phosphorylation of amyloid precursor protein (APP). When phosphory- lated at threonine 668 (T668), APP undergoes conformational changes affecting its intracellular sorting and trafficking, which in turns impact proteolytic cleavage and increases A b production. The role of APP T668 phosphorylation and obesity in AD are not well understood and indi- cate a critical need to understand the mechanism(s) linking obesity and cognitive decline. Methods: Obesity is induced in C57Bl/6 mice using a high fat diet (54% kCal from fat) for 24 wk. APP and tau phosphorylation is examined from cortex lysates. Cortical neurons are prepared from E15 rat embryo and cultured in vitro for 7 days before insulin and/or IGF-I treat- ment. Results: Obese mice displayed significant cognitive impairment at 24 wk in parallel with the increased tau and T668-APP phosphorylation in the cortex. We previously reported that embryonic cortical neurons (eCN) develop neuronal IR with decreased insulin and IGF-I signaling following chronic insulin treatment. IGF-I treatment of eCN decreased T668-APP phosphorylation. Insulin also decreased APP phosphorylation but the effect was much weaker compared to IGF-I. Chronic treatment of eCN with insu- lin increased basal T668-APP phosphorylation. IGF-I was still able to reduce T668 phosphorylation after chronic insulin treatment; insulin itself was unable to reduce APP phosphorylation. These effect was reversed with the simultaneous treatment of chronic insulin with PI3-K inhibitor, suggesting chronic hyperactivation of Akt is responsible for IR-induced APP phosphorylation. Conclusions: Our results suggest IR-induced in- creases in T668 phosphorylation of APP as a possible link between obesity and cognitive impairment. Furthermore our data reveal a potential and bene- ficial effect of IGF-I signaling as a therapeutic target. This work is supported by the Program for Neurology Research and Discovery (www.med.umich. edu/PNRD). P1-068 A PHYSIOLOGICAL ROLE FOR AMYLOID BETA IN CYCLIC AMP-STIMULATED LONG TERM POTENTIATION Ernesto Fedele 1 , Daniela Puzzo 2 , Olga Bruno 1 , Elisa Canepa 1 , Elena Gardella 1 , Daniela Rivera 1 , Lucia Privitera 2 , Cinzia Domenicotti 1 , Barbara Marengo 1 , Umberto Marinari 1 , Agostino Palmeri 2 , Maria Pronzato 1 , Ottavio Arancio 3 , Roberta Ricciarelli 1 , 1 University of Genoa, Genoa, Italy; 2 University of Catania, Catania, Italy; 3 Columbia University, New York, New York, United States. Contact e-mail: fedele@ pharmatox.unige.it Background: Cyclic adenosine monophosphate (cAMP) regulates long- term potentiation (LTP) and ameliorates memory in healthy and diseased brain. Increasing evidence also shows that, under physiological conditions, low concentrations of amyloid b (A b) are necessary for LTP expression and memory formation. Based on these evidences, we tested the hypothesis of a functional correlation between cAMP, A b and LTP, in an attemptto reveal novel molecular mechanisms for memory formation. Methods: In neuronal cultured cells and rat hippocampal slices, expression of the A b precursor protein (APP) was measured by RT-PCR and immunoblotting, whereas A b 42 was analyzed using specific ELISA. Electrophysiological LTP record- ings were performed in hippocampal slices from wild-type and APP knockout mice. Results: Our study shows, for the first time, that cAMP en- hances LTP by stimulating the synthesis of APP and, in turn, the production of A b. In particular, our results indicate that PKA but not EPAC is involved in the cAMP-induced increase of APP and A b 42. Moreover, we demon- strate that cAMP requires translation, but not transcription, in order to in- crease APP and A b levels. Finally, we show that the reinforcing effects of cAMP on LTP are abolished in APP knockout mice, where A b cannot be produced, and are prevented in wild-type animals when the extracellular peptide is depleted by anti-A b antibodies. Conclusions: The present data demonstrate that endogenous cAMP requires APP and A b to boost hippo- campal LTP. Collectively, our study has revealed a novel cAMP/PKA/APP/ A b