- Email: [email protected]
HDAC activity suppresses amyloid β-42 accumulation and toxicity through multiple mechanisms
Poster Presentations P2 RCT to ApoA1 or HDL. Unlike bCECs, no modification of ABCG1 expression was observed in pericytes, suggesting that oxysterols have a cell type-dependent effect. Conclusions: Altogether, these results suggest involvement of 24S and 27-OHC in BBB-RCT and highlight a new role of pericytes and bCECs in brain cholesterol homeostasis. As BBB is also involved in Aß peptides exchanges between brain and blood, we will next investigate effects of these oxysterols on Aß peptides transport across the BBB. P2-265
AMYLOID-BETA43 IS THE POTENT PATHOLOGICAL ACCELERATOR FOR ALZHEIMER’S DISEASE
Takashi Saito, Takaomi Saido, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. Background: Presenilin-1 (PS1) associated with familial Alzheimer’s disease (FAD)-causing mutations increase the production or the ratio of Amyloid Beta peptide with residues 42 (ABeta42), the potent pathogenic agent for Alzheimer’s disease (AD). Recently, a R278I misssense mutation in PS1 gene was found to generate a high level of ABeta43 in a cell-based study. This mutation altered the gamma-secretase activity and increased the production of ABeta43. To investigate the pathogenic role of ABeta43, it is necessary to understand the mechanism for amlyoid pathology formation. Methods: To address this issue, we generated a PS1-R278I knock-in (KI) mice. The mice were analyzed by biochemical, immunohistochemical, behavioral study. Results: The PS1-R278I mutation increase the production of ABeta43 at the expense of a decrease of ABeta40 production, and PS1R278I KI heterozygous mice crossbred with APP transgenic (Tg) mouse showed accelerated ABeta depositions in the cortical and hippocampal region. In such mice brain, both the amounts and the ratios of ABeta42 and ABeta43, particularly ABeta43, were significantly increased. Generated ABeta43 leads to accelerated amyloid pathology due to the formation of mature amyloid plaques in the transgenic mice brain as well as in AD brain. In addition, ABeta43 affected the short-term memory formation in APP Tg x PS1-R278I KI mice, prior to the plaque formation. Furthermore, ABeta43 markedly promoted ABeta toxicity and aggregation in vitro more than ABeta40 and ABeta42. We also found that other pathogenic mutations in presenilin-1 cause overproduction of ABeta43 in a manner correlating with ABeta42 and with age of onset. Conclusions: These findings indicate that ABeta43 acts as a potent pathological accelerator for amyloidgenesis in the brain. Our findings also suggest that facilitation of the ABeta43-toABeta40 conversion that takes place in the gamma-secretase complex would be beneficial for prevention of ABeta amyloidosis by decreasing ABeta43. P2-266
AN EFFECT OF I143T PSEN1 MUTATION ON THE GENERATION OF AMYLOID B IN CULTURE CELLS
Nobuo Sanjo1, Temu Qina1, Takumi Hori1, Paul Fraser2, Hidehiro Mizusawa1, 1Tokyo Medical and Dental University, Tokyo, Japan; 2 University of Toronto, Toronto, Ontario. Background: Preseninlin-1 is the catalytic subunit of the g-secretase complex which cleaves amyloid b precursor protein (bAPP) to form b Amyloid peptide (Ab). Mutations in Presenilin gene (PSEN1 and PSEN2) have been demonstrated to be the major account for the Early Onset Familial Alzheimer Disease (EOFAD). Recently, both Ab42 and 43 were reported to be present in plaque cores in the brain of the patients with I143T mutation, which is one of the earliest onset causative genes of EOF AD. (We analyzed an effect of various PSEN mutations on the production of Ab42 and 43 using culture cells. Methods: In order to investigate the effect of PSEN1 mutations on the production of Ap, HEK293 cells stably over-expressing APPswe were transiently transfected with several PSEN1 mutations, and after 24-hour incubation, quantitative change of amount of Ab40, Ab42 and Ab43 in cell culture medium was measured by ELISA. Results: Cells transfected with PSEN1 I143T produced the highest ratio of Ab42, but the same ratio of Ab43 to Ab40, compared with cells transfected with wild type PSEN1, wild type PSEN2, other PSEN1 mutations and PSEN2 mutations.
