- Email: [email protected]
Structural basis for amyloid beta binding to neuronal cells
Poster Presentations P1
S252
considered as a candidate target gene for drug discovery and development. P1-269
STRUCTURAL BASIS FOR AMYLOID BETA BINDING TO NEURONAL CELLS
Lorena Perrone1, Dirk Bartnik2, Honoree Marzaguil3, Susanne A. Funke2, Paolo Gubellini4, Marie L. Maddelein3, Dieter Willbold2, Michel Khrestchatisky1, 1NICN UMR6184, CNRS, Marseille, France; 2Forschungszentrum Ju¨lich, ISB-3, 52425, Ju¨lich, Germany; 3IPBS, CNRS, Toulouse, France; 4IBDM, CNRS, Marseille, France. Contact e-mail: loperron@ hotmail.com Background: Alzheimer’s disease (AD) is characterized by the accumulation of amyloid beta (Abeta) peptides in amyloid deposits in the cerebral tissue. Current evidence indicates that intraneuronal accumulation of Abeta is an early pathological biomarker for the onset of AD and may contribute to a cascade of neurodegenerative events. Many reports found strong evidence that intermediates in the aggregation process called ‘oligomers’ are the principal pathogenic species that drive neuronal dysfunction rather than the large amyloid aggregates. There are studies suggesting that cellular membranes play a major role in Abeta oligomerization process and that Abeta uptake in neuronal cells is responsible for Abeta-induced neuronal dysfunction and toxicity. Objective: We investigate the link between in vitro amyloid structure and the capability of Abeta peptides to be internalized in neuronal cells and induce toxicity. Methods: We analyzed in vitro the aggregation properties of an Abeta mutant with impaired aggregation properties by dynamic light scattering (DLS) and thioflavin-T binding. We also measured its capability to alter the aggregation properties of wild-type Abeta. Next, we analyzed the uptake and aggregation of this Abeta mutant in neuronal cells and its capability to alter the uptake of wild-type Abeta. By confocal imaging and Congo red staining, we analyzed the binding at the plasma membrane, internalization and further elongation of wild-type oligomers (Ab42) compared to the mutant Abeta oligomers Results: We found that adhesion at the plasma membrane of Abeta peptides is inversely correlated with their capability to elongate. These results were finally correlated with the capability of mutant and wild-type Abeta to induce cell toxicity. Conclusions: These studies give insight on the structural basis for Abeta-induced neuronal toxicity and open the way for new therapeutic strategies aimed at selecting Abeta mutants that alter the uptake and the toxicity induced by wild-type Abeta. P1-270
NOTCH SIGNALING IMPACTS ON AMYLOID b METABOLISM BY MODULATING IDE AND BACE1
Laura Morelli1, Ezequiel I. Surace1, Maria C. Leal1, Maria P. Holgado1, Carina C. Ferrari1, Rodolfo Tarelli1, Fernando Pitossi1, Thomas Wisnieswsky2, Miguel Riudavets3, Eduardo M. Castan˜o1, 1Leloir Institute Foundation, Ciudad de Buenos Aires, Argentina; 2New York University School of Medicine., New York 10016, NY, USA; 3Department of Neuropathology. FLENI, Ciudad de Buenos Aires, Argentina. Contact e-mail: [email protected] Background: Cerebral amyloid b (Ab) accumulation is pathogenically associated with sporadic Alzheimer‘s disease (SAD). BACE-1 is the rate-limiting enzyme in Ab generation while insulin-degrading enzyme (IDE) has a major role in the extracellular clearance of naturally secreted Ab. Vulnerable neurons in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. The mechanisms underlying these observations remain mostly unknown. Other common feature in SAD brains is the overexpression of Notch-1. The role of Notch in neuronal differentiation during development and adult neurogenesis is well established. We hypothesize that Notch pathway activation is implicated in the transcriptional deregulation of genes involved in Ab metabolism. Methods: With RSAT software we detected HES/Hey-1 (Notch target genes) putative binding sites on IDE promoter. By immunohistochemistry and QRT-PCR we determined Notch activation in brain samples. 125I-insulin and a fluorogenic substrate (R&Dcat#ES004) were used to assay IDE and BACE-1 activities, respec-
