a transport adaptor of the Golgi-localized gamma-ear-containing ARF- binding (GGA) family, has been shown to interact with BACE via its VHS-domain. We analyzed the differential roles of GGA1, GGA2 and GGA3 upon co-localization and interaction with BACE and effects upon APP-processing. In addition we tested the hypothesis that serine-phosphor- ylation of GGA1 and GGA3 affects BACE-interaction. Methods: We ap- plied confocal imaging and fluorescence lifetime imaging microscopy (FLIM) for colocalization and interaction studies, an electrochemilumines- cence-based assay to measure the influence of all three GGA upon APP processing, westernblotting to measure intracellular levels of APP cleavage fragments and insitu-hybridization to visualize correlated expression of GGAs and BACE in rat brain. Results: All three GGAs colocalize with BACE1 at perinuclear compartments. FLIM revealed a donor-lifetime de- crease indicating interaction between all three GGAs and BACE. Control experiments with GGA and BACE mutants show that the VHS domain of the GGA proteins and the DXXLL-motive in the BACE protein are nec- essary for this interaction. Mutants of GGA1 and GGA3 which represent non-phosphorylated forms of these proteins showed decreased lifetime whereas autoinhibited pseudo-phosphorylated mutants reversed this. Elisa and Westernblotting revealed an increase of intracellular sAPP upon over- expression of any GGA. However a decrease in sAPP secretion was ob- served. This effect was neither reversible with D-VHS mutants nor with GGA1/3 phosphorylation-mutants.Insitu-hybridization revealed spatial and time correlated expression of GGAs and BACE in postnatal and adult rats. Conclusions: These results indicate that all GGAs have related func- tions on BACE interaction, controlled by concentration, location and phos- phorylation. Beside the interaction with BACE, we suggest additional BACE-independent influence of GGAs on APP transport and processing, and therefore an essential role in APP cleavage and subsequent Abeta generation. P2-142 ANALYSIS OF THE DIMERIZATION AND METABOLISM OF DIFFERENT APP ISOFORMS Naouel Ben Khalifa, Stefan N. Constantinescu, Bernadette Tasiaux, Joanne Van Hees, Pierre J. Courtoy, Patrick Vandersmissen, Jean- Christophe Renauld, Steven O. Smith, Jean-Noe ¨l Octave, Pascal Kienlen- Campard, Universite ´ Catholique de Louvain, Brussels, Belgium. Contact e-mail: Naouel.Benkhalifa@uclouvain.be Background: Proteolytic processing of the Amyloı ¨d Precursor Protein (APP) generates the b-amyloı ¨d peptide (Ab) and the APP Intracellular C-ter- minal Domain (AICD), which was reported to control expression of APP tar- get genes. Ab and AICD are produced by sequential proteolytic cleavages involving b and g-secretases. Several studies have evidenced that Ab pro- duction depends on APP dimerization. Dimerization can be controlled by motifs present in the ectodomain or the transmembrane/juxtamembrane do- main of the protein. Methods: The majors isoforms of APP (neuronal APP695 and peripheric APP751) differ by a sequence insertion in the extra- cellular domain. To investigate potential homodimeric interactions of APP751 and APP695, we have quantified the formation of APP dimers by flow cytometry using bimolecular fluorescence complementation (BiFC) analysis. This was coupled to the study of APP processing by biochemical approaches. Results: BiFC assay was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to either APP751 or APP695. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP751 ho- modimer produced a higher fluorescent signal than APP695, suggesting that APP751 isoform forms more homodimers. The results were confirmed by fluorescence microscopy in living cells. To further investigate the homodi- meric interactions of APP, we are currently developing a Fluorescence Res- onance Energy Transfer (FRET) and co-patching approaches to analyze the presence of APP dimers at the cell surface. Ab triplex assays are developed to investigate the relation existing between the dimerization state of these APP isoforms and amylodogenic processing. Conclusions: These findings could provide new insight on how APP dimerization controls its metabolism and function, and highlight important differences between neuronal APP and APP from other cell types. P2-143 EFFECTS OF SECRETASE INHIBITORS ON APP PROCESSING IN PRIMARY CULTURES OF EMBRYONIC CHICKEN CORTICAL NEURONS Stefan