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Conclusions: The ratio of Ab42 to Ab40, but not of Ab43, is possibly the important factor for the age at onset of EOF AD I143T, despite the deposition in the brain of the patients mainly consisting of both types of Abs. P2-267
MITOCHONDRIAL DYNAMICS IN ALZHEIMER’S DISEASE
Karen Schmitt1, Steven Brown2, Stephan Frank3, Anne Eckert4, 1 Neurobiology Lab for Brain Aging & Mental Health, Universit€are Psychiatrische Kliniken Basel, Basel, Switzerland; 2Chronobiology and Sleep Research Group Institut f€ur Pharmakologie und Toxikologie, Zurich, Switzerland; 3Institute of Pathology Department of Neuropathology Basel University Hospital, Basel, Switzerland; 4Neurobiology Laboratory For Brain Aging and Mental Health Assoc. Res. Group Dept. Biomedicine Univ. of Basel Psychiatric University Clinics Basel, Basel, Switzerland. Background: Mitochondrial dysfunction is a hallmark of amyloid-beta (Aß)-induced neuronal toxicity in Alzheimer’s disease (AD). Aß triggers mitochondrial dysfunction through several pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production and alteration of mitochondrial dynamics. Particularly, the control and maintenance of mitochondrial dynamics requires a tightly regulated equilibrium between opposing mitochondrial fusion and fission activities. Importantly, disturbances of fusion and fission activities or their imbalance result in mitochondrial impairment that might play a crucial role in neurodegeneration. The aim of this study is to better understand the key role of mitochondrial dynamics in AD. Methods: For this purpose, we first characterized the mitochondrial network by confocal microscope in cell cycle-controlled human primary skin fibroblasts. Then, we analysed the expression levels of mitochondrial fusion (MFN1, MFN2, and OPA1) and fission (DNML1, Fis1) genes and the expression of ascertained genes involved in the mitochondrial respiration. Results: We found that the mitochondrial network is characterized by 3 distinct states (fragmented, intermediate and tubular), during one cell cycle. Our preliminary data show that the expression of mitochondrial fusion genes is enhanced in the tubular mitochondrial network whereas expression of mitochondrial fission genes does not change between tubular and fragmented mitochondrial network. In addition, an increase of the expression of complex V subunits involved in ATP production was observed in fragmented mitochondrial network but mot in tubular network. Plus, in between the switch of tubular to fragmented mitochondrial network, we observed a transient increase in ATP level. Conclusions: Currently, we investigate changes induced by Aß in the regulation of mitochondrial dynamics and energy homeostasis. We expect from these experiments new insights into the vicious cycle between abnormal mitochondrial dynamics, mitochondrial function and energy production in AD. Acknowledgements: This work was supported by grants from the Swiss National Science Foundation (#31000_122572) and Synapsis Foundation. P2-268
INHIBITION OF SIN3A/HDAC ACTIVITY SUPPRESSES AMYLOID b-42 ACCUMULATION AND TOXICITY THROUGH MULTIPLE MECHANISMS
Michiko Sekiya1, Kanae Iijima-Ando2, Koichi Iijima2, 1Thomas jefferson University, Philadelphia, Pennsylvania, United States; 2Thomas Jefferson University, Philadelphia, Pennsylvania, United States. Background: Sin3A is a transcriptional co repressor, which associates with histone deacetylases (HDACs) to regulate the accessibility of chromatin. Although underlying mechanisms are not fully understood, recent studies have revealed that inhibition of Sin3A/HDACs functions in neurons is protective against polyglutamine- and a-synuclein induced toxicity in animal models of Huntington’s and Parkinson’s diseases. These results suggest that Sin3A/ HDAC may be a common therapeutic target for several human neurodegenerative diseases. Methods: Here we provide evidence that inhibition of Sin3A/HDACs activity is protective against human Aß42 toxicity using a transgenic Drosophila model. Results: Knockdown of Sin3A and SAP130, a binding partner of Sin3A, suppressed Aß42-induced behavioral deficits and neurodegeneration. We found that Sin3A/SAP130 knockdown significantly reduced Aß42 accumulation in fly brains by increasing
