P4-075

Poster P4:: Wednesday Posters A␤, and it is thought that clustering of LRs may represent a physiological mechanism for the modulation of A␤ production. Thus, insulin may modulate amyloidogenesis, at least in part, by affecting the dynamics of LR clustering. Objective(s): Accordingly our aim is to investigate whether the mechanism whereby insulin effectors regulate A␤ generation involve changes in lipid raft clustering dynamics. Methods: We have treated CHO cells, SH-SY5Y neuroblastoma cells and primary mouse neurons with insulin or IGF-1, and monitored the presence of relevant lipid raft markers under basal and stimulated conditions. To assess the role of specific downstream effectors of insulin-mediated A␤ generation and lipid raft clustering, we have transfected wildtype, dominant-negative or constitutively active forms of relevant small GTPases. Results: We have determined the effects of different insulin effectors on lipid raft homeostasis and amyloidogenesis. Conclusions: Our studies address the role of specific insulin signalling pathways on amyloidogenesis and membrane dynamics, and contribute to our understanding of how insulin may play a role in the pathology of AD. P4-073

BACE AND C99 PROTEIN LEVELS IN PRIMARY ASTROCYTES FROM THE FRONTAL CORTEX OF ALZHEIMER’S DISEASE AND NORMAL CONTROL BRAINS

Yoshihiro Konishi1, Yong Shen2, 1National Tottori Medical Center, Tottori, Japan; 2Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: [email protected] Background: The deposition of amyloid ␤ peptide (A␤) is one of the predominant pathological hallmarks of Alzheimer’s disease (AD). A␤ derives from the sequential cleavage of amyloid precursor protein (APP) by ␤- and ␥-secretases. BACE (beta-site APP-cleaving enzyme) was cloned and characterized as the most possible candidate of the long soughtafter ␤-secretase. However, the mechanisms under the A␤ deposition in AD patients, particularly sporadic cases remain unclear. We have recently demonstrated that BACE mRNA and protein expression is elevated, and the elevation of BACE enzymatic activity is correlated with A␤ production in the temporal cortex and hippocampus of sporadic AD brain. Although the major cellular source of BACE in brain is extensively believed to be neurons, in vitro studies using rodent primary astrocytes and the human astrocytoma cell line have recently revealed that activated astrocytes can express BACE. Objective: The purpose of this study is to address the issue that glial cells, i.e. astrocytes, express BACE, and if do they, how glial BACE participates in A␤ production in AD brain. Methods: By using primary astrocytes cultured from brain autopsy samples of AD and nondemented elderly control (ND) subjects that enrolled in the Sun Health Research Institute Brain Donation Program, we conducted Western blot analysis, immunoprecipitation, ELISA, and enzymatic activity assay for comparative determination of BACE and C99 (a product cleaved by BACE) levels, and immunocytochemistry for comparison of subcellular localization of BACE, between AD and normal brains. Results and Conclusions: Our initial experiments demonstrated that activated astrocytes from AD brain are more capable of participating in A␤ production than those from ND brain. The result suggests that glial BACE does play a role in A␤ production in AD brain. P4-074

ANALYSIS OF THE RELATIONSHIP BETWEEN AD PATHOLOGY AND THE LEVELS AND LOCALIZATION OF BACE1 AND BACE2 WITH RESPECT TO UNDERSTANDING INCREASED ␤SECRETASE ACTIVITY IN AD

John Stockley1, Rivka Ravid2, Cora O’Neill1, 1BioSciences Institute, Cork, Ireland; 2The Netherlands Brain Bank, Amsterdam, The Netherlands. Contact e-mail: [email protected] Background: Understanding ␤-secretase activity in the human brain is central to elucidating the role of the enzyme in A␤ production in AD. The aspartyl proteases BACE1 and BACE2 have been identified to have

