Poster Presentations P4 1 University of Munich, Munich, Germany; 2University of Leipzig, Leipzig, Germany. Contact e-mail: stefan.lichtenthaler@med.uni-muenchen.de Background: The alpha-secretase is a proteolytic activity, which cleaves within the amyloid ß (Aß) sequence and, thus, prevents Aß generation. The identity of alpha-secretase is unknown, but several proteases, including ‘‘a disintegrin and metalloproteases’’, have been proposed to be candidate alpha-secretases. Methods: To systematically characterize proteases with alpha-secretase activity, we first mapped the alpha-secretase cleavage sites within the Aß domain of APP and then screened for proteases cleaving at these sites. Results: We found that the main cleavage site is between amino acids 16 and 17 of the Aß sequence, which we refer to as major alpha-secretase cleavage site. Additional cleavage sites were identified at nearby peptide bonds, but only represent a smaller amount of total APP alphasecretase shedding. In order to selectively detect the secreted APP ending at the major alpha-secretase cleavage site, we generated a monoclonal antibody detecting the neoepitope arising through this cleavage. Next, we individually knocked-down different proteases and evaluated their contribution to the major alpha-secretase cleavage site of APP in neuronal cells. Surprisingly, we found that the knock-down of a single metalloprotease was sufficient to completely suppress APP shedding at the major cleavage site. This reveals that other proteases were not able to compensate for this loss of cleavage. Conclusions: In summary, our data show that a) distinct proteases contribute to total alpha-secretase cleavage of APP, but that b) only a single metalloprotease seems to mediate APP shedding at the major cleavage site. P4-148 MERGING AMYLOID-BETA CASCADE AND ACTIVITY-DEPENDENT PLASTICITY IN HIPPOCAMPUS: A POSSIBLE ROLE FOR ADAM10 Elena Marcello1, Fabrizio Gardoni1, Hugo Vara-Rivera2, Roberta Epis1, Flaminio Cattabeni1, Barbara Borroni3, Alessandro Padovani3, Maurizio Giustetto2, Isabel Perez-Otaño4, Monica Di Luca1, 1Dept. Pharmacological Sciences - University of Milano, Milano, Italy; 2Dept. of Anatomy, Pharmacology and Forensic Medicine and Istituto Nazionale di Neuroscienze, Torino, Italy; 3Department of Neurological Sciences, University of Brescia, Brescia, Italy; 4Cellular Neurobiology Laboratory, Neuroscience Dept, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain. Contact e-mail: elena.marcello@unimi.it Background: Molecular pathogenesis of neurodegenerative diseases, such as Alzheimer Disease, is the result of a complex interplay of several crossing pathways. Accumulating evidence indicates the amyloid cascade as a pivotal player in the pathogenesis. Besides this process, synaptic abnormalities occurring early in the course of the disease have been described. Recently, we have demonstrated a clear relation between the amyloid cascade and synaptic plasticity. We reported that ADAM10, the protagonist of the non-amyloidogenic pathway of the amyloid cascade, interacts directly with SAP97, a protein involved in trafficking of glutamate receptors. This interaction is required for ADAM10 localization at postsynaptic membranes and enzymatic activity. In fact, NMDA receptor activation increased SAP97-mediated membrane trafficking of ADAM10 and increased APP cleavage at the alpha-secretase site. Methods: Experiments were performed in two different cellular models: primary neuronal cultures and hippocampal slices. In primary cultures, chemical treatments were used to induce long-term potentiation (LTP) and long-term depression (LTD), while in hippocampal slices LTP and LTD were induced by electrophysiological stimulation. ADAM10 synaptic localization and surface expression were assessed by means of biochemical fractionation, BS3 treatment and surface biotinylation. Results: Here we addressed the effect of activity-dependent synaptic plasticity on ADAM10 trafficking long-term potentiation (LTP) and long-term depression (LTD) were induced in acute hippocampal slices using electrophysiological stimulation, and in primary hippocampal cultures using chemical treatments, and then ADAM10 localization was analysed. Induction of LTP caused