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A cell-based model for presenilin 1 early-onset familial Alzheimer's disease (EOFAD): Dominant negative effects and relative stabilities of presenilin 1 with EOFAD mutations
P242
Oral Sessions: O2-04: Cellular and Molecular Mechanisms: Amyloid I
erlin-2, that resides in DRMs and affects Ab production with less effect on Notch processing. O2-04-04
THE METALLOPROTEASE MEPRIN BETA GENERATES AMINO-TERMINALLY TRUNCATED BETA-AMYLOID PEPTIDE SPECIES
Jessica Bien1, Tamara Jefferson2, Mirsada Causevic1, Thorsten Jumpertz3, Lisa M. Munter4, Gerd Multhaup4, Sascha Weggen3, Christoph BeckerPauly2, Claus Pietrzik1, 1University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany; 2Christian-AlbrechtsUniversity Kiel, Kiel, Germany; 3Heinrich-Heine University, Dusseldorf, Germany; 4Free University, Berlin, Germany. Background: The amyloid b (Ab) peptide, which is abundantly found in the brains of patients suffering from Alzheimer’s disease is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin b cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates, but not in meprin b-/- mouse brain lysates. Methods: Based on a novel proteomics technique termed TAILS (Terminal Amine Isotopic Labeling of Substrates), APP was identified as a substrate for meprin b. Subsequent analysis of APP processing using ELISA, SDS-PAGE, and mass spectrometry revealed cleavage events by meprin b close to the regular BACE1 cleavage site in APP. Results: In this work, we present evidence that meprin b can also process APP in a manner reminiscent of b-secretase. We identified cleavage sites of meprin b in the amyloid b sequence of the wild type and Swedish mutant of APP at positions p1, p2, and p3, thereby generating Ab variants starting at the first, second or third amino acid residue. Importantly, we provide strong evidence that meprin b produces an Ab peptide starting with the glutamate in the third position, indicating that this protease might produce an amino-terminally truncated Ab species. We observed even higher kinetic values for meprin b than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin b on APP and Ab generation was also observed in the absence of BACE1 and 2, indicating that meprin b acts independently of b-secretase. Conclusions: Our data indicate that meprin b is an enzyme capable of cleaving APP in a b-secretase like manner, probably also at position 674 (Abp3) to expose a glutamate residue, which might be subsequently modified by a glutaminylcyclase to generate pyroglutamate-Ab peptides. Although BACE1 acts as the major b-secretase in vivo generating most of the Ab 1-40/42 peptides, we suggest that meprin b might act as one enzyme responsible for the release of N-terminal truncated Ab species. O2-04-05
A CELL-BASED MODEL FOR PRESENILIN 1 EARLY-ONSET FAMILIAL ALZHEIMER’S DISEASE (EOFAD): DOMINANT NEGATIVE EFFECTS AND RELATIVE STABILITIES OF PRESENILIN 1 WITH EOFAD MUTATIONS
Anthony Stevens1, Carolyn Collins1, Evan Katz1, Gary Lee1, Vernon Alford2, Hadis Williams1, Darlene Guillen2, Xiaoyang Wu1, John Flanagan1, Eric Sjoberg1, Sam Gandy3, David J. Lockhart1, Brandon Wustman1, LB Dungan1, 1Amicus Therapeutics, Cranbury, New Jersey, United States; 2Amicus Therapeutics, La Jolla, California, United States; 3Mount Sinai, New York City, New York, United States. Background: The most frequent cause of Early-onset familial Alzheimer’s disease (EOFAD) is the autosomal dominant inheritance of a missense mutation in the presenilin 1 (PS1) gene (PSEN). Since EOFAD results from a mutation in a single allele of PSEN, both wild type and the PSEN variant gene products are co-expressed. The potential for the mutant PS1 protein to affect the function of wild-type PS1 (e.g., a dominant negative effect) and the relative amount of wild type and mutant PS1 incorporated into the gamma secretase complex are important factors that are relevant to the understanding and the treatment of PS1 EOFAD. Methods: We have generated five PS1 EOFAD cell lines (M146V, A431E, H163R, delta9, E280A and WT