S535

␤-secretase activity. BACE1 is the major ␤-secretase responsible for A␤ production, whereas BACE2 preferentially cleaves APP between Phe19 and Phe20 or Phe20 and Ala21of A␤, and may inhibit ␤-site APP cleavage. Objective(s): This study aims to examine the relationship between ␤-secretase enzyme kinetics, BACE1/2 protein levels and BACE1/2 cellular and subcellular distribution in normal brain and in AD. Results: We performed a comparative analysis of ␤-secretase enzyme kinetics in control (n ⫽ 7) and AD (n ⫽ 9) mid-temporal cortex, graded for the severity of AD pathology, using increasing concentrations (1- 50 ␮M) of the wild type APP peptide substrate, MCA-EVKMDAEFK-DNP. Enzyme activity was saturable and followed Michaelis-Menten kinetics in both AD and control groups. No significant difference was detected between Km values when comparing AD and control groups. However, Vmax values were significantly increased in the AD cases (5.8 ⫹/- 0.66nM/min) compared to controls (3.5 ⫹/- 0.41nM/min). Vmax values for ␤-secretase activity did not correlate with the protein levels of either BACE1 or BACE2 in AD or control samples, after analysis and quantification using western immunoblotting. This discrepancy indicates that increased ␤-secretase activity in AD may be associated with altered conformation of BACE1 and possibly BACE2, and/or changes in their cellular/subcellular localization. To understand this we have first examined the regional distribution and cellular localization of BACE1 and APP in adult mouse hippocampal formation. BACE1 and APP are detected primarily in neurons of the dentate gyrus, CA2, CA3 and the entorhinal cortex. BACE1 is present throughout the cell soma and co-localizes with APP near the plasma membrane. However, not all APP positive cells contain BACE1. Some BACE1 immunoreactivity colocalized with GFAP positive glia in the stratum oriens and molecular layer. We are characterizing the localization of APP, BACE1 and BACE2 and their relationship to A␤ and tau pathology in AD. This work will be presented, and it contributes to further understanding ␤-secretase function in AD pathogenesis. P4-075

FIBRILLAR A␤ TRIGGERS INSULIN DEGRADING ENZYME (IDE) OVER-EXPRESSION BY GLIAL CELLS IN OLD TG2576 MICE

Maria C. Leal1, Veronica B. Dorfman1, Agata C. Fernandez Gamba1, Blas Frangione2, Thomas Wisniewski3, Eduardo M. Castan˜o1, Einar M. Sigurdsson4, Laura Morelli1, 1Fundacion Instituto Leloir, Buenos Aires, Argentina; 2Departments of Psychiatry and Pathology, New York University School of Medicine, New York, NY, USA; 3 Departments of Psychiatry, Pathology and Neurology, New York University School of Medicine, New York, NY, USA; 4Departments of Psychiatry and Pathology, New York, NY, USA. Contact e-mail: [email protected] Background: It was proposed that IDE participates in the clearance of amyloid ␤ (A␤) in the brain and its low expression or activity may be relevant for the progression of Alzheimer’s disease (AD). Objective(s): To study the profile of IDE during the neurodegenerative process in transgenic mice (Tg2576) brains, which carry the amyloid precursor protein with the Swedish mutation. Methods: Tg2576 mice and non-Tg (NTg) littermates of 4.5, 11 and 16 months of age (m) (n⫽4/group) were used to determine IDE protein and activity levels by ELISA, western-blot (WB) and immunoprecipitation assays. IDE transcripts were quantified by competitive RT-PCR. Brain sections were subjected to immunohistochemistry with anti-IDE, anti-A␤, anti-GFAP and L. esculentum lectin. Results: At 16 m Tg2576 mice showed a significant 2-fold increment in IDE protein levels as compared to 11 m (612 ⫾ 54.3 vs. 291.96 ⫾ 23.7 ng/mg, respectively) that was fully active using 125I-insulin as a substrate. No significant differences were found in the amount of IDE transcripts in whole brain homogenates, suggesting that IDE increment was not due to a widespread increase in its mRNA levels. The peak of soluble IDE was in synchrony with a sharp accumulation of SDS-soluble A␤40 (355.046 ng/mg ⫾ 2.096), and massive A␤ deposition into plaques. Moreover, at this stage, IDE appeared surrounding amyloid deposits within GFAP-positive astrocytes and activated microglia. This suggested that IDE was locally over-ex-