Czvitkovich, Birgit Hutter-Paier, Robert Wronski, Evelyn Schauer, Else Mathiesen, Manfred Windisch, JSW-Research, Grambach, Austria. Contact e-mail: sczvitkovich@jswresearch.com Background: Secretases involved in APP processing are recognized as po- tential therapeutic targets to counteract brain amyloid pathology in AD. This study was carried out to evaluate and compare the efficacy of different sec- retase inhibitors in an assay using primary cultures of cortical chicken neu- rons. All tested drugs have been already characterized in different cell culture systems or assay systems based on recombinant enzymes. Methods: Two gamma- and one beta-secretase inhibitor, two non-steroidal anti-inflam- matory drugs, and one calpain inhibitor were investigated on dissociated E8 telencephalon cultures. ELISAs specific for Aß1-38, Aß1-40 and Aß1-42 were performed of cell supernatants and MTT viability assay of cells. Re- sults: DAPT, one of the gamma-secretase inhibitors, did not influence neu- ronal survival but significantly reduced Aß1-38, Aß1-40 and Aß1-42 levels in a dose dependent manner in chicken neurons. All the tested secretase in- hibitors reduced or increased Aß peptides as described in already published data. Conclusions: For the first time, diferent secretase inhibitors were com- pared in one cell culture system. Primary cultures of embryonic chicken neu- rons confirmed the IC50 values of inhibitors from already published data and hence provide a valid system for analysing the activity of secretase inhibitors. In addition, as primary cells they have a biological advantage over cell lines ectopically expressing hAPP. P2-144 INTRABODIES AGAINST Ab OLIGOMERS Giovanni Meli 1 , Agnese Lecci 1 , Antonino Cattaneo 1,2 , 1 EBRI - European Brain Research Institute, Roma, Italy; 2 Scuola Normale Superiore, Pisa, Italy. Contact e-mail: g.meli@ebri.it Background: The concept of intracellular antibody (intrabody) exploits the specific targeting of an antibody to subcellular compartments where it can bind the target protein (Biocca and Cattaneo, 1995). Intrabodies represent a versatile option to modulate and neutralize protein function in mammalian cells, although not all intrabodies are able to fold well under conditions of intracellular expression. A great improvement was achieved when a new strategy, the intracellular antibody capture technology (IACT), was estab- lished, allowing the direct selection of antibodies on the basis of their ability to bind the protein antigen under conditions of intracellular expression (Vis- intin et al., 1999; 2002). Intrabodies can be equipped with effector functions that allow the neutralization or silencing of intracellular proteins or peptides, in the relevant cellular compartment (silencing intrabody technology (SIT)) (Melchionna and Cattaneo, 2007). IACT was exploited to select intrabodies specific for AbOligomers (AbOs), in the form of single chain Fv fragments, that are conformation-sensitive and sequence-specific (Meli et al., 2009). We describe now the use of these anti-AbOs intrabodies for selective subcellular targeting and intracellular knock-down/silencing of AbOs. Methods: Differ- ent constructs for anti-AbOs intrabody expression and cellular targeting, also based on adeno-associated vectors, were prepared, and used for expression in neuronal cells lines, primary cultured neurons and in well established cell models for AbOs production (7PA2 cells). Results: The intracellular expres- sion and the subcellular targeting the anti-AbOs intrabodies in neuronal cells was well characterized. Different strategies for intrabody-mediated AbOs si- lencing were investigated, and their efficacy to interfere with intracellular AbO formation and effects are currently under investigation. Interesting fea- tures of intrabodies appear to be linked to the specific epitope recognized (C- terminus versus N-terminus of Ab). Conclusions: The use of specific intra- bodies raised against Ab would allow to selectively target Ab and/or Ab olig- omers (AbOs) inside cells at different points in their intracellular traffic, for mechanistic as well as for interference purposes. The intrabody approach, in conjunction with the intrabody protein-silencing approach, is therefore a promising way for interfering with the activity of different intracellular Ab assemblies, in ways that would not be possible by RNA interference aproaches. Poster Presentations P2 P303