P3-422

S500

Poster P3:: Tuesday Posters

sulindac sulfone. Based on these data, we hypothesized that Fbxo11 might function in the molecular pathway of APP proteolytic processing at the level of ␥-secretase. Knockdown of Fbxo11 expression by siRNA in 293 cells had no effect on the levels of APP full length, APP-CTFs, secreted APP, or presenilin 1 (PS1) fragments but increased AICD generation by ⬃2-fold. Paradoxically, Fbxo11 siRNA decreased total A␤ secretion, with A␤42 showing a greater reduction than A␤40, indicating shifts in the sites of ␥- and ⑀-cleavages in APP. Moreover, Fbxo11 siRNA transfection markedly reduced SDS-resistant high molecular weight species (100-130 kDa) of PS1 in multi-ubiquitin immune complexes, suggesting a role of Fbxo11 in the ubiquitination of PS1. A similar dose-dependent reduction in PS1 ubiquitination was seen by the treatment of ibuprofen, an A␤42 lowering NSAID. In transient transfection experiments, myc-tagged Fbxo11 co-precipitated with PS1 in an expression-dependent manner. Conclusions: These results suggest that Fbxo11 alters A␤ generation by modifying PS1 structure and function. Further studies are being carried out to decipher the precise manner in which Fbxo11 modifies presenilin-dependent ␥-secretase activity. P3-421

IDENTIFICATION OF STRUCTURAL DETERMINANTS ON PS-1 AND PS-2 THAT AFFECT INHIBITOR POTENCY

Guriqbal Basi, Mei Yu, Marty Neitzel, Jennifer Marugg, Jacek Jagodzsinski, Jowell Go, Susanna Hemphill, Paul Shapiro, Stephen Freedman, Ted Yednock, Dale Schenk, Byron Zhao, Elan Pharmaceuticals, South San Francisco, CA, USA. Contact e-mail: [email protected] Background: Production and deposition of A␤ peptide in amyloid plaques contributes the hallmark pathology of Alzheimer’s disease in brains of affected patients. Two presenilin homologues, referred to as PS1 and PS2, comprise the catalytic core of gamma secretase, a critical enzyme in A␤ production. Gamma secretase also mediates intramembrane cleavage of Notch and other substrates. Inhibition of Notch signaling by non-selective gamma secretase inhibitors is an important issue for the clinical development of this class of therapeutics. The observation that PS1 KO mice exhibit perinatal lethality, whereas PS2 KO mice are viable suggests that PS2 selective inhibitors may spare Notch signaling while lowering A␤. Objective(s): To identify PS selective inhibitors, and to map the underlying structural basis, Methods: We employed murine cell lines lacking PS1 and PS2. We performed transient transfections on these cells with Swedish APP plus either PS1, or PS2, or a chimerical presenilin molecule derived from portions of PS1 and PS2. Results: We observed that sulfonamides, as represented by BMS 299897, were significantly more potent inhibitors of A␤ production from PS1-gamma secretase than PS2-gamma secretase for A␤ production. In contrast, other classes of gamma inhibitors we tested were largely equipotent for PS1- or PS2-gamma secretase. Conclusions: Using the combination of chimerical constructs and point mutants, we identified specific amino acid residues that can account for differences in inhibition potency of PS1 vs. PS2 gamma secretase by BMS299897, as well as related compounds. P3-422

INFLUENCE OF CHOLESTEROL ON NEUROTOXICITY OF ␤-AMYLOID PEPTIDE AND EXPRESSION OF NICOTINIC RECEPTORS IN RAT BRAIN

Zhi-Zhong Guan1,2, Ru-Yu Liu1, Ran Gu1, 1Guiyang Medical College, Guiyang, China; 2Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: The possibility that cholesterol is a risk factor in the development of Alzheimer’s disease (AD) has been supported by epidemiological studies. However, the mechanism regarding how cholesterol participates the pathogenesis of AD is still elusive. Objective(s): In order to investigate the influence of cholesterol on effect of ␤-amyloid peptide (A␤) and expression of neuronal nicotinic acetylcholine receptors (nAChRs),

Wistar rats received a single intracerebroventricular injection of A␤1-42 or were fed a diet containing a high content of cholesterol or a combination of both treatments. Methods: Three weeks after treatments, cognitive function of animals was detected in the Morris water maze test. After the animals were killed, the brain sections were observed by histopathological examination; expressions of nAChRs at protein and mRNA levels were detected by Western blotting and RT-PCR. Results: The results showed that A␤ injection together with high content of cholesterol in diet induced significant impairment in rat memory; deposition of amyloid plaques and increased activated astrocytes were found in the cortex and hippocampus of the rats treated by A␤ injection or A␤ injection together feeding high content of cholesterol; the ␣7 and ␣4 nAChR subunits at protein level were decreased in rat brains by A␤ injection or A␤ injection plus high content cholesterol diet, but no change of ␤2 was observed; the ␣7 mRNA level was increased in the brains by A␤ injection or A␤ injection together with feeding high content of cholesterol; among above detections, more obvious changes were observed in the group treated by A␤ injection plus high content of cholesterol in diet. Conclusions: These data indicated that cholesterol can raise the neurotoxicity of A␤ and modify expression of nAChRs by certain connections, which might give an important explanation to that elevated level of plasma cholesterol induces an increased susceptibility to AD. P3-423

MOLECULAR DISSECTION OF THE INTERACTION BETWEEN APP AND ITS NEURONAL TRAFFICKING RECEPTOR SORLA/ LR11

Olav M. Andersen1, Vanessa Schmidt1, Robert Spoelgen2, Joergen Gliemann3, Joachim Behlke1, Denise Galatis4, William McKinstry5, Michael Parker5, Colin Masters4, Bradley Hyman2, Roberto Cappai6, Thomas Willnow1, 1Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany; 2Harvard Medical School, Charlestown, USA; 3Department of Medical Biochemistry, Aarhus, Denmark; 4The Mental Health Research Institute of Victoria, Parkville, Australia; 5St. Vincent’s Institute of Medical Research, Victoria, Australia; 6The Mental Health Resarch Institute of Victoria, Parkville, Australia. Contact e-mail: [email protected] Background: SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid ␤-peptide (A␤), the principal component of senile plaques in Alzheimer’s disease (AD). Objective(s): To gain insights into the molecular mechanisms governing sorLA/LR11 and APP interaction, we dissect the respective protein interacting domains. Methods: Using a fluorescence resonance energy transfer (FRET)-based assay of protein proximity we identified binding sites in the extracellular regions of both proteins. Surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA/LR11 fragments further narrowed down the binding domains. Conclusions: We showed that the cluster of complement-type repeats in sorLA/LR11 forms a 1:1 stoichiometric complex with part of the carbohydrate-linked domain of APP, and that this interaction is sufficient to promote association of both proteins in neurons. These data shed new light on the molecular determinants of neuronal APP trafficking and processing. P3-424

EXPRESSION OF NICOTINIC RECEPTORS ON ASTROCYTES INFLUENCED BY A␤ 1-42, LOVASTATIN AND CHOLESTEROL

Jin Xiu, Agneta Nordberg, Zhizhong Guan, Karolinska Institute, Stockholm, Sweden. Contact e-mail: [email protected] Background: Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in the pathogenesis of Alzheimer’s disease (AD). A␤ is the major constituent of the senile plaques, which play a role in triggering nerve cell