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Poster Presentations P2
transcription of genes involved in protein quality control and degradation pathways as well as fly homologue of Aß degrading enzymes. In addition, genes known to be induced by stresses were up regulated by Sin3A knockdown, and overexpression of these genes suppressed Aß42 toxicity without altering Aß accumulation. We further demonstrated that these genes were up regulated in human neuroblastoma SH-SY5Y cells treated with HDAC inhibitors. Conclusions: Our results indicate that multiple mechanisms underlie protective effects of inhibition of Sin3A/HDACs activity against Aß42induced toxicity. P2-269
INDUCTION OF CEREBRAL AMYLOIDOSIS AND MEMORY IMPAIRMENTS BY BLOOD TRANSFUSION IN MICE MODELS OF ALZHEIMER’S DISEASE
Rodrigo Morales1, Claudia Duran-Aniotz1, Akihiko Urayama1, Lisbell D Estrada2, Diego Morales-Scheihing1, Claudio Soto1, 1University of Texas Medical School at Houston, Houston, Texas, United States; 2 Universidad Catolica de Chile, Santiago, Chile. Background: Aß misfolding and oligomerization is a hallmark event in Alzheimer’s disease (AD) pathogenesis, however the factors that initiate this pathological process are not understood. Recent, exciting reports have shown that intra-cerebral or intra-peritoneal injection of brain homogenates containing Aß aggregates promotes the formation of cerebral Aß plaques in mice models of AD. However, it remains to be demonstrated if this phenomenon can occur by more relevant routes of administration. The goal of this study was to assess whether AD pathogenesis can be induced intravenously, mimicking the known transmission of prion diseases through blood transfusion. Methods: Young (2 months old) Tg2576 mice were injected with different boosts of blood obtained from 12 months old Tg2576 mice containing cerebral Ab deposits. Mice were tested for spatial memory impairment using Barnes Maze test and were sacrificed at 250 days old, a time in which these animals scarcely develop Aß deposits. Brains were analyzed by immunohistochemistry for Aß deposition, brain inflammation, and synaptic and neuronal damage. In addition, we performed a similar experiment in a double transgenic mice model which develops Aß plaques at an accelerated rate (4 months old) Results: We observed that infusion of blood from old Tg2576 mice significantly enhances Aß aggregation in the brain of treated mice. Indeed, whereas a substantial Aß accumulation was observed in this group of animals, Tg2576 mice no treated or injected with blood from wild type littermates showed barely any detectable Aß lesions. Plaque deposition was mainly present in cortex and hippocampus. In addition, we observed a decrease in memory in mice challenged with Aß containing blood. Other features such as brain inflammation and neuro-synaptic damage were also measured. Importantly, similar results were obtained in a second and independent experiment performed in a double transgenic mouse model. Conclusions: Our results indicate that an AD-like pathogenesis can be induced by transfusion of blood from animals bearing cerebral amyloid pathology, presumably through induction of protein misfolding in a similar way as prion diseases. These findings may open a new avenue to understand the origin of sporadic AD and may provide new strategies for disease intervention and prevention. P2-270
A NEW IMMUNOASSAY BASED ON SURFACE PLASMON RESONANCE FOR DETECTION OF b-AMYLOID OLIGOMERS
Matteo Stravalaci1, Mara Canovi1, Margherita Romeo1, Laura Tapella1, Marten Beeg2, Alfredo Cagnotto1, Mario Salmona1, Roberto Chiesa1, Luisa Diomede1, Marco Gobbi1, 1Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy; 2ETH-Zurich / Swiss Federal Institute of Technology-Zurich, Zurich, Switzerland. Background: Recent evidence suggests that soluble oligomers formed by the aggregation of amyloid-beta peptide (Aß) may play a key function in Alzheimer’s disease. Detection of these oligomeric species and analysis of the kinetics of their formation is required for a better insight into their pathogenic role. However, this is a difficult task because of their
