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321 Phosphorylation of Alzheimer's β-amyloid protein precursor — What is the physiological function of APP phosphorylation ?
FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE
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Effect of presenilin mutations on 6 A P P processing in presenilin transfected ceils M. Citron*, W. Xia, T.S. Diehl, M.B. Podlisny, R. Sherrington 1, C. Haass 2, p. Seubert3, P.Hyslop I and D.J. Selkoe Center for Neurologie Diseases and Harvard Medical School, Boston, MA, USA 1University of Toronto, Canada; 2Zentralinstitut ftir Seelisehe Gesundheit, Mannheim, Germany; 3Athena Neurosciences, USA Two highly homologous presenilin genes on chromosome 14 (PSi) and chromosome 1 (PS 2) were recently identified as the cause of the most common and aggressive forms of early onset familial Alzheimer's disease (FAD). The normal biological roles of the presenilins and the mechanism by which the FADassociated mutations exert their effect are unknown. However, a collaborative study (Scheuner et al., 1996) on primary fibroblasts and plasma from some of these FAD kindreds has suggested that FAD linked mutations in both PS 1 and PS2 lead to a specific increase in the production of A1342,the most pathogenic form of A13.Here, we analyze cell lines transfected with the wild type and mutant forms of both presenilin genes. Such cell lines are an essential tool for understanding the role of the presenilin genes and how their mutated forms lead to an increase in A1342production. We have raised several polyclonal antibodies specific for PSI and/or PS2. Using these antibodies we have initially characterized the presenilin proteins. In both cos and 293 human embryonic kidney ceils the presenilins were transiently overproduced from expression constructs and localized predominantly within the endoplasmic reticulum with lesser amounts in the Golgi and no detectable staining on the plasma membrane. By immunoprecipitation and Western blotting the presenilin proteins were identified as 43-50 kDa proteins, consistent with their predicted molecular weight. We did not observe significant changes in the quantity and size of the presenilin bands when some of the FAD-linked mutant constructs were expressed. We have also produced several cell lines stably transfeeted with both wild type or mutant 13APP and wild type or mutant presenilin genes. These lines generally show lower levels of presenilin expression than the transiently transfected cells. Using these cell lines, we are currently analyzing the effects of presenilin mutations on the production of A1340 and A1342 using end specific antibodies in ELISA and immunoprecipitation assays. We are also analyzing the other secreted 13APP metabolites as well as the membrane bound full-length gAPP and its C-terminal fragments. If these lines show the same effect as primary fibroblasts, we will use various 13APP and presenilin constructs to address directly the mechanism by which the mutations lead to increased A1342 production, e.g., by subtle changes in 13APP trafficking and proteolytic processing.
INHIBITORS OF CATHEPSIN D BLOCK [ 3 - S E C R E T A S E - L I K E CLEAVAGE IN HUMAN CELLS Chevallier1 N., Marambaud 1 P., Vizzavona2 I., Baur C.P3., Spillantini M 3, Fulcrand2 P., Martinez2 J., Goedert, M3 and Vincent 1 J.P.and Checlerl F*. 1. UPR411 CNRS, Sophia-Antipolis, France. 2. URA CNRS 1845, Montpellier, France. 3. MRC Molecular Biology, Cambridge, U.K. A13formation is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Aspl) of A13is liberated from the amyloid precursor protein (13APP)by 13-secretase(s). 13-secretase(s) aodvity(ies) appear(s) highly increased by the presence (N-terminally to residue 1 of A13)of a double mutation (KM .-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal" (N peptide) and mutated (aNL peptide) sequences targeted by 13secretase(s), we have detected a human brain protease displaying preferred affinity for the ANt. peptide than for the non mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PCI2 cells and primary cultured astrocytes. Interestingly, a series of novel inhibitors designed to block the cathepsin D (CD) catalytic activity displays IC50 values towards this purified enzyme that fully correlate with those observed on the ANL peptide-hydrolysing activity present in human brain membranes. Therefore, CD was examined as a putative }-seeretase-like candidate. CD displays higher catalytic parameters for the ANL peptide than for the non mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. By contrast, CD does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block AI3 secretion. Furthermore, CD hydrolyses recombinant baeuloviral td,~LI3APP751at a sixfold higher rate than 13APP751and gives rise to a 12 kDa Cterminal product that is recognized by antibodies fully specific of the Nterminus of A13.HK293 ceils overexpressing ANL13APP751secrete high amounts of A13and produce an intracellular 12 kDa fragment, the formation of which is drastically inhibited by the prior treatment of the ceils with CD inhibitors. Among other endosomal/lysosomal proteases, cathepsin B and G did not exhibit the 13-secretase-like specificity towards both synthetic peptides and recombinant ANL[3APP751. Finally, the two endopeptidases 3.4.24.11 and 3.4.24.15 do not cleave ANL13APP751, even after prolonged incubation times. Altogether, our study indicates that cathepsin D displays several in vitro 13secretase-like properties that suggest that this protease could fulfill such a role in human brain. Therefore, this enzyme could be considered as a potential therapeutic target of a strategy aimed at preventing A13 formation that occurs during neurodegenerative process associated with Alzheimer's disease.
