- Email: [email protected]
Aggregation of the Aβ(1-40) peptide: Secondary structure change from oligomers to fibrils
Poster Presentations P1
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age matched controls) and individuals with TBI (survival time 0-24 hours n ¼ 18, 1-28 days, n ¼ 6, 1-32 years n ¼ 13) underwent 4G8 immunohistochemistry to detect amyloid- b and AT8 immunostaining to detect neurofibrillary tangles. Sections underwent semi-quantitative assessment of intraneuronal amyloid and tau immunostaining using a scoring system of 0-3 in the dentate gyrus, CA4, CA3, CA1 and subiculum. Results: Intraneuronal amyloid was detected in both control cases and in response to mild brain injury. There was a significant increase in intraneuronal amyloid in all fields of the hippocampus in the TBI cases compared with controls (p ¼ 0.0027 -0.0135). Intense immunostaining (grade 3) was present in 0-15% of control cases and 45-55% of TBI cases. Intraneuronal amyloid increased with increasing survival time following TBI and was most extensive in individuals who survived longest after injury (p ¼ 0.0035 - 0.0087 compared to controls). There was no evidence of increased neurofibrillary tangles in the head injury as compared to controls. Conclusions: This study indicates that mild brain injury is sufficient to induce alterations in amyloid and highlights the need to understand more about the mechanisms by which injury can lead to AD. P1-235
AGGREGATION OF THE Ab(1-40) PEPTIDE: SECONDARY STRUCTURE CHANGE FROM OLIGOMERS TO FIBRILS
Rabia Sarroukh1, Emilie Cerf1, Sylvie Derclaye2, Yves F. Dufrene2, JeanMarie Ruysschaert1, Erik Goormaghtigh1, Vincent Raussens1, 1Universite Libre de Bruxelles, Bruxelles, Belgium; 2Universite Catholique de Louvain, Louvain, Belgium. Contact e-mail: [email protected] Background: Amyloid-b peptide (Ab) is the primary component of the senile plaques and plays an important, but not completely understood, role in the pathogenesis of Alzheimer’s disease. Ab can form different entities: monomeric, large soluble entities collectively called oligomers and insoluble fibrils. In the early stages of aggregation, Ab forms oligomers, which are considered as the most toxic forms. Continued aggregation gives rise to fibrils formation. The mechanisms leading to accumulation of misfolded peptide and the fibrils formation still remain unknown. We have previously shown, in our laboratory, that the oligomeric species and fibrils of Ab42 display distinct spectral features by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Whereas fibrils display spectral features of parallel b-sheet, oligomers adopt an antiparallel b-sheet structure. Methods: ATR-FTIR: Attenuated Total Reflection-Fourier Transform Infrared Spectrocopy Results: Based on these observations, we followed the aggregation of Ab40 using ATR-FTIR. We observed that Ab40 polymerized, in our case, first into antiparallel b-sheet oligomers which are recognized by a conformational antibody specific to these species (A11). Ab moves then from an antiparallel to a parallel b-sheet structure. This shift from oligomers to fibrils observed by ATR-FTIR was in perfect agreement with the marked increase in ThT fluorescence upon fibrils formation. This conformational change supposes a reorganization of the peptide upon aggregation, which is also observed using monoclonal region-specific antibodies used to study the accessibility of the aggregates. Conclusions: We demonstrated that ATR-FTIR is a powerful tool for protein aggregation studies: ATR-FTIR (i) can discriminate between Ab oligomers and fibrils from a structural point of view (ii) shows a conformational change in the structure of the peptide upon aggregation, perfectly correlated to the formation of fibrils, as probed by fluorescence. P1-236
E2012: A NOVEL GAMMA-SECRETASE MODULATOR-PHARMACOLOGY PART
Toshihide Hashimoto1, Akira Ishibashi1, Hiroaki Hagiwara1, Yoshiyuki Murata1, Osamu Takenaka1, Takehiko Miyagawa2, 1Eisai Co. Ltd., Tsukuba, Japan; 2Eisai Co. Ltd. Discovery Research, London, United Kingdom. Contact e-mail: [email protected] Background: Cerebral amyloid-beta (Ab) deposition is believed to play a central role in the pathogenesis of Alzheimer’s disease (AD). Ab is produced by the sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Gamma-secretase is also responsible for the intramembraneous cleavage of Notch and other substrates. While in-