control in H4 neuroglioma cells) in which recombinant mutant PS1 expression, in the presence of endogenous wild type PS1, is under the control of the TETrepressor. Results: We measured Ab42and Ab40 levels as a function of the ratio of wild type to mutant PS1 in the gamma secretase complex and demonstrated that all EOFAD PS1 mutants tested exhibit dominant negative behavior for Ab42 and total Ab production. In contrast, over-expression of wild type PS1 had no effect on Ab42 or total Ab production. Although equal gene dosage of mutant and wild type PS1 is expected in EOFAD, the actual ratio of mutant to wild type PS1 in the gamma secretase complex will be dependent on the relative stability (i.e., multiple factors such as mutant holoPS1 stability, its ability to interact with other gamma secretase components and the stability of the resulting gamma secretase complex). In PS1 EOFAD, the relative stability of the PS1 variant could impact the degree of a dominant negative effect and thus make a significant contribution to disease severity and age of onset. We determined the relative stability of the PS1 mutants by measuring the amount of mutant PS1 incorporated into the gamma secretase complex as a function of recombinant mRNA levels. Conclusions: Our results suggest that increasing the ratio of wild type to mutant PS1 may promote normalization of gamma secretase function and could be a promising therapeutic approach for PS1 EOFAD. O2-04-06
THE ROLE OF PRESENILIN 1 FAMILIAL ALZHEIMER’S DISEASE MUTATIONS FOR GAMMA-SECRETASE ACTIVITY AND CHANGED STRUCTURAL CONFORMATION
Patricia Lara Vasquez1, Johanna Wanngren2, Silvia Maioli2, Lars Tjernberg3, Bengt Winblad3, Johan Lundkvist4, IngMarie Nilsson5, Helena Karlstr€om2, 1Department of Biochemistry & Biophysics, Stockholm, Sweden; 2Karolinska Institutet, Stockholm, Sweden; 3Karolinska Institutet, Huddinge, Sweden; 4AstraZeneca, S€odert€alje, Sweden; 5Stockholm University, Stockholm, Sweden. Background: The Alzheimer disease (AD) related amyloid b-peptide (Ab) is generated from the amyloid b-precursor protein (APP) by g-secretase. g-Secretase cleaves APP at two different sites, g- and ^Im-sites, generating Ab-peptides and the APP intracellular domain. g-Sectretase is composed of presenilin (PS), nicastrin, Pen-2 and Aph-1. PS is a highly conserved membrane protein with nine transmembrane domains (TMDs) and harbours the catalytic site with two conserved aspartate residues, located in TMD6 and 7. Currently, 185 autosomal dominantly inherited familial AD (FAD) mutations have been identified in PS1. The scattered distribution of the mutations in the TMDs, and that they change the complex’s cleavage preference, i.e. yielding an increase in Ab42/Ab40 ratio, suggest that they may cause a conformational change of PS1. In this project we have investigated if the FAD mutations cause an alteration in the membrane integration of the PS1 TMDs, or if they influence structural changes in the catalytic site. Methods: Cultured medium from PS1/ 2-/- cell lines expressing 12 different PS1-FAD mutations have been monitored for Ab peptides by Meso Scale Discovery technology. An assay based on N-linked glycosylation and pull down with a g-secretase transition state analogue inhibitor coupled to biotin (GCB) have been used to examine the TMDs membrane integration efficiency and the catalytic site conformation, respectively. Results: When comparing the Ab42/Ab40 ratio to membrane integration as well as for age of AD onset, we observe that i) very early onset FAD mutations have high Ab42/Ab40 ratio and in general no altered membrane integration, ii) the FAD mutations with later onset have less increase in Ab42/Ab40 ratio but changed membrane integration. These results indicate that altered membrane integration by a PS1-FAD mutation may be a cause for altered Ab42/Ab40 ratio, but that other mechanisms are underlying the increased ratio and subsequently earlier AD onset. One mechanism could be changed conformation in the catalytic site, which currently is under investigation. Conclusions: By understanding the mechanisms by which the FAD mutations cause an increased Ab42/Ab40 ratio, we will gain more knowledge about the g-secretase biology. Moreover, these parameters are of significance when considering g-secretase as a target for pharmaceutical intervention in AD.