tively. N2aSW (neuroblastoma cells stably expressing human Ab40) were transfected with HA-Notch Intracellular Domain (NICD) or HA-Hey-1 cDNAs and assessment of IDE and BACE-1 transcript levels (by QRTPCR), extracellular Ab (by ELISA) and promoter activity (by LUC reporter assay) was performed. Cells were exposed to Jagged (JAG, a Notch ligand) and the parameters evaluated above were determined in the presence of a Notch-specific siRNA. Intracranial injection of JAG in Tg2576 mice, a well-established plaque pathology animal model of AD, was used for Notch signaling activation in vivo. Results: We identified over-expression of NICD and Notch target genes and alterations of BACE-1 and IDE activities in the hippocampus of SAD brains. We demonstrated that over-expression of NICD in N2aSW increased Hey-1 and BACE-1 and reduced IDE mRNA levels, promoting extracellular Ab accumulation. We showed an opposite effect of NICD on IDE and on BACE promoters and on their proteolytic activities which were reverted by silencing Notch. We reproduced the molecular and pathological effect of Notch activation in Tg2576 mice. Conclusions: Our results support that a Notch-dependent IDE and BACE transcriptional modulation impacts on Ab metabolism providing a novel functional link between Notch signaling and the amyloidogenic pathway in SAD. P1-271
GAMMA-SECRETASEeASSOCIATED PROTEINS IN DETERGENT RESISTANT MEMBRANES FROM BRAIN
Ji-Yeun Hur, Yasuhiro Teranishi, Masakazu Hashimoto, Waltteri Hosia, Hedvig Welander, Bengt Winblad, Susanne Frykman, Lars O. Tjernberg, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: Characteristic hallmarks of Alzheimer’s disease (AD) are the depositions of amyloid beta-peptide (Abeta) in senile plaques, and the hyperphosphorylated tau protein in neurofibrillary tangles in the brain. Sequencial cleavages by beta-secretase and gamma-secretase of the amyloid precursor protein (APP) produce soluble APP, the APP intracellular domain (AICD) and Abeta. Gamma-secretase is an aspartyl protease complex, containing at least four transmembrane proteins (PS, nicastrin, Aph-1, Pen-2). These four proteins appear to be sufficient for gamma-secretase activity, but it is possible that other proteins could have a regulatory role. For instance, recent studies reported TMP21, CD147, and other proteins as possible regulators of gamma-secretase. The lipid membrane environment can affect the activity of proteins. Previously we showed that active gamma-secretase is localized to lipid rafts in brain. Lipid rafts are enriched in cholesterol and sphingolipids and are important for cell signaling, membrane protein sorting and transport. Lipid rafts are biochemically studied by treatment with detergents such as Triton X-100 at 4 C followed by centrifugation. As a result, the insoluble parts of the lipid membranes are isolated and are called detergent resistant membranes (DRMs). In this study, we have identified novel gamma-secretase associated proteins from DRMs by nanoscale liquid chromatography - tandem mass spectrometry (LC-MS/MS). Methods: We studied gammasecretase in DRMs prepared from rat brain. DRMs were isolated from brain by sucrose gradient centrifugation. The DRM-associated-gamma-secretase was incubated with biotinylated gamma-secretase inhibitor in the absence or presence of an excess amount of a competing inhibitor, L-685,458, followed by pulldown using streptavidin-conjugated magnetic beads. After removing non-specific binding proteins by washing, the samples were eluted, trypsinized, and analyzed by LC-MS/MS. The non-specific binding proteins (in the presence of L-685,458) were subtracted from the specific binding proteins (in the absence of L-685,458). Results: Western blot analysis showed that the pulldown was efficient and specific. We could detect lipid raft marker proteins and other possible gamma-secretase associated proteins. Potential gamma-secretase associated proteins will be presented. Conclusions: We identified possible and novel gamma-secretase associated protein candidates from DRMs. P1-272
TARGETING INTRACELLULAR Ab OLIGOMERS THROUGH CONFORMATIONAL INTRABODIES
Giovanni Meli1,2, Agnese Lecci2, Antonino Cattaneo1,2, 1Scuola Normale Superiore, Pisa, Italy; 2European Brain Research Institute (EBRI), Rome, Italy. Contact e-mail: [email protected]