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P h o s p h o r y l a t i o n o f A l z h e i m e r ' s I~--amylold protein precursor - What is the p h y s i o l o g i c a l function o f APP p h o s p h o r y l a t i o n ? Toshiharu Suzuki Laboratory of Neurobiophysics, Faculty of Pharmaceutical Sciences, University of Tokyo. Tokyo 113, Japan. In the history of research for Alzheimer d~sease (AD), an evaluation of APP phosphorylation study has changed often. Abnormalites of brain protein phosphorylation were expected to play primary roles in AD pathogenesis because the alterations of protein phosphorylation systems in AD were reported at the dawn of biochemical researchs of AD. Earliest studies demonstrated phosphorylation of cytoplasmic domain peptides from APP with protein kinase C (PKC) and soon it was demonstrated that APP is a phosphoprotein. Cell biological studies pointed out a possibility that APP phosphorylation through an aberrant regulation of PKC may be a cause of amyloidogenic processing of APP. Regardless of effort by researchers, a direct relationship of intracellularAPP phosphorylation by PKC has not beeen reported, thus phosphorylation of APP appeared to be of no significance fo~"',!'.~ercgu!uticn of~ a:nyloid pr~.Iuc.tie,l~,. However, a new phosphorylation site was identified through the phosphorylation study of APP. The site, Thr668 (numbering for [~APP~95isoform), was phosphorylated by ede2 kinase in a cell cycle-dependent manner. It has not been elucidated whether this novel regulation of APP phosphorylation has some significance on APP metabolism and/or biological function because the cdc2 kinase is not active in post-mitotic neurons. Recent development of phosphorylation statespecific antibody raised to phospho-Thr668 (AbpThr668) revealed that the phosphorylation of APP at Thr668 is a physiological and brain specific event. Immunohist~hemical studies utilizing AbpThr668 may suggest some role of APP phosphorylation in brain specific functions. Furthermore, a recent report for APP binding protein indicates an importance of the amino acid sequences including the phosphorylation site in APP cytoplasmic domain.
Regulation of AIzheimer B-Amyloid Protein Precursor Metabolism in Cell-free Systems. H. Xu .1, 2, D. Sweeney l , R. Wang 3, S. Sisodia4, P. Greengard 2, and S. Gandy 1. 1 Department of Neurology and Neuroscience, Cornell University Medical College, New York NY 10021; 2 Laboratory of Molecular and Cellular Neuroscience; and 3 Laboratory for Mass Spectrometry,. The Rockefeller University, New York NY 10021:4 Neuropathology Laboratory, The Johns Hopkins University, Baltimore, MD 21205 The relative utilization of alternative processing pathways for the 13amyloid protein precursor (13APP) can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A, leads to a relative increase in utilization of the nonamyloidogenic, "a-secretase" cleavage pathway for 13APP processing at the expense of other pathways. Recently, we have demonstrated in a reconstituted cell-free system that activation of endogenous PKC increases formation from the trans-Golgi network (TGN) of "constitutive" secretory vesicles containing 13APP and that this effect can be mimicked by purified PKC. The results demonstrated directly that PKC is involved in regulation of secretory vesicle formation, revealing a major mechanism by which 13APP metabolism is controlled. Unfortunately, from a therapeutic standpoint, PKC activation can stimulate 13APP transcription, indirectly increasing 13-amyloid (A13) formation. Since there are many examples wherein multiple protein kinases can exert similar regulatory effects, we tested whether protein kinase A (PKA) might also regulate 13APP metabolism. PKA-activating reagents increased sBAPP secretion from intact PC12 cells and, by using an in vitro reconstitution system, purified PKA also stimulated formation of 13APP-containing vesicles from the TGN. Although PKA and PKC converge at the level of formation from the TGN of BAPP-containing vesicles, additional evidence (e.g., sensitivity to GTP~/S or okadaic acid) indicates that the regulatory mechanisms involved are distinct. In addition, we have successfully reconstituted AI3 formation using a cell-free system previously developed to reconstitute proteolytic cleavage of prohormones. This cell-free system can enable us to dissect as separate events, either A13 generation or the formation of nascent TGN vesicles containing AB or BAPP. The detailed characterization of this cellfree system will be presented.
