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Amyloid metabolism in Niemann-Pick type C disease
e22
Developing Topics
biochemical instability of neurotoxic aggregates in vitro is a problem for research on the molecular biology of AD. While the conformation adopted by Aß within these aggregates is not known, a ß-hairpin conformation is known to be accessible to monomeric Aß. The ß-hairpin is for instance observed in a complex with Affibody binding protein. Methods: To test the hypothesis that the ß-hairpin is a building block of toxic oligomers, we have engineered a double-cysteine mutant of Aß (called AßCC) in which the ß-hairpin conformation is stabilized by an intramolecular disulfide bond. AßCC oligomers were characterized to determine if they represent a stable model of wild type neurotoxic aggregates. Results: Aß40CC and Aß42CC form stable protofibrils, but are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aß aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in AßCC oligomers. Stable oligomers are potent inducers of neuronal apoptosis. Conclusions: Stable neurotoxic AßCC oligomers/protofibrils might facilitate studies of their structure and role in the pathogenesis of AD. Stable protofibril preparations might potentially also be used for therapy development based on active or passive immunization.
P4-317
AMYLOID METABOLISM IN NIEMANN-PICK TYPE C DISEASE
Mikael Gustavsson1, Niklas Mattsson2, Maria Olsson3, Maria Blomqvist3, Erik Portelius2, Ulf Andreasson2, Monica Christiansson3, Asa K€allen3, 3 3 4 ansson3, Dzemila Secic , Sara Hullberg , Charles Vite , Jan-Erik M 1 Sahlgrenska University Hospital, G€oteborg, Sweden; 2Institute of Neuroscience and Fysiology, M€olndal, Sweden; 3Institute of Neuroscience and Fysiology, Gothenburg, Sweden; 4Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States. Background: There is accumulating evidence for lysosomal dysfunction in Alzheimer’s disease (AD). One of the hallmarks of AD is abnormal metabolism of synaptotoxic and neurotoxic amyloid beta (Abeta). Interestingly, amyloid metabolism is also abnormal in the lysosomal storage disorder Niemann-Pick type C (NPC). NPC patients have increased cerebrospinal fluid (CSF) levels of Abeta isoforms and Abeta levels correlate with clinical disease intensity. However, the precise mechanism of the altered amyloid metabolism is unclear. We here tested the hypothesis that NPC phenotype shifts cellular amyloid metabolism into an alternative processing pathway. Methods: We used cell media from SH-SY5Y cells, treated with U18666A to induce NPC phenotype, and CSF from NPC-cats. Cell media and CSF were analyzed for different isoforms of Abeta peptides and other APP metabolites, using immunoassays and immunoprecipitation (IP)MALDI-TOF mass spectrometry (MS) assays. Peak identifications were done by FT-ICR-MS/MS and MALDI-TOF-TOF-MS/MS. Results: U18666A treatment resulted in altered amyloid metabolism, resembling previous findings in patient CSF. Treated samples had increased cell media levels of AbetaX-40/42 but reduced levels of Abeta1-40/42 They also had decreased levels of the N-terminal soluble APP fragment sAPP-beta, but increased levels of sAPP-alpha, indicating a shift from beta-secretase cleavage to alpha-secretase cleavage. The increased levels of AbetaX-40/42 was partly inhibited by adding a Cathepsin B-inhibitor, but not by adding a BACE-inhibitor. IP-MS identified increased levels of novel short Abeta peptides in cell media from treated cells. Analyses of the cat CSF revealed a 14 Da mass shift of Abeta1-40 in cats compared to humans, and MS/MS analyses identified an amino acid substitution in the cat Abeta sequence. When compared to wild type cats and cats heterozygous for the NPC-mutation, homozygous cats had an altered Abeta isoform pattern. Conclusions: Human neuronal cells treated to NPC phenotype have up-regulation of a BACE-independent pathway resulting in the release of novel short Abeta isoforms which may be partly inhibited by Cathepsin B-inhibition. NPC cats also have an altered amyloid profile. These findings shed new light on amyloid metabolism in NPC, and on amyloid processing in general. This may also increase the understanding of AD, in particular regarding its relation to lysosomal dysfunction.
