P1-461: The role of cholesterol level alterations upon APP and BACE proximity and APP cleavage

Poster Presentations P1 *P⬍0.001 vs. Control and PBN) but also oxidative stress (⬃40% reduction, *P⬍0.001 vs. Control). The MMP inhibitors did not have direct antioxidant effects. Treatment of animals with the antioxidant PBN or MMP inhibitor minocycline did not have a significant effect on CAA progression rates measured with longitudinal imaging when compared to untreated mice (untreated⫽0.51%, PBN⫽0.51, minocycline⫽0.54 % vessel segment affected per week). Conclusions: These data offer insights on the molecular pathways activated by vascular amyloid and suggest a close association between A␤-related MMP activation and ROS generation in vivo. They also raise the possibility that therapeutic treatment with MMP inhibitors may have beneficial effects in the A␤ amyloidosis by indirectly reducing the oxidative stress associated with CAA Acknowledgements: NIH EB000768, AG020570, AG024688, AG021084, Harvard Neurodiscovery Center (R.A.B.), Leffler fellowship (CP), Bugher Foundation fellowship (M.G-A). P1-461

THE ROLE OF CHOLESTEROL LEVEL ALTERATIONS UPON APP AND BACE PROXIMITY AND APP CLEAVAGE

Christine A. F. von Arnim1, Bjo¨rn von Einem1, Daniel Schwanzar1, Petra Weber2, Michael Wagner2, Herbert Schneckenburger2, 1Ulm University, Ulm, Germany; 2University of Applied Sciences, IAF, Aalen, Germany. Contact e-mail: [email protected] Background: BACE-cleavage of APP is the first proteolytic step in the production of Amyloid beta, which accumulates in senile plaques in Alzheimer’s disease. There is a large body of evidence that this happens in endosomes. However there is controversial data whether APP and BACE come into close proximity and interact on the cell surface. Methods: To examine whether APP and BACE come into close proximity on the cell surface and /or within the cell in living cells we developed a novel technique by combining FRET-measurements by lifetime imaging with total internal reflection microscopy (TIRET) offering excellent axial resolution. Membrane fluidity was observed by fluorescence microscopy. To assess functional data upon APP cleavage we employed a novel electrochemiluminescent based assay to detect sAPP␣ and sAPP␤ simultaneously. Results: Our data show that BACE and APP are both present on the cell surface but interact only within the cell and not on the cell surface. This interaction is dependent upon cholesterol level. Membrane fluidity is also dependent upon cholesterol level. To analyze the impact of alterations in cholesterol level upon BACE-cleavage we measured sAPP secretion. sAPP beta secretion increased upon cholesterol enrichment and decreased upon depletion. sAPP␣ increased upon cholesterol depletion and decreased upon cholesterol enrichment. Conclusions: Thus the FRET approach developed here utilizing FLIM under TIRF conditions (TIRET) may be broadly applicable to the issue of identifying the localization of specific protein-protein proximity on the cell surface with exquisite spatial resolution, since membrane selective optical excitation is combined with ultrasensitive and time-resolving fluorescence detection. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity. P1-462

RCK/P54 UPREGULATES AMYLOID PRECURSOR PROTEIN MESSENGER RNA AS PART OF A NOVEL RNA-BINDING PROTEIN COMPLEX

Oleg Broytman, Pamela R. Westmark, Zafer G. Gurel, James S. Malter, University of Wisconsin - Madison, Madison, WI, USA. Contact e-mail: [email protected] Background: Overproduction of Amyloid Precursor Protein (APP) and beta-amyloid likely contribute to neurodegeneration seen in Alzheimer’s Disease (AD). Elevated APP mRNA levels are positively correlated with downstream beta-amyloid production both in AD and Down’s Syndrome. APP mRNA contains several cis-acting regulatory elements, one of which (52sce) has been recently shown to complex with uncharacterized cytoplasmic proteins. Methods: 52sce-binding proteins were isolated by affin-