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Effect of presenilin mutations on 6 A P P processing in presenilin transfected ceils M. Citron*, W. Xia, T.S. Diehl, M.B. Podlisny, R. Sherrington 1, C. Haass 2, p. Seubert3, P.Hyslop I and D.J. Selkoe Center for Neurologie Diseases and Harvard Medical School, Boston, MA, USA 1University of Toronto, Canada; 2Zentralinstitut ftir Seelisehe Gesundheit, Mannheim, Germany; 3Athena Neurosciences, USA Two highly homologous presenilin genes on chromosome 14 (PSi) and chromosome 1 (PS 2) were recently identified as the cause of the most common and aggressive forms of early onset familial Alzheimer's disease (FAD). The normal biological roles of the presenilins and the mechanism by which the FADassociated mutations exert their effect are unknown. However, a collaborative study (Scheuner et al., 1996) on primary fibroblasts and plasma from some of these FAD kindreds has suggested that FAD linked mutations in both PS 1 and PS2 lead to a specific increase in the production of A1342,the most pathogenic form of A13.Here, we analyze cell lines transfected with the wild type and mutant forms of both presenilin genes. Such cell lines are an essential tool for understanding the role of the presenilin genes and how their mutated forms lead to an increase in A1342production. We have raised several polyclonal antibodies specific for PSI and/or PS2. Using these antibodies we have initially characterized the presenilin proteins. In both cos and 293 human embryonic kidney ceils the presenilins were transiently overproduced from expression constructs and localized predominantly within the endoplasmic reticulum with lesser amounts in the Golgi and no detectable staining on the plasma membrane. By immunoprecipitation and Western blotting the presenilin proteins were identified as 43-50 kDa proteins, consistent with their predicted molecular weight. We did not observe significant changes in the quantity and size of the presenilin bands when some of the FAD-linked mutant constructs were expressed. We have also produced several cell lines stably transfeeted with both wild type or mutant 13APP and wild type or mutant presenilin genes. These lines generally show lower levels of presenilin expression than the transiently transfected cells. Using these cell lines, we are currently analyzing the effects of presenilin mutations on the production of A1340 and A1342 using end specific antibodies in ELISA and immunoprecipitation assays. We are also analyzing the other secreted 13APP metabolites as well as the membrane bound full-length gAPP and its C-terminal fragments. If these lines show the same effect as primary fibroblasts, we will use various 13APP and presenilin constructs to address directly the mechanism by which the mutations lead to increased A1342 production, e.g., by subtle changes in 13APP trafficking and proteolytic processing.
INHIBITORS OF CATHEPSIN D BLOCK [ 3 - S E C R E T A S E - L I K E CLEAVAGE IN HUMAN CELLS Chevallier1 N., Marambaud 1 P., Vizzavona2 I., Baur C.P3., Spillantini M 3, Fulcrand2 P., Martinez2 J., Goedert, M3 and Vincent 1 J.P.and Checlerl F*. 1. UPR411 CNRS, Sophia-Antipolis, France. 2. URA CNRS 1845, Montpellier, France. 3. MRC Molecular Biology, Cambridge, U.K. A13formation is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Aspl) of A13is liberated from the amyloid precursor protein (13APP)by 13-secretase(s). 13-secretase(s) aodvity(ies) appear(s) highly increased by the presence (N-terminally to residue 1 of A13)of a double mutation (KM .-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal" (N peptide) and mutated (aNL peptide) sequences targeted by 13secretase(s), we have detected a human brain protease displaying preferred affinity for the ANt. peptide than for the non mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PCI2 cells and primary cultured astrocytes. Interestingly, a series of novel inhibitors designed to block the cathepsin D (CD) catalytic activity displays IC50 values towards this purified enzyme that fully correlate with those observed on the ANL peptide-hydrolysing activity present in human brain membranes. Therefore, CD was examined as a putative }-seeretase-like candidate. CD displays higher catalytic parameters for the ANL peptide than for the non mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. By contrast, CD does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block AI3 secretion. Furthermore, CD hydrolyses recombinant baeuloviral td,~LI3APP751at a sixfold higher rate than 13APP751and gives rise to a 12 kDa Cterminal product that is recognized by antibodies fully specific of the Nterminus of A13.HK293 ceils overexpressing ANL13APP751secrete high amounts of A13and produce an intracellular 12 kDa fragment, the formation of which is drastically inhibited by the prior treatment of the ceils with CD inhibitors. Among other endosomal/lysosomal proteases, cathepsin B and G did not exhibit the 13-secretase-like specificity towards both synthetic peptides and recombinant ANL[3APP751. Finally, the two endopeptidases 3.4.24.11 and 3.4.24.15 do not cleave ANL13APP751, even after prolonged incubation times. Altogether, our study indicates that cathepsin D displays several in vitro 13secretase-like properties that suggest that this protease could fulfill such a role in human brain. Therefore, this enzyme could be considered as a potential therapeutic target of a strategy aimed at preventing A13 formation that occurs during neurodegenerative process associated with Alzheimer's disease.