P4-318
AMYLOID BETA (AB) NANOPARTICLES EMPLOYED TO STUDY INTRACELLULAR TAU PROTEIN INTERACTION
Syed Hasan, Garth Hall, University of Massachusetts-Lowell, Lowell, Massachusetts, United States. Background: The relationship between abnormal processing of amyloid precursor protein (APP) to beta amyloid (Abeta) and the misprocessing of tau protein is of central importance in the pathogenesis of Alzheimer’s disease (AD), but is as yet poorly understood. In particular, it is unclear whether and to what extent interactions between beta amyloid and tau protein are involved in AD cytopathogenesis. Processing of APP to Abeta involves endosomal trafficking pathways, and Abeta secretion appears to occur via an exosome-mediated mechanism, and we recently showed that tau can also be exosomally secreted. Here we use a nanoparticle-based approach to investigate the possibility that tau misprocessing and secretion may be modulated by interactions between Abeta and tau in the endosomes and trafficking vesicles required for the processing of exosomes and other classes of secreted microvesicles. Methods: We electrostatically loaded 5-10 nm polystyrene nanoparticles with AB (1-42) and incubated them with human neuroblastoma (M1C) cells induced to overexpress and secrete the 4R0N isoform of human tau protein. Confocal imaging and colocalization analysis were used to identify interactions between intracellularly derived tau and endocytosed Abeta, which were labeled using ThioflavinS (Abeta) and anti-tau immunolabel (tau), respectively. Results: We show that M1C cells accumulate tau in a subset of EEA-positive endosomes after Tet-off induction of tau expression and that such cells readily endocytose 5 and 10 nm Abeta-nanoparticles within 5 minutes of application. At this time, we found that tau and nanoparticle-bound A beta were localized to EEAþ endosome-like vesicles, with a subset of these vesicles showing colocalized Abeta and tau immunolabel. Endosomes with the highest A beta levels also contained the highest tau levels. Conclusions: Our results show that human neuroblastoma (M1C) cells readily endocytose AB-nanoparticles and suggest that the presence of Abeta causes tau to become enriched in Abeta-containing endosomes, either via direct Abeta-tau interaction or via a synergisitic effect on endocytosis. We propose that Abeta and tau colocalization in trafficking vesicles may reflect synergistic misprocessing of both proteins and may be relevant to misprocessing of other exosomally secreted, aggregation-prone proteins (e.g. alpha synuclein, prion protein) in neurodegenerative disease. P4-319
FACILE SYNTHESIS OF BETA-AMYLOID BY SOLID-PHASE PEPTIDE SYNTHESIS
Jun Seok Lee, Hye Yun Kim, Dong Jin Kim, YoungSoo Kim, Korea Institute of Science and Technology, Seoul, South Korea. Background: The beta-amyloid(1-42) sequence has long been recognized as a challenging target for solid-phase peptide synthesis. Methods: We demonstrate a hitherto-unreported on-resin reduction of the sulfoxide "aggregation protecting group" to allow for standard cleavage protocols, obviating a separate solution-phase sulfoxide reduction step. Results: We found that the known disaggregating role of Met-35 sulfoxide could be capitalized during stepwise solid-phase assembly of the A beta(1-42) peptide chain to mitigate on-resin peptide chain aggregation, a presumed major source of synthetic difficulties. Conclusions: The use of a biologically inspired sulfoxide protection strategy encompassing a side-chain protecting group and coupling solvent designed to suppress aggregation of the resin-bound peptide chain is recommended for the synthesis of Abeta isoforms and analogues and has potentially broad applicability in the synthesis of similar large, structure-ridden peptide targets. P4-320
LRP10, A NEW REGULATOR OF APP TRAFFICKING AND PROCESSING
Julie Brodeur, Caroline Theriault, Christine Lavoie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada. Background: Accumulation of extracellular amyloid peptide (Aß), generated from amyloid precursor protein (APP) processing by ß- and g-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer’s disease
Developing Topics
biochemical instability of neurotoxic aggregates in vitro is a problem for research on the molecular biology of AD. While the conformation adopted by Aß within these aggregates is not known, a ß-hairpin conformation is known to be accessible to monomeric Aß. The ß-hairpin is for instance observed in a complex with Affibody binding protein. Methods: To test the hypothesis that the ß-hairpin is a building block of toxic oligomers, we have engineered a double-cysteine mutant of Aß (called AßCC) in which the ß-hairpin conformation is stabilized by an intramolecular disulfide bond. AßCC oligomers were characterized to determine if they represent a stable model of wild type neurotoxic aggregates. Results: Aß40CC and Aß42CC form stable protofibrils, but are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aß aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in AßCC oligomers. Stable oligomers are potent inducers of neuronal apoptosis. Conclusions: Stable neurotoxic AßCC oligomers/protofibrils might facilitate studies of their structure and role in the pathogenesis of AD. Stable protofibril preparations might potentially also be used for therapy development based on active or passive immunization.