S536

Poster P4:: Wednesday Posters

pressed by glial cells in the context of an inflammatory response triggered by fibrillar A␤. To explore this possibility, primary rat astrocytes were used to test IDE levels after exposure to soluble or fibrillar A␤. After treatment with 5 ␮M fibrillar A␤, IDE protein and transcripts increased 2-fold as detected by WB, immunofluorescence and RT-PCR compared to soluble A␤ or control cultures. Conclusions: Our results suggest that the defective clearance of A␤ due to IDE lower expression or activity is not an accelerating factor in Tg2576 mice amyloid deposition. Moreover, IDE overexpression associated to late-stages of the neuropathologic process in this animal model may be part of an inflammatory response, suggesting a new role for IDE in the brain that deserves future investigation. P4-076

IMMOBILIZATION OF ␤-AMYLOID PEPTIDE ON A CHIP AND CHARACTERIZATION OF ITS INTERACTION WITH PROTEINS VIA ATOMIC FORCE MICROSCOPY AND MASS SPECTROMETRY

Ste´phanie Boussert1,2, 1Barcelona Science Park, Barcelona, Spain; Institut de Recerca Biome`dica - Universitat de Barcelona, Barcelona, Spain. Contact e-mail: [email protected] 2

Here we describe a strategy using self assembled monolayer (SAM), atomic force microscopy (AFM) and mass spectrometry (MS) to identify attachment of peptide on gold chip and specific peptide-protein interaction. The ␤-amyloid is a self-aggregating protein found in Alzheimer’s disease and is thought to play a major role in the disease process. The methodology of self-assembled monolayers was employed to functionalize gold surfaces with alkanethiol. In this study, ␤-amyloid (1-40) was synthesized with N-terminal cystein residue in order to achieve a specific attachment to the alkanethiol modified gold surface. Attachment of ␤-amyloid was characterized using AFM methodology. A modification of the surface corresponding to the peptide attachment was clearly observed. Size measurement of the molecule corresponded to the theoretical expected size of the ␤-amyloid peptide. Besides, the exact mass of the ␤-amyloid peptide was observed by MS experiment directly on the gold surface. This peptide array was used to characterize specific interactions between ␤-amyloid-antibody. Images from the interaction of ␤-amyloid -antibody were taken by AFM. Profile of the images and size measurement of the complex showed clear differences. Chemiluminescence was used to confirm the presence of the antibody and so the formation of the complex. We are studying now the ability of this ␤-amyloid-chip to interact specifically with proteins in complex protein extract. Moreover, we would try to identify specific interaction by MS as such an affinity capture surface should avoid protein loss and considerably simplify sample preparation for mass spectrometry analysis. High sensitivity of mass spectrometry should be well suited to this on-chip study. P4-077

MODULATION OF AMYLOID PRECURSOR PROTEIN-PROCESSING BY OXYSTEROLBINDING PROTEIN

Joanna M. Cordy1, Celina V. Zerbinatti2, Ci-Di Chen1, Maria Guillily1, William J. Ray2, Guy R. Seabrook2, Carmela R. Abraham1, Benjamin Wolozin1, 1Boston University School of Medicine, Boston, MA, USA; 2Merck & Co, West Point, PA, USA. Contact e-mail: [email protected] Background: The mechanisms by which cholesterol modulates amyloid precursor protein (APP) processing to generate the Alzheimer’s disease amyloid-␤ (A␤) peptide are poorly understood. Oxysterol-binding protein 1 (OSBP1) is a member of a family of sterol-binding proteins with roles in lipid metabolism and signal transduction. OSBP1 has binding sites for cholesterol, oxysterols, and protein phosphatases; the binding characteristics of OSBP1 suggest a role in coupling cholesterol metabolism with signal transduction. When bound to cholesterol, OSBP1 exists in the

