amyloid-lowering ability in mouse neuroblastoma (N2a) cells transfectedwith human Swedish APP695 (N2aAPPswe). Objective(s): To evaluateanti-amyloidogenic efficacy of aged garlic extract in N2aAPPswe cells.Methods: Swedish APP695 (N2aAPPswe) cells, N2a cells transfected withwild type human APP695 (N2aAPPwt) and non-transfected N2a cells(N2a) were propagated and maintained in 45% DMEM, 45% Opti-MEMand 10% FBS, containing 200�g/ml G418 and 1% Penn/Strep at 37oC.G418 was omitted for N2a. Cells were seeded at a density of 106 cells/well,and cell viability determined with Trypan Blue dye exclusion. 24h prior toAGE treatment, cells were incubated with lipoprotein-free serum, and thentreated with different concentrations of AGE (1/2/4/8/16mg/ml) in a serum-free medium for 24h. After treatment, conditioned media were harvested,reacted with protease inhibitor cocktail, centrifuged at 10,000g at 4oC for10 min, supernatant protein-concentration determined (BCA, Pierce).100�l aliquots containing equal amount of protein were subjected tosandwich ELISA for A�40/42 (Signet), values expressed as pg/100�l.Results: [1] AGE treatment between 1-8mg/ml concentration rangeshowed cell viability above �95%, while at the highest AGE concentration(16mg/ml) cell viability declined to �75%; [2] Both N2aAPPwt andN2aAPPswe cells showed release of A�40/42 peptides in conditionedmedia with the predominance of A�40, which were decreased after AGEtreatment, but more robust reductions were observed in N2aAPPswe cells;[3] AGE treatment produced dose-dependent reduction in the secretedlevels of A�40 by 12.9% (1mg), 23.1% (2mg), 40.8% (4mg), and 40.3%(8 and 16 mg), and reduction in the secreted levels of A�42 by 7.5% (1mg),16.1% (2mg), 21.5% (4mg), 21.5% (8mg), and 20.4% (16 mg) inN2aAPPswe cells. Conclusions: AGE treatment reduced secreted levels ofA� peptides in a dose-dependent manner peaking at 4mg/ml optimumconcentration in N2aAPPswe cells while highest concentration of AGE iscytotoxic. Although preliminary, this is the first report showing amyloid-lowering effects of AGE on N2aAPPswe cells.

P4-089 SYNAPSE LOSS AND �-AMYLOID DEPOSITIONIN THE TG2576 MOUSE—A QUANTITATIVEELECTRON MICROSCOPY STUDY

Hongxin Dong1, Maureen V. Martin2, Shawn Chambers2,John G. Csernansky2, 1Washington University School of Medicine, St.Louis, MO, USA; 2Washington University School of Medicine, St. Louis,USA. Contact e-mail: dongh@psychiatry.wustl.edu

Background: The deposition of �-amyloid plaques throughout the brain isone of the neuropathological hallmarks of Alzheimer’s disease. However,the relationship between the presence of � -amyloid (oligomers andplaques), and the degeneration of neurons and their processes is poorlyunderstood. Although the evidence indicates that � -amyloid impairs syn-aptic function, there is little evidence whether amyloid influences synapticnumber at the ultra-structure level. Objective(s): In this study, we assessedsynaptic density within the entorhinal cortex and hippocampus in Tg2576mice. Methods: Forty-three Tg2576 mice at 6 to 18 months of age usinglight and electron microscopy with non-biased quantitative methods forthis study. Synaptophysin positive boutons and synaptic density in layers IIand III of the entorhinal cortex and in the stratum radiatum of the hip-pocampus were analyzed. Conclusions: We found that the density ofsynaptophysin positive boutons related to the proximity of � -amyloidplaques. More specifically, the density of synaptophysin positive boutonswas significantly decreased (p�0.05) within 100-200�m from � -amyloidplaques, but not at greater distances from plaques in these areas. Usingelectron microscopy, we examined synaptic number in the regions 62, 124,186 and 248 �m from the edges of � -amyloid plaques. We found asignificant overall effect of sampling region (F� 21.672, df � 3, 39, p �0.0001) and a significant sampling region by plaque status interactioneffect (F � 8.045, df � 3,30, p � 0.001) on synapse density in layers II andIII of the entorhinal cortex and in the stratum radiatum of the hippocampus.That is, the synaptic density was decreased as a function of distance fromplaque. Furthermore, synaptic density was decreased in the entorhinalcortex at 18 months old (p�0.05) but not at 6-9 months age of Tg2576