P4-317
AMYLOID METABOLISM IN NIEMANN-PICK TYPE C DISEASE
Mikael Gustavsson1, Niklas Mattsson2, Maria Olsson3, Maria Blomqvist3, Erik Portelius2, Ulf Andreasson2, Monica Christiansson3, Asa K€allen3, 3 3 4 ansson3, Dzemila Secic , Sara Hullberg , Charles Vite , Jan-Erik M 1 Sahlgrenska University Hospital, G€oteborg, Sweden; 2Institute of Neuroscience and Fysiology, M€olndal, Sweden; 3Institute of Neuroscience and Fysiology, Gothenburg, Sweden; 4Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States. Background: There is accumulating evidence for lysosomal dysfunction in Alzheimer’s disease (AD). One of the hallmarks of AD is abnormal metabolism of synaptotoxic and neurotoxic amyloid beta (Abeta). Interestingly, amyloid metabolism is also abnormal in the lysosomal storage disorder Niemann-Pick type C (NPC). NPC patients have increased cerebrospinal fluid (CSF) levels of Abeta isoforms and Abeta levels correlate with clinical disease intensity. However, the precise mechanism of the altered amyloid metabolism is unclear. We here tested the hypothesis that NPC phenotype shifts cellular amyloid metabolism into an alternative processing pathway. Methods: We used cell media from SH-SY5Y cells, treated with U18666A to induce NPC phenotype, and CSF from NPC-cats. Cell media and CSF were analyzed for different isoforms of Abeta peptides and other APP metabolites, using immunoassays and immunoprecipitation (IP)MALDI-TOF mass spectrometry (MS) assays. Peak identifications were done by FT-ICR-MS/MS and MALDI-TOF-TOF-MS/MS. Results: U18666A treatment resulted in altered amyloid metabolism, resembling previous findings in patient CSF. Treated samples had increased cell media levels of AbetaX-40/42 but reduced levels of Abeta1-40/42 They also had decreased levels of the N-terminal soluble APP fragment sAPP-beta, but increased levels of sAPP-alpha, indicating a shift from beta-secretase cleavage to alpha-secretase cleavage. The increased levels of AbetaX-40/42 was partly inhibited by adding a Cathepsin B-inhibitor, but not by adding a BACE-inhibitor. IP-MS identified increased levels of novel short Abeta peptides in cell media from treated cells. Analyses of the cat CSF revealed a 14 Da mass shift of Abeta1-40 in cats compared to humans, and MS/MS analyses identified an amino acid substitution in the cat Abeta sequence. When compared to wild type cats and cats heterozygous for the NPC-mutation, homozygous cats had an altered Abeta isoform pattern. Conclusions: Human neuronal cells treated to NPC phenotype have up-regulation of a BACE-independent pathway resulting in the release of novel short Abeta isoforms which may be partly inhibited by Cathepsin B-inhibition. NPC cats also have an altered amyloid profile. These findings shed new light on amyloid metabolism in NPC, and on amyloid processing in general. This may also increase the understanding of AD, in particular regarding its relation to lysosomal dysfunction.