S252
considered as a candidate target gene for drug discovery and development. P1-269
STRUCTURAL BASIS FOR AMYLOID BETA BINDING TO NEURONAL CELLS
Lorena Perrone1, Dirk Bartnik2, Honoree Marzaguil3, Susanne A. Funke2, Paolo Gubellini4, Marie L. Maddelein3, Dieter Willbold2, Michel Khrestchatisky1, 1NICN UMR6184, CNRS, Marseille, France; 2Forschungszentrum Ju¨lich, ISB-3, 52425, Ju¨lich, Germany; 3IPBS, CNRS, Toulouse, France; 4IBDM, CNRS, Marseille, France. Contact e-mail: loperron@ hotmail.com Background: Alzheimer’s disease (AD) is characterized by the accumulation of amyloid beta (Abeta) peptides in amyloid deposits in the cerebral tissue. Current evidence indicates that intraneuronal accumulation of Abeta is an early pathological biomarker for the onset of AD and may contribute to a cascade of neurodegenerative events. Many reports found strong evidence that intermediates in the aggregation process called ‘oligomers’ are the principal pathogenic species that drive neuronal dysfunction rather than the large amyloid aggregates. There are studies suggesting that cellular membranes play a major role in Abeta oligomerization process and that Abeta uptake in neuronal cells is responsible for Abeta-induced neuronal dysfunction and toxicity. Objective: We investigate the link between in vitro amyloid structure and the capability of Abeta peptides to be internalized in neuronal cells and induce toxicity. Methods: We analyzed in vitro the aggregation properties of an Abeta mutant with impaired aggregation properties by dynamic light scattering (DLS) and thioflavin-T binding. We also measured its capability to alter the aggregation properties of wild-type Abeta. Next, we analyzed the uptake and aggregation of this Abeta mutant in neuronal cells and its capability to alter the uptake of wild-type Abeta. By confocal imaging and Congo red staining, we analyzed the binding at the plasma membrane, internalization and further elongation of wild-type oligomers (Ab42) compared to the mutant Abeta oligomers Results: We found that adhesion at the plasma membrane of Abeta peptides is inversely correlated with their capability to elongate. These results were finally correlated with the capability of mutant and wild-type Abeta to induce cell toxicity. Conclusions: These studies give insight on the structural basis for Abeta-induced neuronal toxicity and open the way for new therapeutic strategies aimed at selecting Abeta mutants that alter the uptake and the toxicity induced by wild-type Abeta. P1-270
NOTCH SIGNALING IMPACTS ON AMYLOID b METABOLISM BY MODULATING IDE AND BACE1
Laura Morelli1, Ezequiel I. Surace1, Maria C. Leal1, Maria P. Holgado1, Carina C. Ferrari1, Rodolfo Tarelli1, Fernando Pitossi1, Thomas Wisnieswsky2, Miguel Riudavets3, Eduardo M. Castan˜o1, 1Leloir Institute Foundation, Ciudad de Buenos Aires, Argentina; 2New York University School of Medicine., New York 10016, NY, USA; 3Department of Neuropathology. FLENI, Ciudad de Buenos Aires, Argentina. Contact e-mail: [email protected] Background: Cerebral amyloid b (Ab) accumulation is pathogenically associated with sporadic Alzheimer‘s disease (SAD). BACE-1 is the rate-limiting enzyme in Ab generation while insulin-degrading enzyme (IDE) has a major role in the extracellular clearance of naturally secreted Ab. Vulnerable neurons in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. The mechanisms underlying these observations remain mostly unknown. Other common feature in SAD brains is the overexpression of Notch-1. The role of Notch in neuronal differentiation during development and adult neurogenesis is well established. We hypothesize that Notch pathway activation is implicated in the transcriptional deregulation of genes involved in Ab metabolism. Methods: With RSAT software we detected HES/Hey-1 (Notch target genes) putative binding sites on IDE promoter. By immunohistochemistry and QRT-PCR we determined Notch activation in brain samples. 125I-insulin and a fluorogenic substrate (R&Dcat#ES004) were used to assay IDE and BACE-1 activities, respec-