intrinsic instability, and the lack of knowledge of their precise physicochemical features. Here we report the characterization of a new immunological method for Aß oligomer detection, based on Surface Plasmon Resonance (SPR). Methods: Anti-Aß antibody 4G8 and an oligomer-specific antibody were immobilized on the surface of a sensor chip. Synthetic Aß1-42 (100 mM) was incubated at either 22 C or 37 C to allow oligomerization, and aliquots were taken at different time points (from t ¼ ^ 0 to t ¼ 72h), diluted to 1 AmM and flowed over the immobilized antibodies. In the second part of the study, we flowed lysates obtained from a transgenic C. elegans strain, engineered to inducibly express and accumulate Aß1-42 oligomers. Results: The sensor grams (time course of the SPR binding signal) obtained with 4G8 could be fitted by a model involving the presence of two flowing Aß species, discernible because of their different binding constants. The first was preponderant at t ¼ 0 (freshly prepared Aß solution) and decreased during the incubation, suggesting that it corresponded to “monomers”. Their half-life was temperature-dependent (1.1h at 37 C and 10.3h at 22 C). The second component, characterized by a very low dissociation rate (suggestive of multivalent interactions), showed a bell-shaped time-course, with peaktimes of 1-5h (at 37 C and 22 C) and disappearance at 24h. The same transient species were also specifically bound by the oligomer-specific antibody. Protofibrils and fibrils are not detected in this assay. Native Aß142 oligomers from C. elegans lysates were bound by both 4G8 and the oligomer-specific antibody, with very low dissociation constants. Conclusions: We described here a novel SPR-based immunoassay useful to dissect the kinetics of Aß oligomers formation and to measure their levels in biological samples. It may have important applications, for example to study the effects of mutations or pharmacological treatments on the process of Aß oligomerization. P2-271
MUTATIONS IN CODON 716 OF THE AMYLOID PRECURSOR PROTEIN DIFFERENTIALLY AFFECT APP PROCESSING
Marc Suarez-Calvet1, Cristina Guardia-Laguarta1, Marta Pera1, Nahuai Badiola1, Daniel Alcolea1, Nuria Seto-Salvia1, Oriol Dols1, Jordi Magrane2, Jordi Clarimon1, Rafael Blesa1, Alberto Lleo1, 1Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2Weill Medical College of Cornell University, New York, New York, United States.
AMYLOID-BETA43 IS THE POTENT PATHOLOGICAL ACCELERATOR FOR ALZHEIMER’S DISEASE
Takashi Saito, Takaomi Saido, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. Background: Presenilin-1 (PS1) associated with familial Alzheimer’s disease (FAD)-causing mutations increase the production or the ratio of Amyloid Beta peptide with residues 42 (ABeta42), the potent pathogenic agent for Alzheimer’s disease (AD). Recently, a R278I misssense mutation in PS1 gene was found to generate a high level of ABeta43 in a cell-based study. This mutation altered the gamma-secretase activity and increased the production of ABeta43. To investigate the pathogenic role of ABeta43, it is necessary to understand the mechanism for amlyoid pathology formation. Methods: To address this issue, we generated a PS1-R278I knock-in (KI) mice. The mice were analyzed by biochemical, immunohistochemical, behavioral study. Results: The PS1-R278I mutation increase the production of ABeta43 at the expense of a decrease of ABeta40 production, and PS1R278I KI heterozygous mice crossbred with APP transgenic (Tg) mouse showed accelerated ABeta depositions in the cortical and hippocampal region. In such mice brain, both the amounts and the ratios of ABeta42 and ABeta43, particularly ABeta43, were significantly increased. Generated ABeta43 leads to accelerated amyloid pathology due to the formation of mature amyloid plaques in the transgenic mice brain as well as in AD brain. In addition, ABeta43 affected the short-term memory formation in APP Tg x PS1-R278I KI mice, prior to the plaque formation. Furthermore, ABeta43 markedly promoted ABeta toxicity and aggregation in vitro more than ABeta40 and ABeta42. We also found that other pathogenic mutations in presenilin-1 cause overproduction of ABeta43 in a manner correlating with ABeta42 and with age of onset. Conclusions: These findings indicate that ABeta43 acts as a potent pathological accelerator for amyloidgenesis in the brain. Our findings also suggest that facilitation of the ABeta43-toABeta40 conversion that takes place in the gamma-secretase complex would be beneficial for prevention of ABeta amyloidosis by decreasing ABeta43. P2-266