hibition of gamma-secretase by classical gamma-secretase inhibitors results in reduced neurotoxic Ab species, inhibiting the cleavage of other gammasecretase substrates, including signaling molecules such as Notch, has been associated with undesirable side effects. Therefore, compounds that inhibit the production of toxic Ab species without affecting Notch cleavage represent a promising therapeutic approach for AD. Here we provide the first report on E2012, a novel compound with gamma-secretase modulatory activity. Methods: Ab40, 42 production in rat cortical neuron culture and Notch cleavage in the SUP-T1 human T-lymphoblast cell line were assayed before and after exposure to E2012. Ab40, 42 content in rat cerebrospinal fluid (CSF), brain, and plasma were measured after treatment with E2012 (3, 10, 30mg/kg for 3 days). Results: E2012 reduced the production of Ab40 and Ab42 in the rat cortical neuron culture dose-dependently without significant cytotoxicity. IC50 values of E2012 for Ab40 and Ab42 were 330 nM and 92 nM, respectively. E2012 decreased the levels of Ab40 and Ab42 in rat CSF, brain and plasma in vivo in a dose dependent manner. Especially, in rat CSF, E2012 significantly decreased Ab42 levels by 16.6% and 47.2% at doses of 10 and 30 mg/kg, respectively. The reduction in Ab42 levels in brain at doses of 10 and 30 mg/kg were 17.1% and 42.9%, respectively. Ab42 levels in plasma was decreased by 59.2, 90.8, and 96.1% at doses of 3, 10, and 30 mg/kg, respectively. The compound had no effect on Notch processing in vitro up to 3 mM. Conclusions: Our results suggest that E2012 is a novel and potent gamma-secretase modulator that reduces Ab40 and Ab42 levels without affecting Notch cleavage. P1-237
FIBRILLOGENESIS AND TOXICITY OF IOWA-Ab AMYLOID IS PRIMARILY DRIVEN BY THE D23N MUTATION
Agueda Rostagno, Silvia Fossati, Jorge Ghiso, NYU School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Mutations within the amyloid-b (Ab) sequence, especially those clustered at residues 21-23, are linked to early onset familial Alzheimer’s disease (AD) and primarily associated with cerebral amyloid angiopathy (CAA). Biochemical analysis of brain tissue deposits in AbD23N Iowa cases indicate the presence of complex mixtures of mutated and non-mutated Ab species, highly heterogeneous at both N- and C-termini, and showing partial Asp isomerization at positions 1, 7, and 23. Methods: Our study aimed to assess the role of the IsoAsp modifications and the D23N substitution in Ab aggregation/fibrillization as well as in the induction of cell toxicity in neuronal and microvascular endothelial cells. Peptide conformations and fibrillization profiles were monitored by circular dichroism, Western blot, thioflavin-T binding, and electron microscopy studies. Toxicity of the different Ab species for SH-SY5Y and microvascular endothelial cells was assessed by evaluation of nucleosome formation via Cell Death ELISA together with LDH release. Results: The presence of isoAsp had an enhancing -albeit modest- effect on fibrillization, without significantly affecting the lag phase of wild-type Ab40. On the contrary, the D23N mutation strikingly changed the peptide behavior yielding a dramatically accelerated fibrillization profile. Notably, AbD23N homologues bearing Asp residues rendered overlapping results with the mutant peptide containing IsoAsp. Evaluation of cell death mechanisms correlated with the fibrillization tendency, being AbD23N species highly toxic and inducing comparable apoptotic and LDH-release levels irrespective of their isoAsp content. Conclusions: The high fibrillization proclivity of AbD23N species containing isoAsp modifications, as found in the Iowa brain deposits, is primarily driven by the presence of the mutation and correlates with exacerbated neuronal and endothelial cell toxicity that could explain the early onset of the disease in the Iowa kindred. P1-238
SOLUBLE Ab FROM PSAPP MICE IS DIFFERENT TO THAT EXTRACTED FROM ALZHEIMER PATIENTS
Warren D. Hirst1, Elie Needle1, Roland G. W. Staal1, Robert Crozier1, Nathalie Breysse1, Reka Nagy1, Amy Sung1, Michael Monaghan1, David R. Riddell1, Jeannie Chin1, Dominic M. Walsh2, Dominic M. Walsh2, Menelas N. Pangalos1, Peter H. Reinhart1, J. S. Jacobsen1, 1Pfizer Global Research and Development, Groton, CT, USA; 2Conway Institute of