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P h o s p h o r y l a t i o n o f A l z h e i m e r ' s I~--amylold protein precursor - What is the p h y s i o l o g i c a l function o f APP p h o s p h o r y l a t i o n ? Toshiharu Suzuki Laboratory of Neurobiophysics, Faculty of Pharmaceutical Sciences, University of Tokyo. Tokyo 113, Japan. In the history of research for Alzheimer d~sease (AD), an evaluation of APP phosphorylation study has changed often. Abnormalites of brain protein phosphorylation were expected to play primary roles in AD pathogenesis because the alterations of protein phosphorylation systems in AD were reported at the dawn of biochemical researchs of AD. Earliest studies demonstrated phosphorylation of cytoplasmic domain peptides from APP with protein kinase C (PKC) and soon it was demonstrated that APP is a phosphoprotein. Cell biological studies pointed out a possibility that APP phosphorylation through an aberrant regulation of PKC may be a cause of amyloidogenic processing of APP. Regardless of effort by researchers, a direct relationship of intracellularAPP phosphorylation by PKC has not beeen reported, thus phosphorylation of APP appeared to be of no significance fo~"',!'.~ercgu!uticn of~ a:nyloid pr~.Iuc.tie,l~,. However, a new phosphorylation site was identified through the phosphorylation study of APP. The site, Thr668 (numbering for [~APP~95isoform), was phosphorylated by ede2 kinase in a cell cycle-dependent manner. It has not been elucidated whether this novel regulation of APP phosphorylation has some significance on APP metabolism and/or biological function because the cdc2 kinase is not active in post-mitotic neurons. Recent development of phosphorylation statespecific antibody raised to phospho-Thr668 (AbpThr668) revealed that the phosphorylation of APP at Thr668 is a physiological and brain specific event. Immunohist~hemical studies utilizing AbpThr668 may suggest some role of APP phosphorylation in brain specific functions. Furthermore, a recent report for APP binding protein indicates an importance of the amino acid sequences including the phosphorylation site in APP cytoplasmic domain.
Regulation of AIzheimer B-Amyloid Protein Precursor Metabolism in Cell-free Systems. H. Xu .1, 2, D. Sweeney l , R. Wang 3, S. Sisodia4, P. Greengard 2, and S. Gandy 1. 1 Department of Neurology and Neuroscience, Cornell University Medical College, New York NY 10021; 2 Laboratory of Molecular and Cellular Neuroscience; and 3 Laboratory for Mass Spectrometry,. The Rockefeller University, New York NY 10021:4 Neuropathology Laboratory, The Johns Hopkins University, Baltimore, MD 21205 The relative utilization of alternative processing pathways for the 13amyloid protein precursor (13APP) can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A, leads to a relative increase in utilization of the nonamyloidogenic, "a-secretase" cleavage pathway for 13APP processing at the expense of other pathways. Recently, we have demonstrated in a reconstituted cell-free system that activation of endogenous PKC increases formation from the trans-Golgi network (TGN) of "constitutive" secretory vesicles containing 13APP and that this effect can be mimicked by purified PKC. The results demonstrated directly that PKC is involved in regulation of secretory vesicle formation, revealing a major mechanism by which 13APP metabolism is controlled. Unfortunately, from a therapeutic standpoint, PKC activation can stimulate 13APP transcription, indirectly increasing 13-amyloid (A13) formation. Since there are many examples wherein multiple protein kinases can exert similar regulatory effects, we tested whether protein kinase A (PKA) might also regulate 13APP metabolism. PKA-activating reagents increased sBAPP secretion from intact PC12 cells and, by using an in vitro reconstitution system, purified PKA also stimulated formation of 13APP-containing vesicles from the TGN. Although PKA and PKC converge at the level of formation from the TGN of BAPP-containing vesicles, additional evidence (e.g., sensitivity to GTP~/S or okadaic acid) indicates that the regulatory mechanisms involved are distinct. In addition, we have successfully reconstituted AI3 formation using a cell-free system previously developed to reconstitute proteolytic cleavage of prohormones. This cell-free system can enable us to dissect as separate events, either A13 generation or the formation of nascent TGN vesicles containing AB or BAPP. The detailed characterization of this cellfree system will be presented.