molecular pathway through which the second messenger positively in- fluences the cellular mechanisms of memory formation and adds further ev- idence for a physiological role of A b. Research supported by grants from Alzheimer’s Association (NIRG-07-59597 to D.P. and IIRG-11-208306 to O.B.) and Fondazione CARIGE (to M.A.P.). P1-069 A PRESENILIN 1 MUTATIONALTERS APP LOCALIZATION IN HUMAN NEURONS Grace Woodruff, Sol Reyna, Lawrence Goldstein, UCSD, La Jolla, California, United States. Contact e-mail: gwoodruff@ucsd.edu Background: Presenilin 1 (PS1) is the catalytic core of the g-secretase com- plex that cleaves the Amyloid Precursor Protein (APP) and other type 1 transmembrane proteins. Mutations in PS1 are the most common cause of early onset Alzheimer’s disease (AD). Despite extensive research into the mechanisms by which PS1 mutations cause AD, there is still uncertainty in the precise mechanisms by which PS1 mutations initiate disease. Methods: We utilized isogenic induced pluripotent stem cell (iPSC) derived neurons that harbor the PS1 De9 mutation to investigate neuronal pheno- types caused by mutant PS1. Additionally, we used quantitative immunoflu- orescence to measure APP localization and APP colocalization with endocytic markers. Results: We report that the PS1 De9 mutation alters localization of APP such that there is decreased APP in axons and increased APP in the cell body. Additionally we found that there is decreased colocalization of APP with the early enodcytic marker Rab5 in axons. Conclusions: Our results suggest that abnormal APP localization may be an early event in progression of familial AD. P1-070 DEVELOPMENT OF AN IMPROVED IMMUNOASSAY FOR DETECTION OF SORLA IN CELLS AND BIOLOGICAL SAMPLES Ishita Guha Thakurta, Mark J. West, Olav M. Andersen, Aarhus University, Aarhus, Denmark. Contact e-mail: ishita.g.thakurta@biokemi.au.dk Background: SorLA (Sorting - related receptor with A- type repeats) is a 250 kDa type I transmembrane protein, belonging to the VPS10P (vacu- olar protein sorting 10 protein) family of neuronal receptors. It is impli- cated in the development of AD (Alzheimer’s Disease), atherosclerosis, diabetic retinopathy, and acute leukemia. Despite the overwhelming evi- dence regarding the role of sorLA in various diseases, there has been a lack of technologies which can precisely quantitate the levels of sorLA in various complex biological matrices. The methods are either qualitative like immunohistochemistry, or traditional sandwich ELISA assays which are time consuming and less sensitive. Hence, the purpose of the present study is to develop a new assay called AlphaLISA which is fast and very sensitive, to measure sorLA in extremely small volumes of cells and bio- logical samples. Methods: The AlphaLISA is a homogenous bead based assay using donor and acceptor beads. The donor bead is coated with strep- tavidin that captures a biotinylated antibody while the acceptor bead is coated with analyte-specific antibody. When brought into proximity through binding to the analyte and excited by laser at 680 nm, the donor bead re- leases singlet oxygen which triggers a series of chemical reactions in the acceptor beads causing a sharp peak of light emission at 615 nm. A series of experiments were designed to optimize the assay by conjugation of the beads to various anti-sorLA antibodies, cross titrations of the antibodies, spike and recovery experiments to check matrix interference, signal to noise ratio determined for the counts, and comparison of our novel immunoassay in terms of sensitivity with existing methods. Results: Our results show that as compared to traditional methods, AlphaLISA is a sensitive and rapid assay, which can be automated suitably for determination of sorLA in large sample batches. It also shows high recovery and signal to noise ratio. Conclusions: The results support the development of an improved method for measuring sorLA quantitatively, which could further prove as an impor- tant tool in investigation and establisment of sorLA as a potential biomarker in AD. Poster Presentations: P1 P328