Oral Sessions: O2-04: Cellular and Molecular Mechanisms: Amyloid I
erlin-2, that resides in DRMs and affects Ab production with less effect on Notch processing. O2-04-04
THE METALLOPROTEASE MEPRIN BETA GENERATES AMINO-TERMINALLY TRUNCATED BETA-AMYLOID PEPTIDE SPECIES
Jessica Bien1, Tamara Jefferson2, Mirsada Causevic1, Thorsten Jumpertz3, Lisa M. Munter4, Gerd Multhaup4, Sascha Weggen3, Christoph BeckerPauly2, Claus Pietrzik1, 1University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany; 2Christian-AlbrechtsUniversity Kiel, Kiel, Germany; 3Heinrich-Heine University, Dusseldorf, Germany; 4Free University, Berlin, Germany. Background: The amyloid b (Ab) peptide, which is abundantly found in the brains of patients suffering from Alzheimer’s disease is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin b cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates, but not in meprin b-/- mouse brain lysates. Methods: Based on a novel proteomics technique termed TAILS (Terminal Amine Isotopic Labeling of Substrates), APP was identified as a substrate for meprin b. Subsequent analysis of APP processing using ELISA, SDS-PAGE, and mass spectrometry revealed cleavage events by meprin b close to the regular BACE1 cleavage site in APP. Results: In this work, we present evidence that meprin b can also process APP in a manner reminiscent of b-secretase. We identified cleavage sites of meprin b in the amyloid b sequence of the wild type and Swedish mutant of APP at positions p1, p2, and p3, thereby generating Ab variants starting at the first, second or third amino acid residue. Importantly, we provide strong evidence that meprin b produces an Ab peptide starting with the glutamate in the third position, indicating that this protease might produce an amino-terminally truncated Ab species. We observed even higher kinetic values for meprin b than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin b on APP and Ab generation was also observed in the absence of BACE1 and 2, indicating that meprin b acts independently of b-secretase. Conclusions: Our data indicate that meprin b is an enzyme capable of cleaving APP in a b-secretase like manner, probably also at position 674 (Abp3) to expose a glutamate residue, which might be subsequently modified by a glutaminylcyclase to generate pyroglutamate-Ab peptides. Although BACE1 acts as the major b-secretase in vivo generating most of the Ab 1-40/42 peptides, we suggest that meprin b might act as one enzyme responsible for the release of N-terminal truncated Ab species. O2-04-05
A CELL-BASED MODEL FOR PRESENILIN 1 EARLY-ONSET FAMILIAL ALZHEIMER’S DISEASE (EOFAD): DOMINANT NEGATIVE EFFECTS AND RELATIVE STABILITIES OF PRESENILIN 1 WITH EOFAD MUTATIONS
Anthony Stevens1, Carolyn Collins1, Evan Katz1, Gary Lee1, Vernon Alford2, Hadis Williams1, Darlene Guillen2, Xiaoyang Wu1, John Flanagan1, Eric Sjoberg1, Sam Gandy3, David J. Lockhart1, Brandon Wustman1, LB Dungan1, 1Amicus Therapeutics, Cranbury, New Jersey, United States; 2Amicus Therapeutics, La Jolla, California, United States; 3Mount Sinai, New York City, New York, United States. Background: The most frequent cause of Early-onset familial Alzheimer’s disease (EOFAD) is the autosomal dominant inheritance of a missense mutation in the presenilin 1 (PS1) gene (PSEN). Since EOFAD results from a mutation in a single allele of PSEN, both wild type and the PSEN variant gene products are co-expressed. The potential for the mutant PS1 protein to affect the function of wild-type PS1 (e.g., a dominant negative effect) and the relative amount of wild type and mutant PS1 incorporated into the gamma secretase complex are important factors that are relevant to the understanding and the treatment of PS1 EOFAD. Methods: We have generated five PS1 EOFAD cell lines (M146V, A431E, H163R, delta9, E280A and WT