cytoplasm/ER where it sequesters two phosphatases causing increased activation of extracellular signal-regulated kinase (ERK). Binding of 25hydroxycholesterol (25OHC) or removal of cholesterol induces translocation of OSBP1 to the Golgi, reducing ERK activity. Objectives: The role of OSBP1, cholesterol and oxysterols in the regulation of APP processing has been investigated. Methods: We transiently transfected OSBP1 into H4 glioma cells stably over-expressing APP, and examined the effects on the production of soluble APP␣/␤ (sAPP␣/␤), APP C-terminal fragments (CTF) and A␤. Results: Expression of OSBP1 increased ␣-secretase cleavage and decreased ␤-secretase cleavage of APP and these effects could be prevented by addition of 25OHC. Conversely, knockdown of OSBP1 using siRNA increased ␤-CTFs and secreted A␤ levels. Next, we examined whether OSBP1 or oxysterols affected APP cellular distribution, which might regulate exposure to ␣- and ␤-secretases. Neither over-expressing OSBP1 nor 25OHC treatment changed the apparent cellular distribution of APP. However, using bi-molecular fluorescence (BiFC) we observed that OSBP1 appears to modulate the distribution of APP dimers (APP-APP or APP-Notch2). Over-expressing OSBP1 caused sequestration of APP dimers in the Golgi. The addition of 25OHC reversed the process, inducing translocation of OSBP1 from the cytosol/ER to the Golgi and translocation of APP dimers from the Golgi to the ER. Conclusions: These data suggest that APP dimers might be excluded from organelles containing high levels of OSBP1, possibly due to competition between APP and OSBP1 for binding to a trafficking protein. Prior studies indicate that APP dimers strongly regulate A␤ production, therefore the ability of OSBP1 to alter the distribution of APP dimers within the secretory pathway demonstrates a new mechanism by which cholesterol and oxysterols may regulate APP processing. P4-078

MATRIX METALLOPROTEASES AND A␤ CLEARANCE

Yasushi Tomidokoro1, Tammaryn Lashley2, Tamas Revesz2, Steven M. Greenberg3, Blas Frangione1, Agueda Rostagno1, Jorge Ghiso1, 1NYU School of Medicine, New York, NY, USA; 2Institute of Neurology, London, United Kingdom; 3Massachusetts General Hospital, Boston, MA, USA. Contact e-mail: [email protected] Background: The composition of amyloid deposits is highly heterogeneous. Besides the existence of a variety of post-translational modifications, N- and C-terminal truncations of the deposited amyloid subunits are commonly found in sporadic and familial forms of cerebral amyloidosis. It is debatable whether they reflect the process of amyloid deposition or simply clearance mechanisms. Objective(s): To analyze the role of N- and C-terminal A␤ truncations in the process of amyloid formation and/or clearance and probe potential proteases involved. Methods: A␤ species were sequentially extracted in water-based buffers, detergent and formic acid from autopsy cases of sporadic Alzheimer’s disease, familial forms of the disease (APP Iowa mutant and the PS1⌬I83-M84 variant) and familial Danish dementia (FDD) in the presence or absence of bacterial collagenase. Analysis was carried out via immunoprecipitation, mass spectrometry and western blot analysis. Synthetic homologues to the wild-type A␤1-40 peptide and the A␤N23 Iowa mutant were used for in vitro degradation experiments using human matrix metalloproteinases MMP-2 and MMP-9 as well as bacterial collagenase. Results: Water-soluble extracts contained A␤1-34 and A␤4-34 either as the major or the sole components. However, these truncated A␤ forms were not found in amyloid deposits, questioning whether they play any role in amyloid formation. Interestingly, A␤⫻-34 species were also generated by the introduction of bacterial collagenase in the extraction protocol and mimicked in vitro with synthetic peptides by purified MMP-2, MMP-9 and bacterial collagenase. Western blot analysis indicated that monomers as well as small oligomers of A␤1-40 (dimers and trimers) were susceptible for degradation whereas those derived from the A␤N23 mutant were more resistant to proteolysis. Mass spectrometry analysis revealed that Leu34-Met35 peptide bond was the primary cleavage site of MMP-9 and a secondary site for MMP-2. For broadly specific