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age matched controls) and individuals with TBI (survival time 0-24 hours n ¼ 18, 1-28 days, n ¼ 6, 1-32 years n ¼ 13) underwent 4G8 immunohistochemistry to detect amyloid- b and AT8 immunostaining to detect neurofibrillary tangles. Sections underwent semi-quantitative assessment of intraneuronal amyloid and tau immunostaining using a scoring system of 0-3 in the dentate gyrus, CA4, CA3, CA1 and subiculum. Results: Intraneuronal amyloid was detected in both control cases and in response to mild brain injury. There was a significant increase in intraneuronal amyloid in all fields of the hippocampus in the TBI cases compared with controls (p ¼ 0.0027 -0.0135). Intense immunostaining (grade 3) was present in 0-15% of control cases and 45-55% of TBI cases. Intraneuronal amyloid increased with increasing survival time following TBI and was most extensive in individuals who survived longest after injury (p ¼ 0.0035 - 0.0087 compared to controls). There was no evidence of increased neurofibrillary tangles in the head injury as compared to controls. Conclusions: This study indicates that mild brain injury is sufficient to induce alterations in amyloid and highlights the need to understand more about the mechanisms by which injury can lead to AD. P1-235
AGGREGATION OF THE Ab(1-40) PEPTIDE: SECONDARY STRUCTURE CHANGE FROM OLIGOMERS TO FIBRILS
Rabia Sarroukh1, Emilie Cerf1, Sylvie Derclaye2, Yves F. Dufrene2, JeanMarie Ruysschaert1, Erik Goormaghtigh1, Vincent Raussens1, 1Universite Libre de Bruxelles, Bruxelles, Belgium; 2Universite Catholique de Louvain, Louvain, Belgium. Contact e-mail: [email protected] Background: Amyloid-b peptide (Ab) is the primary component of the senile plaques and plays an important, but not completely understood, role in the pathogenesis of Alzheimer’s disease. Ab can form different entities: monomeric, large soluble entities collectively called oligomers and insoluble fibrils. In the early stages of aggregation, Ab forms oligomers, which are considered as the most toxic forms. Continued aggregation gives rise to fibrils formation. The mechanisms leading to accumulation of misfolded peptide and the fibrils formation still remain unknown. We have previously shown, in our laboratory, that the oligomeric species and fibrils of Ab42 display distinct spectral features by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Whereas fibrils display spectral features of parallel b-sheet, oligomers adopt an antiparallel b-sheet structure. Methods: ATR-FTIR: Attenuated Total Reflection-Fourier Transform Infrared Spectrocopy Results: Based on these observations, we followed the aggregation of Ab40 using ATR-FTIR. We observed that Ab40 polymerized, in our case, first into antiparallel b-sheet oligomers which are recognized by a conformational antibody specific to these species (A11). Ab moves then from an antiparallel to a parallel b-sheet structure. This shift from oligomers to fibrils observed by ATR-FTIR was in perfect agreement with the marked increase in ThT fluorescence upon fibrils formation. This conformational change supposes a reorganization of the peptide upon aggregation, which is also observed using monoclonal region-specific antibodies used to study the accessibility of the aggregates. Conclusions: We demonstrated that ATR-FTIR is a powerful tool for protein aggregation studies: ATR-FTIR (i) can discriminate between Ab oligomers and fibrils from a structural point of view (ii) shows a conformational change in the structure of the peptide upon aggregation, perfectly correlated to the formation of fibrils, as probed by fluorescence. P1-236
E2012: A NOVEL GAMMA-SECRETASE MODULATOR-PHARMACOLOGY PART
Toshihide Hashimoto1, Akira Ishibashi1, Hiroaki Hagiwara1, Yoshiyuki Murata1, Osamu Takenaka1, Takehiko Miyagawa2, 1Eisai Co. Ltd., Tsukuba, Japan; 2Eisai Co. Ltd. Discovery Research, London, United Kingdom. Contact e-mail: [email protected] Background: Cerebral amyloid-beta (Ab) deposition is believed to play a central role in the pathogenesis of Alzheimer’s disease (AD). Ab is produced by the sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Gamma-secretase is also responsible for the intramembraneous cleavage of Notch and other substrates. While in-
