aggregation and facilitating the degradation of misfolded proteins. The age-dependent decline of the chaperones-assisted mechanisms is thought to contribute to proteostasis diseases, including amyloidoses. Clusterin is a chaperone implicated in the processing of misfolded and aggregated extracellular proteins, with a mechanism involving a preferential binding to the hydrophobic regions exposed on early oligomeric assemblies, regardless of their identity. Clusterin also binds Ab1-40 oligomers, providing a molecular basis for the genetic link between clusterin and Alz- heimer’s disease. The binding of clusterin may prevent Ab oligomers toxicity by facilitating their proteolysis and/or improving their degradation and/or rendering oligomers less toxic by shielding of hydrophobic patches. This study focus on the effects of clusterin on toxic Ab1-42 oligomers and on the kinetics of Ab1-42 fibril formation. Methods: Fibril formation and elongation was followed by in-situ Thioflavin-T (ThT) kinetic experiments. Toxic oligomers, and their formation, were detected by two assays recently developed in our labs (Stravalaci et al., JBC, 2012), i.e. a Surface Plasmon Resonance (SPR)-based immunoassay and a new behavioural assay in C. el- egans. For these studies, we used synthetic Ab 1-42, produced in home using the "depsi-peptide" technique (Beeg et al. Anal Biochem 2011), which reli- ably produces seed-free starting solutions, a fundamental prerequisite for analysis with highly aggregating peptides. Results: SPR studies indicated: i) a direct and high-affinity interaction of clusterin with toxic A b 1-42 olig- omers; ii) a decrease of oligomers-dependent signal when clusterin was added to preformed assemblies; and iii) an inhibition of oligomer formation, when clusterin was co-incubated with A b 1-42 monomers. This latter effect was confirmed by studies in C. elegans, showing that the co-incubation with clusterin prevents the formation of toxic A b 1-42 assemblies. ThT experi- ments indicated that clusterin dose-dependently increases the lag-phase for fibril formation, possibly because of its interaction with early oligomeric species. Conclusions: Overall, these data show that clusterin prevents the formation and induces the disaggregation of toxic A b 1-42 oligomers, and delay the formation of A b 1-42 fibrils. These effects are likely due to the high affinity binding of clusterin to A b 1-42 oligomers. P3-069 A NOVEL REAGENT MODULATES CN/CX43 INTERACTIONS DURING THE PROGRESSION OF ALZHEIMER’S DISEASE Melanie M. Pleiss 1 , Jennifer L. Furman 1 , Hafiz Mohmmad Abdul 1 , Christopher Mark Norris 2 , 1 University of Kentucky, Lexington, Kentucky, United States; 2 University of Kentucky College of Medicine, Lexington, Kentucky, United States. Contact e-mail: melanie.pleiss@uky.edu Background: Alzheimer’s disease (AD) is characterized by microglial and astroglial activation that arises in the early stages of the disease. Upon acti- vation, astrocytes upregulate calcineurin (CN), a protein phosphatase involved in inflammatory signaling and implicated in cognitive decline with AD. Several inflammatory mediators related to CN appear to disrupt gap junction (GJ) coupling between astrocytes through impairment of GJ proteins, including the most abundant astrocytic GJ protein connexin 43 (Cx43). CN has also been shown to directly dephosphorylate Cx43 at Ser 368 in primary astrocytes. The purpose of these studies is to determine the functional implications of CN/Cx43 interactions during the progression of AD. Methods: Total and dephosphorylated Cx43 levels were measured and correlated with CN activity in human hippocampal membrane fractions from non-demented subjects (n¼10) and subjects with mild cognitive impair- ment (MCI) (n¼14) or AD (n¼21). Western blot analysis and dye-coupling assays were also performed in rat primary cultures to determine the direct ef- fects of CN on Cx43 and GJ coupling. The Ca 2+ mobilizers ionomycin and phorbol ester (Ion/PE), or the pro-inflammatory cytokine IL-1b were used to activate CN. The CN inhibitor cyclosporine and novel Cx43 mimetic peptides were used to disrupt CN/Cx43 interactions. Results: Levels of dephosphory- lated Cx43 were significantly elevated in MCI cases relative to non- demented and AD cases (p<0.01), and strongly related to increased CN signaling. In primary astrocytes, Ion/PE and IL-1b caused a significant in- crease in dephosphorylated Cx43 levels (p<0.05), which was blocked by cyclosporine