L. Masters and Konrad T. Beyreuther Center for Molecular Biology, University of Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-6900 Heidelberg 1, FRG lEuropean Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRO 2Department of Pathology, University of Melbourne, Parkville, Victoria 3052. Australia. Depositions of amyloid A4 protein are one characteristic feature of Alzheimer's Disease. This protein is derived from t r a n s m v m b r a n e p r e c u r s o r proteins w h i c h c a r r y a c y t o p l a s m i c domain. We started an a n a l y s i s o f the p o s t t r a n s l a t i o n a l modifications of this e n d o d o m a i n with a synthetic c y t o p l a s m i c domain in order to improve our understanding of the physiological functions of these precursors. Using the protein tyrosinc kinase pp60V'src we find the synthetic peptide to be s p e c i f i c a l l y p h o s p h o r y l a t c d on t y r o s i n ¢ 682 ( n u m b e r i n g a c c o r d i n g to A P P 6 9 5 ) . The Km-valu¢ of this reaction is o f the same order of magnitude as that of the major phosphory!ation site of pp60 v - s r c , which is p h o s p h o r y l a t e d on t y r o s i n ¢ u n d e r p h y s i o l o g i c a l conditions. Tyrosine 682 is flanked by a total of eight amino acids which are homologous to the putative cytoplasmic domain of the D r o s o p h i l a ventral nervous system c o n d e n s a t i o n defective (end) gene transcript. The end geoc region was shown to be essential for the c o n d e n s a t i o n of the nervous system of the fruit fly. According to our results, the Alzheimer precursor proteins contain an evolutionary conserved functional accepter site for p h o s p h o r y l a t i o n through protein tyrosin¢ kinases. Since tyroaine phosphorylation in c n d o d o m a i n s is a s i g n a l w h i c h m a y be involved in s i g n a l t r a n s d u c t i o n and r e c e p t o r r e c y c l i n g , our findings s u g g e s t t h a t the a m y t o i d p r e c u r s o r p r o t e i n s of A l z h e i m e r ' s Disease may participate in signal transduction or may u n d e r g o an e n d o g e n o u s enzymatic regulation.
131
THE NEURONALANTIGENA68 RECOGNIZEDBY ALZ-50 IN AD BRAINS IS AN ABNORMALLYPHOSPHORYLATEDTAU PROTEIN. S.Flament (1), *L.Buee (1-2), A.Delacourte ( i ) . (I) Lab.INSERM of Neurosciences, Faculte de Medecine de L i l l e , 59045 FRANCE. (2) The Mount Sinai Medical Center, Department!of Geriatrics and Adult Development, New York, NY, 10029-6574 USA.