AN EFFECT OF I143T PSEN1 MUTATION ON THE GENERATION OF AMYLOID B IN CULTURE CELLS
Nobuo Sanjo1, Temu Qina1, Takumi Hori1, Paul Fraser2, Hidehiro Mizusawa1, 1Tokyo Medical and Dental University, Tokyo, Japan; 2 University of Toronto, Toronto, Ontario. Background: Preseninlin-1 is the catalytic subunit of the g-secretase complex which cleaves amyloid b precursor protein (bAPP) to form b Amyloid peptide (Ab). Mutations in Presenilin gene (PSEN1 and PSEN2) have been demonstrated to be the major account for the Early Onset Familial Alzheimer Disease (EOFAD). Recently, both Ab42 and 43 were reported to be present in plaque cores in the brain of the patients with I143T mutation, which is one of the earliest onset causative genes of EOF AD. (We analyzed an effect of various PSEN mutations on the production of Ab42 and 43 using culture cells. Methods: In order to investigate the effect of PSEN1 mutations on the production of Ap, HEK293 cells stably over-expressing APPswe were transiently transfected with several PSEN1 mutations, and after 24-hour incubation, quantitative change of amount of Ab40, Ab42 and Ab43 in cell culture medium was measured by ELISA. Results: Cells transfected with PSEN1 I143T produced the highest ratio of Ab42, but the same ratio of Ab43 to Ab40, compared with cells transfected with wild type PSEN1, wild type PSEN2, other PSEN1 mutations and PSEN2 mutations.
S397
Conclusions: The ratio of Ab42 to Ab40, but not of Ab43, is possibly the important factor for the age at onset of EOF AD I143T, despite the deposition in the brain of the patients mainly consisting of both types of Abs. P2-267
MITOCHONDRIAL DYNAMICS IN ALZHEIMER’S DISEASE
Karen Schmitt1, Steven Brown2, Stephan Frank3, Anne Eckert4, 1 Neurobiology Lab for Brain Aging & Mental Health, Universit€are Psychiatrische Kliniken Basel, Basel, Switzerland; 2Chronobiology and Sleep Research Group Institut f€ur Pharmakologie und Toxikologie, Zurich, Switzerland; 3Institute of Pathology Department of Neuropathology Basel University Hospital, Basel, Switzerland; 4Neurobiology Laboratory For Brain Aging and Mental Health Assoc. Res. Group Dept. Biomedicine Univ. of Basel Psychiatric University Clinics Basel, Basel, Switzerland. Background: Mitochondrial dysfunction is a hallmark of amyloid-beta (Aß)-induced neuronal toxicity in Alzheimer’s disease (AD). Aß triggers mitochondrial dysfunction through several pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production and alteration of mitochondrial dynamics. Particularly, the control and maintenance of mitochondrial dynamics requires a tightly regulated equilibrium between opposing mitochondrial fusion and fission activities. Importantly, disturbances of fusion and fission activities or their imbalance result in mitochondrial impairment that might play a crucial role in neurodegeneration. The aim of this study is to better understand the key role of mitochondrial dynamics in AD. Methods: For this purpose, we first characterized the mitochondrial network by confocal microscope in cell cycle-controlled human primary skin fibroblasts. Then, we analysed the expression levels of mitochondrial fusion (MFN1, MFN2, and OPA1) and fission (DNML1, Fis1) genes and the expression of ascertained genes involved in the mitochondrial respiration. Results: We found that the mitochondrial network is characterized by 3 distinct states (fragmented, intermediate and tubular), during one cell cycle. Our preliminary data show that the expression of mitochondrial fusion genes is enhanced in the tubular mitochondrial network whereas expression of mitochondrial fission genes does not change between tubular and fragmented mitochondrial network. In addition, an increase of the expression of complex V subunits involved