and Cx43 peptides. Preliminary work reveals that mimetic pep- tides significantly reduce levels of dephosphorylated Cx43 (p<0.001), but do not interfere with other CN substrates (e.g. NFAT), suggesting that effects may be selective for CN/Cx43 interactions. Conclusions: Our results suggest that increased CN activity leads to Cx43 dephosphorylation during MCI, and may contribute to early clinical progression of AD through disruption of as- trocytic gap junctions. Additionally, our results suggest that our mimetic pep- tide may selectively interfere with CN/Cx43 interactions, which could serve as important new reagent for assessing the impact of astrocyte signaling in AD. The specificity of these peptides and their impact on astrocyte function are the subject of ongoing studies. P3-070 DETECTION OF PRE-PLAQUE AMYLOID AGGREGATION USING FTIR Oxana Klementieva 1 , Anders Engdahl 2 , Mathilde Faideau 1 , Katarina Ingrid Will en 1 , Per Uvdal 2 , Gunnar K. Gouras 1 , 1 Lund University, Lund, Sweden; 2 MAX IV Laboratory, Lund, Sweden. Contact e-mail: oxana. klementieva@med.lu.se Background: Alzheimer’s disease (AD) is characterized by misfolding and aggregation of naturally occurring beta-amyloid peptides (Ab). These ag- gregates are thought to be pathogenic to neurons, although the conformation of the pathogenic Ab species remains unclear. Biochemical extraction methods and different microscopy techniques (TEM, confocal) can be used to identify pathogenic Ab species in the brain, although such methods can alter protein conformation or are n ot designed to determine structural details of protein assemblies. The dominant hypothesis in AD has been that Ab initiates the disease via toxicity from secreted, extracellular Ab ag- gregates. More recently, an alternative hypothesis has emerged focusing on a pool of Ab that accumulates early on within AD vulnerable neurons of the brain. Although the topic of intraneuronal Ab has been of major interest in the field, the mechanisms whereby Ab misfolds and becomes pathogenic remain unclear. Elucidating the molecular structure of intraneuronal Ab should provide important insights into the mechanisms of Ab-induced syn- apse dysfunction, which is crucial for understanding, diagnosing, and treat- ing AD. Methods: We use synchrotron based FTIR microspectroscopy to visualize neurons in their natural state, which is critical for the determina- tion of the real structure of proteins. Using this technique we study protein aggregation in primary neurons and brains of APP/PS1 mutant transgenic mice. This technique allows the determination of the secondary structure of proteins by analyzing the amide I region of the infrared spectrum. The aim is to obtain Infrared spectra from neurons using a method that sensitive to the formation of different b structures. Results: Synchrotron based FTIR microspectroscopy demonstrated b aggregated proteins within intracellular regions i n AD transgenic neurons. From the comparison of the spectral in- tensity of the alpha and beta bands we deduce that overexpressed Ab starts to misfold within neurons. Conclusions: These data support the growing awareness that misfolded intraneuronal Ab can be a key pathogenic species involved in causing synapse dysfunction in AD. P3-071 A GENOME-WIDE META-ANALYSIS OF PLASMA CLUSTERIN LEVELS IN THE CHARGE CONSORTIUM Vincent Antoine Chouraki 1 , Johanna Jakobsdottir 2 , Karen Mather 3 , Hieab Adams 4 , Jennifer Mollon 5 , Christopher Oldmeadow 6 , Anbupalam Thalamuthu 3 , Toshiko Tanaka 7 , Rodney Scott 6 , Daniel Levy 8 , Liz Holliday 6 , Fei Song 9 , Madhav Thambisetty 10 , Anne Poljak 11 , Gudny Eiriksdottir 2 , Perminder S. Sachdev 12 , Veer Bala Gupta 13 , Ralph Martins 14 , Lenore Launer 15 , Richard Dobson 5 , Henry Brodaty 16 , John Attia 6 , Simon Lovestone 17 , Vilmundur Gudnason 18 , Mohammad Ikram 19 , Sudha Seshadri 1 , 1 Boston University School of Medicine, Boston, Massachusetts, United States; 2 Icelandic Heart Association, Kopavagur, Iceland; 3 University of New South Wales, Randwick, Australia; 4 Erasmus MC, Rotterdam, Netherlands; 5 Kings College London, London, United Kingdom; 6 University of Newcastle, Newcastle, Australia; 7 NIA, Baltimore, Maryland, United States; 8 The Framingham Heart Study, Framingham, Massachusetts, United States; 9 University of New South Wales, Sydney, Australia; 10 National Institute on Aging, Baltimore, Maryland, United States; 11 UNSW, Sydney, Australia; Poster Presentations: P3 P652