control in H4 neuroglioma cells) in which recombinant mutant PS1 expression, in the presence of endogenous wild type PS1, is under the control of the TETrepressor. Results: We measured Ab42and Ab40 levels as a function of the ratio of wild type to mutant PS1 in the gamma secretase complex and demonstrated that all EOFAD PS1 mutants tested exhibit dominant negative behavior for Ab42 and total Ab production. In contrast, over-expression of wild type PS1 had no effect on Ab42 or total Ab production. Although equal gene dosage of mutant and wild type PS1 is expected in EOFAD, the actual ratio of mutant to wild type PS1 in the gamma secretase complex will be dependent on the relative stability (i.e., multiple factors such as mutant holoPS1 stability, its ability to interact with other gamma secretase components and the stability of the resulting gamma secretase complex). In PS1 EOFAD, the relative stability of the PS1 variant could impact the degree of a dominant negative effect and thus make a significant contribution to disease severity and age of onset. We determined the relative stability of the PS1 mutants by measuring the amount of mutant PS1 incorporated into the gamma secretase complex as a function of recombinant mRNA levels. Conclusions: Our results suggest that increasing the ratio of wild type to mutant PS1 may promote normalization of gamma secretase function and could be a promising therapeutic approach for PS1 EOFAD. O2-04-06
THE ROLE OF PRESENILIN 1 FAMILIAL ALZHEIMER’S DISEASE MUTATIONS FOR GAMMA-SECRETASE ACTIVITY AND CHANGED STRUCTURAL CONFORMATION
Patricia Lara Vasquez1, Johanna Wanngren2, Silvia Maioli2, Lars Tjernberg3, Bengt Winblad3, Johan Lundkvist4, IngMarie Nilsson5, Helena Karlstr€om2, 1Department of Biochemistry & Biophysics, Stockholm, Sweden; 2Karolinska Institutet, Stockholm, Sweden; 3Karolinska Institutet, Huddinge, Sweden; 4AstraZeneca, S€odert€alje, Sweden; 5Stockholm University, Stockholm, Sweden. Background: The Alzheimer disease (AD) related amyloid b-peptide (Ab) is generated from the amyloid b-precursor protein (APP) by g-secretase. g-Secretase cleaves APP at two different sites, g- and ^Im-sites, generating Ab-peptides and the APP intracellular domain. g-Sectretase is composed of presenilin (PS), nicastrin, Pen-2 and Aph-1. PS is a highly conserved membrane protein with nine transmembrane domains (TMDs) and harbours the catalytic site with two conserved aspartate residues, located in TMD6 and 7. Currently, 185 autosomal dominantly inherited familial AD (FAD) mutations have been identified in PS1. The scattered distribution of the mutations in the TMDs, and that they change the complex’s cleavage preference, i.e. yielding an increase in Ab42/Ab40 ratio, suggest that they may cause a conformational change of PS1. In this project we have investigated if the FAD mutations cause an alteration in the membrane integration of the PS1 TMDs, or if they influence structural changes in the catalytic site. Methods: Cultured medium from PS1/ 2-/- cell lines expressing 12 different PS1-FAD mutations have been monitored for Ab peptides by Meso Scale Discovery technology. An assay based on N-linked glycosylation and pull down with a g-secretase transition state analogue inhibitor coupled to biotin (GCB) have been used to examine the TMDs membrane integration efficiency and the catalytic site conformation, respectively. Results: When comparing the Ab42/Ab40 ratio to membrane integration as well as for age of AD onset, we observe that i) very early onset FAD mutations have high Ab42/Ab40 ratio and in general no altered membrane integration, ii) the FAD mutations with later onset have less increase in Ab42/Ab40 ratio but changed membrane integration. These results indicate that altered membrane integration by a PS1-FAD mutation may be a cause for altered Ab42/Ab40 ratio, but that other mechanisms are underlying the increased ratio and subsequently earlier AD onset. One mechanism could be changed conformation in the catalytic site, which currently is under investigation. Conclusions: By understanding the mechanisms by which the FAD mutations cause an increased Ab42/Ab40 ratio, we will gain more knowledge about the g-secretase biology. Moreover, these parameters are of significance when considering g-secretase as a target for pharmaceutical intervention in AD.