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ity purification with 52sce oligoribonucleotide, and identified through trypsin digestion and mass spectroscopy. APP mRNA was detected in immunoprecipitates of 52sce-binding proteins from human neuroblasts through real-time PCR. Rck/p54, one of the identified proteins, was overexpressed in human neuroblasts by Rck gene transfection or TAT-fusion protein transduction. APP mRNA and protein levels in treated and control cells were assayed with real-time PCR and immunocytochemistry, respectively. Results: We identify six novel proteins that specifically bind to 52sce and interact with APP mRNA in intact human neuroblastoma cells. Overexpression of one of these proteins, rck/p54, upregulated APP mRNA and protein in those cells. Conclusions: These data suggest a novel mechanism for post-transcriptional regulation of APP mRNA. P1-463

A␤ ACCUMULATION IN CHOROID PLEXUS OF HUMAN ALZHEIMER’S DISEASE BRAIN IS ASSOCIATED WITH MITOCHONDRIAL-INDUCED APOPTOSIS AND BLOOD-CSF BARRIER DISRUPTION

Teo Vargas1, Cristina Ugalde1, Carlos Spuch1, Desiree Antequera1, Maria Moran1, Miguel A. Martin1, Isidro Ferrer2, Felix Bermejo3, Eva Carro1, 1Research Center. Hospital 12 de Octubre, Madrid, Spain; 2 Institute of Neuropathology, Service of Pathologic Anatomy. IDIBELLHospital Universitario de Bellvitge, Barcelona, Spain; 3Service of Neurology. Hospital 12 de Octubre, Madrid, Spain. Contact e-mail: [email protected] Background: Abundant evidences indicate that toxic effects of ␤-amyloid (A␤) involve activation of apoptosis pathways. However, which precise signaling pathway mediates apoptosis induced by A␤ remain to be completely defined. A␤ has been implicated as a key molecule in the pathological cascades of Alzheimer⬘s disease (AD). Blood-cerebrospinal fluid (CSF) barrier dysfunction is a potential mechanism involved in progressive damage by brain A␤ accumulation. Recently, we found elevated expression of A␤ in the choroid plexus, which likely impairs physiological functions of choroid plexus epithelium. Methods: Therefore, we decided to investigate the effects of increased expression of A␤ on mitochondrial function of this brain tissue. Results: We studied this hypothesis in choroid plexus from AD patients and APP/PS1 transgenic mice, which exhibited high A␤ levels besides increased nitric oxide (NO) levels and an impairment of the activity and assembly of mitochondrial respiratory chain complexes I and IV. Reactive oxygen species (ROS) and caspases expression were significantly increased in choroid plexus epithelial cells treated with A␤, suggesting that ROS generation could be consequent to A␤induced mitochondrial damage. Our results demonstrate a relationship between A␤, and an increased expression of metalloproteinase MMP-9, accompanied by blood-CSF barrier disruption. Conclusions: We suggest a hypothetical sequence of pathogenic steps linking A␤ and choroid plexus epithelium with enhanced NO production, mitochondrial dysfunction, and up-regulation of MMP-9, finally leading to opening barrier and cell death. Taken together, our results provide new information about the molecular mechanisms involved on A␤ toxicity. P1-464

EXOSOME-DEPENDENT TRAFFICKING OF INSULIN DEGRADING ENZYME MODULATES AMYLOID BETA CLEARANCE

Maria C. Leal1, Ayelen Bulloj1, Huaxi Xu2, Eduardo M. Castan˜o1, Laura Morelli1, 1Fundacion Instituto Leloir, Buenos Aires, Argentina; 2 The Burnham Institute, La Jolla, CA, USA. Contact e-mail: [email protected] Background: IDE regulates the steady-state levels of A␤ in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer’s disease (AD), mainly characterized by A␤ deposition. However, the sub-cellular localization of IDE relevant to A␤ degradation remains poorly understood. IDE is located in cytosol, peroxisomes, plasma membrane and recently we reported a pool of IDE localized in lipid rafts.