in ATP production was observed in fragmented mitochondrial network but mot in tubular network. Plus, in between the switch of tubular to fragmented mitochondrial network, we observed a transient increase in ATP level. Conclusions: Currently, we investigate changes induced by Aß in the regulation of mitochondrial dynamics and energy homeostasis. We expect from these experiments new insights into the vicious cycle between abnormal mitochondrial dynamics, mitochondrial function and energy production in AD. Acknowledgements: This work was supported by grants from the Swiss National Science Foundation (#31000_122572) and Synapsis Foundation. P2-268
INHIBITION OF SIN3A/HDAC ACTIVITY SUPPRESSES AMYLOID b-42 ACCUMULATION AND TOXICITY THROUGH MULTIPLE MECHANISMS
Michiko Sekiya1, Kanae Iijima-Ando2, Koichi Iijima2, 1Thomas jefferson University, Philadelphia, Pennsylvania, United States; 2Thomas Jefferson University, Philadelphia, Pennsylvania, United States. Background: Sin3A is a transcriptional co repressor, which associates with histone deacetylases (HDACs) to regulate the accessibility of chromatin. Although underlying mechanisms are not fully understood, recent studies have revealed that inhibition of Sin3A/HDACs functions in neurons is protective against polyglutamine- and a-synuclein induced toxicity in animal models of Huntington’s and Parkinson’s diseases. These results suggest that Sin3A/ HDAC may be a common therapeutic target for several human neurodegenerative diseases. Methods: Here we provide evidence that inhibition of Sin3A/HDACs activity is protective against human Aß42 toxicity using a transgenic Drosophila model. Results: Knockdown of Sin3A and SAP130, a binding partner of Sin3A, suppressed Aß42-induced behavioral deficits and neurodegeneration. We found that Sin3A/SAP130 knockdown significantly reduced Aß42 accumulation in fly brains by increasing
S398
Poster Presentations P2
transcription of genes involved in protein quality control and degradation pathways as well as fly homologue of Aß degrading enzymes. In addition, genes known to be induced by stresses were up regulated by Sin3A knockdown, and overexpression of these genes suppressed Aß42 toxicity without altering Aß accumulation. We further demonstrated that these genes were up regulated in human neuroblastoma SH-SY5Y cells treated with HDAC inhibitors. Conclusions: Our results indicate that multiple mechanisms underlie protective effects of inhibition of Sin3A/HDACs activity against Aß42induced toxicity. P2-269
INDUCTION OF CEREBRAL AMYLOIDOSIS AND MEMORY IMPAIRMENTS BY BLOOD TRANSFUSION IN MICE MODELS OF ALZHEIMER’S DISEASE
Rodrigo Morales1, Claudia Duran-Aniotz1, Akihiko Urayama1, Lisbell D Estrada2, Diego Morales-Scheihing1, Claudio Soto1, 1University of Texas Medical School at Houston, Houston, Texas, United States; 2 Universidad Catolica de Chile, Santiago, Chile. Background: Aß misfolding and oligomerization is a hallmark event in Alzheimer’s disease (AD) pathogenesis, however the factors that initiate this pathological process are not understood. Recent, exciting reports have shown that intra-cerebral or intra-peritoneal injection of brain homogenates containing Aß aggregates promotes the formation of cerebral Aß plaques in mice models of AD. However, it remains to be demonstrated if this phenomenon can occur by more relevant routes of administration. The goal of this study was to assess whether AD pathogenesis can be induced intravenously, mimicking the known transmission of prion diseases through blood transfusion. Methods: Young (2 months old) Tg2576 mice were injected with different boosts of blood obtained from 12 months old Tg2576 mice containing cerebral Ab deposits. Mice were tested for spatial memory impairment using Barnes Maze test and were sacrificed at 250 days old, a time in which these animals scarcely develop Aß deposits. Brains were analyzed by immunohistochemistry for Aß deposition, brain inflammation, and synaptic and neuronal damage. In addition, we performed a similar experiment in a double transgenic mice model which develops Aß plaques at an accelerated rate (4 months old) Results: We observed that infusion of blood from old Tg2576 mice