an augment of ADAM10 in the postsynaptic compartment that was not accompanied by an increase of ADAM10 insertion in the cell membrane. In contrast, induction of LTD leaded to the delivery of ADAM10 to the postsynapse that was associated with an increase of the P475 surface pool of ADAM10. LTP induction is accompanied by a shift of Amyloid Precursor Protein metabolism towards amyloidogenesis, while LTD induction stimulates the non-amyloidogenica pathway. Conclusions: Our results suggest a strict interdependence between synaptic plasticity processes and ADAM10 trafficking to the postsynapse. Therefore impairments in the regulation of this mechanism may influence APP metabolism and Abeta production. P4-149 MOLECULAR INSIGHTS INTO MECHANISM OF ALPHA SYNUCLEIN EVOKED INHIBITION OF POLY (ADP-RIBOSE) POLYMERASE-1 (PARP-1) ACTIVITY Anna Kaźmierczak, Agata Adamczyk, Grzegorz A. Czapski, Joanna B. Strosznajder, Mossakowski Medical Research Center, Warsaw, Poland. Contact e-mail: aniakazmierczak@gmail.com Background: Alteration of alpha-synuclein (ASN), a small (14 kDa) presynaptic protein abundant in the brain is implicated in the pathogenesis of various neurodegenerative disorders. Our previous data indicated that ASN enhanced oxidative stress and NMDA receptor-mediated nitric oxide synthase (NOS) activity. The DNA-binding poly ADP-ribose polymerase (PARP-1) was suggested to be the nuclear target for free radicals and NMDA receptor-dependent signalling. The aim of the present study was to investigate the time-dependent effect of ASN applied extracellularly on PARP-1 activity and protein level. Methods: The studies were carried out in homogenate from hippocampus and brain cortex together and also in hippocampal cell line (HT22) using radiochemical, spectrophotometrical and Western blot methods. Results: Our results show that short time (30 min.) exposure to ASN (10mM) added extracellularly inhibited PARP-1 activity in the rat brain homogenate by 27% with no effect on PARP-1 protein level. It was also found that ASN significantly stimulated nitric oxide synthase (NOS) in rat brain by 60%, and the inhibition of NOS by the NG-nitro-L-arginine (NNLA, 100mM) partially reversed the effect of ASN on PARP-1 activity. However, long time (24 h) incubation of HT22 cells with extracellularly applied ASN caused significant decrease of PARP-1 protein level. In addition, in the same model ASN increased NOS activity by 62%, that resulted in NO- dependent activation of caspase-3. Moreover, the inhibitors of NOS (NNLA, 100mM) and caspase-3 (Z-DEQD-FMK, 40mM) completely reversed ASN-evoked PARP-1 degradation. Conclusions: In conclusion the results demonstrated that ASN inhibits PARP-1 activity by NO release during short period of action and induces PARP cleavage by NO-mediated caspase-3 activation. Financial support was provided by the Ministry of Science and Higher Education Grant 2PO5A4129. P4-150 A MOLECULAR MECHANISM BY WHICH I2PP2A CLEAVAGE LEADS TO PP2A INHIBITION AND TAU HYPERPHOSPHORYLATION Lisette T. Arnaud, New York Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: lisette.arnaud@omr. state.ny.us Background: Phosphoseryl/phosphothreonyl protein Phosphatase 2A (PP2A), which dephosphorylates most tau phosphorylation sites, has been shown to have a compromised activity in AD cerebral cortex. I2PP2A (also known as SET or TAF-1b) is one of its two known endogenous inhibitors. Although I2PP2A Full Length is localized in the nucleus, a 20kDa N-terminal fragment (I2NTF), product of I2PP2A cleavage is selectively translocated form the nucleus to the neuronal cytoplasm in the affected areas of AD brains.We hypothesized that I2PP2A cleavage induces the loss of the NLS (nuclear localization signal), resulting in the cytoplasmic localization of I2NTF, and consequently the inhibition of PP2A and the hyperphosphorylation of tau. Methods: To understand the mechanisms that link tau hyperphosphorylation and I2PP2A cleavage, we first investigated the direct interaction of I2NTF with PP2A catalytic subunit by GST-pulldown assay. Second, we tested the inhibitory effect of I2NTF on PP2A by transiently transfecting Cos7 cells with the cDNA of human I2NTF and assaying PP2A activity. Third, we transfected Cos7 cells with