P4-318
AMYLOID BETA (AB) NANOPARTICLES EMPLOYED TO STUDY INTRACELLULAR TAU PROTEIN INTERACTION
Syed Hasan, Garth Hall, University of Massachusetts-Lowell, Lowell, Massachusetts, United States. Background: The relationship between abnormal processing of amyloid precursor protein (APP) to beta amyloid (Abeta) and the misprocessing of tau protein is of central importance in the pathogenesis of Alzheimer’s disease (AD), but is as yet poorly understood. In particular, it is unclear whether and to what extent interactions between beta amyloid and tau protein are involved in AD cytopathogenesis. Processing of APP to Abeta involves endosomal trafficking pathways, and Abeta secretion appears to occur via an exosome-mediated mechanism, and we recently showed that tau can also be exosomally secreted. Here we use a nanoparticle-based approach to investigate the possibility that tau misprocessing and secretion may be modulated by interactions between Abeta and tau in the endosomes and trafficking vesicles required for the processing of exosomes and other classes of secreted microvesicles. Methods: We electrostatically loaded 5-10 nm polystyrene nanoparticles with AB (1-42) and incubated them with human neuroblastoma (M1C) cells induced to overexpress and secrete the 4R0N isoform of human tau protein. Confocal imaging and colocalization analysis were used to identify interactions between intracellularly derived tau and endocytosed Abeta, which were labeled using ThioflavinS (Abeta) and anti-tau immunolabel (tau), respectively. Results: We show that M1C cells accumulate tau in a subset of EEA-positive endosomes after Tet-off induction of tau expression and that such cells readily endocytose 5 and 10 nm Abeta-nanoparticles within 5 minutes of application. At this time, we found that tau and nanoparticle-bound A beta were localized to EEAþ endosome-like vesicles, with a subset of these vesicles showing colocalized Abeta and tau immunolabel. Endosomes with the highest A beta levels also contained the highest tau levels. Conclusions: Our results show that human neuroblastoma (M1C) cells readily endocytose AB-nanoparticles and suggest that the presence of Abeta causes tau to become enriched in Abeta-containing endosomes, either via direct Abeta-tau interaction or via a synergisitic effect on endocytosis. We propose that Abeta and tau colocalization in trafficking vesicles may reflect synergistic misprocessing of both proteins and may be relevant to misprocessing of other exosomally secreted, aggregation-prone proteins (e.g. alpha synuclein, prion protein) in neurodegenerative disease. P4-319
FACILE SYNTHESIS OF BETA-AMYLOID BY SOLID-PHASE PEPTIDE SYNTHESIS
Jun Seok Lee, Hye Yun Kim, Dong Jin Kim, YoungSoo Kim, Korea Institute of Science and Technology, Seoul, South Korea. Background: The beta-amyloid(1-42) sequence has long been recognized as a challenging target for solid-phase peptide synthesis. Methods: We demonstrate a hitherto-unreported on-resin reduction of the sulfoxide "aggregation protecting group" to allow for standard cleavage protocols, obviating a separate solution-phase sulfoxide reduction step. Results: We found that the known disaggregating role of Met-35 sulfoxide could be capitalized during stepwise solid-phase assembly of the A beta(1-42) peptide chain to mitigate on-resin peptide chain aggregation, a presumed major source of synthetic difficulties. Conclusions: The use of a biologically inspired sulfoxide protection strategy encompassing a side-chain protecting group and coupling solvent designed to suppress aggregation of the resin-bound peptide chain is recommended for the synthesis of Abeta isoforms and analogues and has potentially broad applicability in the synthesis of similar large, structure-ridden peptide targets. P4-320
LRP10, A NEW REGULATOR OF APP TRAFFICKING AND PROCESSING
Julie Brodeur, Caroline Theriault, Christine Lavoie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada. Background: Accumulation of extracellular amyloid peptide (Aß), generated from amyloid precursor protein (APP) processing by ß- and g-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer’s disease