significantly enhances Aß aggregation in the brain of treated mice. Indeed, whereas a substantial Aß accumulation was observed in this group of animals, Tg2576 mice no treated or injected with blood from wild type littermates showed barely any detectable Aß lesions. Plaque deposition was mainly present in cortex and hippocampus. In addition, we observed a decrease in memory in mice challenged with Aß containing blood. Other features such as brain inflammation and neuro-synaptic damage were also measured. Importantly, similar results were obtained in a second and independent experiment performed in a double transgenic mouse model. Conclusions: Our results indicate that an AD-like pathogenesis can be induced by transfusion of blood from animals bearing cerebral amyloid pathology, presumably through induction of protein misfolding in a similar way as prion diseases. These findings may open a new avenue to understand the origin of sporadic AD and may provide new strategies for disease intervention and prevention. P2-270
A NEW IMMUNOASSAY BASED ON SURFACE PLASMON RESONANCE FOR DETECTION OF b-AMYLOID OLIGOMERS
Matteo Stravalaci1, Mara Canovi1, Margherita Romeo1, Laura Tapella1, Marten Beeg2, Alfredo Cagnotto1, Mario Salmona1, Roberto Chiesa1, Luisa Diomede1, Marco Gobbi1, 1Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy; 2ETH-Zurich / Swiss Federal Institute of Technology-Zurich, Zurich, Switzerland. Background: Recent evidence suggests that soluble oligomers formed by the aggregation of amyloid-beta peptide (Aß) may play a key function in Alzheimer’s disease. Detection of these oligomeric species and analysis of the kinetics of their formation is required for a better insight into their pathogenic role. However, this is a difficult task because of their
intrinsic instability, and the lack of knowledge of their precise physicochemical features. Here we report the characterization of a new immunological method for Aß oligomer detection, based on Surface Plasmon Resonance (SPR). Methods: Anti-Aß antibody 4G8 and an oligomer-specific antibody were immobilized on the surface of a sensor chip. Synthetic Aß1-42 (100 mM) was incubated at either 22 C or 37 C to allow oligomerization, and aliquots were taken at different time points (from t ¼ ^ 0 to t ¼ 72h), diluted to 1 AmM and flowed over the immobilized antibodies. In the second part of the study, we flowed lysates obtained from a transgenic C. elegans strain, engineered to inducibly express and accumulate Aß1-42 oligomers. Results: The sensor grams (time course of the SPR binding signal) obtained with 4G8 could be fitted by a model involving the presence of two flowing Aß species, discernible because of their different binding constants. The first was preponderant at t ¼ 0 (freshly prepared Aß solution) and decreased during the incubation, suggesting that it corresponded to “monomers”. Their half-life was temperature-dependent (1.1h at 37 C and 10.3h at 22 C). The second component, characterized by a very low dissociation rate (suggestive of multivalent interactions), showed a bell-shaped time-course, with peaktimes of 1-5h (at 37 C and 22 C) and disappearance at 24h. The same transient species were also specifically bound by the oligomer-specific antibody. Protofibrils and fibrils are not detected in this assay. Native Aß142 oligomers from C. elegans lysates were bound by both 4G8 and the oligomer-specific antibody, with very low dissociation constants. Conclusions: We described here a novel SPR-based immunoassay useful to dissect the kinetics of Aß oligomers formation and to measure their levels in biological samples. It may have important applications, for example to study the effects of mutations or pharmacological treatments on the process of Aß oligomerization. P2-271
MUTATIONS IN CODON 716 OF THE AMYLOID PRECURSOR PROTEIN DIFFERENTIALLY AFFECT APP PROCESSING
Marc Suarez-Calvet1, Cristina Guardia-Laguarta1, Marta Pera1, Nahuai Badiola1, Daniel Alcolea1, Nuria Seto-Salvia1, Oriol Dols1, Jordi Magrane2, Jordi Clarimon1, Rafael Blesa1, Alberto Lleo1, 1Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2Weill Medical College of Cornell University, New York, New York, United States.