hibition of gamma-secretase by classical gamma-secretase inhibitors results in reduced neurotoxic Ab species, inhibiting the cleavage of other gammasecretase substrates, including signaling molecules such as Notch, has been associated with undesirable side effects. Therefore, compounds that inhibit the production of toxic Ab species without affecting Notch cleavage represent a promising therapeutic approach for AD. Here we provide the first report on E2012, a novel compound with gamma-secretase modulatory activity. Methods: Ab40, 42 production in rat cortical neuron culture and Notch cleavage in the SUP-T1 human T-lymphoblast cell line were assayed before and after exposure to E2012. Ab40, 42 content in rat cerebrospinal fluid (CSF), brain, and plasma were measured after treatment with E2012 (3, 10, 30mg/kg for 3 days). Results: E2012 reduced the production of Ab40 and Ab42 in the rat cortical neuron culture dose-dependently without significant cytotoxicity. IC50 values of E2012 for Ab40 and Ab42 were 330 nM and 92 nM, respectively. E2012 decreased the levels of Ab40 and Ab42 in rat CSF, brain and plasma in vivo in a dose dependent manner. Especially, in rat CSF, E2012 significantly decreased Ab42 levels by 16.6% and 47.2% at doses of 10 and 30 mg/kg, respectively. The reduction in Ab42 levels in brain at doses of 10 and 30 mg/kg were 17.1% and 42.9%, respectively. Ab42 levels in plasma was decreased by 59.2, 90.8, and 96.1% at doses of 3, 10, and 30 mg/kg, respectively. The compound had no effect on Notch processing in vitro up to 3 mM. Conclusions: Our results suggest that E2012 is a novel and potent gamma-secretase modulator that reduces Ab40 and Ab42 levels without affecting Notch cleavage. P1-237
FIBRILLOGENESIS AND TOXICITY OF IOWA-Ab AMYLOID IS PRIMARILY DRIVEN BY THE D23N MUTATION
Agueda Rostagno, Silvia Fossati, Jorge Ghiso, NYU School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Mutations within the amyloid-b (Ab) sequence, especially those clustered at residues 21-23, are linked to early onset familial Alzheimer’s disease (AD) and primarily associated with cerebral amyloid angiopathy (CAA). Biochemical analysis of brain tissue deposits in AbD23N Iowa cases indicate the presence of complex mixtures of mutated and non-mutated Ab species, highly heterogeneous at both N- and C-termini, and showing partial Asp isomerization at positions 1, 7, and 23. Methods: Our study aimed to assess the role of the IsoAsp modifications and the D23N substitution in Ab aggregation/fibrillization as well as in the induction of cell toxicity in neuronal and microvascular endothelial cells. Peptide conformations and fibrillization profiles were monitored by circular dichroism, Western blot, thioflavin-T binding, and electron microscopy studies. Toxicity of the different Ab species for SH-SY5Y and microvascular endothelial cells was assessed by evaluation of nucleosome formation via Cell Death ELISA together with LDH release. Results: The presence of isoAsp had an enhancing -albeit modest- effect on fibrillization, without significantly affecting the lag phase of wild-type Ab40. On the contrary, the D23N mutation strikingly changed the peptide behavior yielding a dramatically accelerated fibrillization profile. Notably, AbD23N homologues bearing Asp residues rendered overlapping results with the mutant peptide containing IsoAsp. Evaluation of cell death mechanisms correlated with the fibrillization tendency, being AbD23N species highly toxic and inducing comparable apoptotic and LDH-release levels irrespective of their isoAsp content. Conclusions: The high fibrillization proclivity of AbD23N species containing isoAsp modifications, as found in the Iowa brain deposits, is primarily driven by the presence of the mutation and correlates with exacerbated neuronal and endothelial cell toxicity that could explain the early onset of the disease in the Iowa kindred. P1-238
SOLUBLE Ab FROM PSAPP MICE IS DIFFERENT TO THAT EXTRACTED FROM ALZHEIMER PATIENTS
Warren D. Hirst1, Elie Needle1, Roland G. W. Staal1, Robert Crozier1, Nathalie Breysse1, Reka Nagy1, Amy Sung1, Michael Monaghan1, David R. Riddell1, Jeannie Chin1, Dominic M. Walsh2, Dominic M. Walsh2, Menelas N. Pangalos1, Peter H. Reinhart1, J. S. Jacobsen1, 1Pfizer Global Research and Development, Groton, CT, USA; 2Conway Institute of