tively. N2aSW (neuroblastoma cells stably expressing human Ab40) were transfected with HA-Notch Intracellular Domain (NICD) or HA-Hey-1 cDNAs and assessment of IDE and BACE-1 transcript levels (by QRTPCR), extracellular Ab (by ELISA) and promoter activity (by LUC reporter assay) was performed. Cells were exposed to Jagged (JAG, a Notch ligand) and the parameters evaluated above were determined in the presence of a Notch-specific siRNA. Intracranial injection of JAG in Tg2576 mice, a well-established plaque pathology animal model of AD, was used for Notch signaling activation in vivo. Results: We identified over-expression of NICD and Notch target genes and alterations of BACE-1 and IDE activities in the hippocampus of SAD brains. We demonstrated that over-expression of NICD in N2aSW increased Hey-1 and BACE-1 and reduced IDE mRNA levels, promoting extracellular Ab accumulation. We showed an opposite effect of NICD on IDE and on BACE promoters and on their proteolytic activities which were reverted by silencing Notch. We reproduced the molecular and pathological effect of Notch activation in Tg2576 mice. Conclusions: Our results support that a Notch-dependent IDE and BACE transcriptional modulation impacts on Ab metabolism providing a novel functional link between Notch signaling and the amyloidogenic pathway in SAD. P1-271
GAMMA-SECRETASEeASSOCIATED PROTEINS IN DETERGENT RESISTANT MEMBRANES FROM BRAIN
Ji-Yeun Hur, Yasuhiro Teranishi, Masakazu Hashimoto, Waltteri Hosia, Hedvig Welander, Bengt Winblad, Susanne Frykman, Lars O. Tjernberg, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: Characteristic hallmarks of Alzheimer’s disease (AD) are the depositions of amyloid beta-peptide (Abeta) in senile plaques, and the hyperphosphorylated tau protein in neurofibrillary tangles in the brain. Sequencial cleavages by beta-secretase and gamma-secretase of the amyloid precursor protein (APP) produce soluble APP, the APP intracellular domain (AICD) and Abeta. Gamma-secretase is an aspartyl protease complex, containing at least four transmembrane proteins (PS, nicastrin, Aph-1, Pen-2). These four proteins appear to be sufficient for gamma-secretase activity, but it is possible that other proteins could have a regulatory role. For instance, recent studies reported TMP21, CD147, and other proteins as possible regulators of gamma-secretase. The lipid membrane environment can affect the activity of proteins. Previously we showed that active gamma-secretase is localized to lipid rafts in brain. Lipid rafts are enriched in cholesterol and sphingolipids and are important for cell signaling, membrane protein sorting and transport. Lipid rafts are biochemically studied by treatment with detergents such as Triton X-100 at 4 C followed by centrifugation. As a result, the insoluble parts of the lipid membranes are isolated and are called detergent resistant membranes (DRMs). In this study, we have identified novel gamma-secretase associated proteins from DRMs by nanoscale liquid chromatography - tandem mass spectrometry (LC-MS/MS). Methods: We studied gammasecretase in DRMs prepared from rat brain. DRMs were isolated from brain by sucrose gradient centrifugation. The DRM-associated-gamma-secretase was incubated with biotinylated gamma-secretase inhibitor in the absence or presence of an excess amount of a competing inhibitor, L-685,458, followed by pulldown using streptavidin-conjugated magnetic beads. After removing non-specific binding proteins by washing, the samples were eluted, trypsinized, and analyzed by LC-MS/MS. The non-specific binding proteins (in the presence of L-685,458) were subtracted from the specific binding proteins (in the absence of L-685,458). Results: Western blot analysis showed that the pulldown was efficient and specific. We could detect lipid raft marker proteins and other possible gamma-secretase associated proteins. Potential gamma-secretase associated proteins will be presented. Conclusions: We identified possible and novel gamma-secretase associated protein candidates from DRMs. P1-272
TARGETING INTRACELLULAR Ab OLIGOMERS THROUGH CONFORMATIONAL INTRABODIES
Giovanni Meli1,2, Agnese Lecci2, Antonino Cattaneo1,2, 1Scuola Normale Superiore, Pisa, Italy; 2European Brain Research Institute (EBRI), Rome, Italy. Contact e-mail: [email protected]