mice. There was no significant change in synaptic density in the stratumradiatum of the hippocampus in Tg2576 mice at either 6-9 or 15-18 monthsof age. These results provide the first quantitative morphological evidenceat the ultra-structure level of a spatial relationship between amyloid plaquedeposition and synaptic degeneration within the entorhinal cortex andhippocampus of Tg2576 mice.

P4-090 LOVASTATIN INHIBITS AMYLOID-PRECURSORPROTEIN (APP) BETA-CLEAVAGE THROUGHREDUCTION OF APP LEVEL IN CAVEOLAE-LIKE LIPID RAFTS: POSSIBLE INVOLVEMENTOF APP ENDOCYTOSIS

Je-Seong Won, Avtar K. Singh, Miguel A. Contreras, Singh Inderjit,Medical University of South Carolina, Charleston, SC, USA. Contacte-mail: singhi@musc.edu

Statins are drugs for hypercholesterolemia that inhibit HMG-CoA reduc-tase. Recent studies showed a reduced prevalence of Alzheimer’s diseasein patients using statins and proposed that statins mediate antiamyloido-genic effect through functional and/or structural modifications of beta-amyloid precursor protein (APP) containing membrane microdomains,such as lipid rafts. However, the detailed mechanisms underlying this arepoorly understood. In hippocampal neurons, we observed three pools ofbeta-amyloid precursor protein (APP) in the membrane. Two were asso-ciated with caveolae-like lipid rafts (CLLR; caveolin-1 and flotillin posi-tive) and glycosylphosphatidylinositol-anchored protein containing lipidrafts (GCLR; NCAM positive), and another was associated with non-lipidrafts membrane (NLRM; CD71 positive) fractions. The GCLR and NLRMfractions included majority of APP levels which were not altered bylovastatin treatment (5microM/36hrs). On the other hand, APP levels inCLLR fractions were relatively small and effectively reduced by lovastatintreatment. In addition to APP levels, lovastatin also inhibited beta-secretaseactivity in CLLR and reduced Abeta generation in a geranylgeranyl-phosphate (GGPP)-dependent manner. APP levels in CCLR were alsoreduced by endocytosis inhibitor (phenylarsine oxide). Moreover, specificreduction of APP levels in endosomal fractions by lovastatin and concom-itant inhibition of phosphatidylinositol 3 kinase (PI3-K) and reduction ofcellular early endosomal antigen 1 (EEA1), key regulators of endocytosisactivity, suggest that lovastatin decreases APP levels in CCLR throughinhibition of EEA1/PI3-K-mediated endocytosis activity. In summary,these results document that lovastatin modulates APP levels in CCLR andAPP beta-cleavage through inhibition of EEA1/PI3-K activity in GGPPdependent manner.

P4-091 MECHANISMS OF NEUROPROTECTIONMEDIATED BY NEPRILYSIN IN APP-TRANSGENIC MICE

Eliezer Masliah1, John Rose1, Edward Rockenstein1, Michael Mante1,Leslie Crews1, Louis Hersh2, Fred Gage3, Inder Verma3, Robert Marr4,1University of California San Diego, La Jolla, CA, USA; 2University ofKentucky, Lexington, KY, USA; 3Salk Institute, La Jolla, CA, USA;4Rosalind Franklin University of Medicine and Science, Chicago, IL,USA. Contact e-mail: emasliah@ucsd.edu

Background: The endopeptidase neprilysin (NEP), has been implicated asa major A� degrading enzyme in mice and humans and decreased NEPactivity might play a role in the pathogenesis of Alzheimer’s disease.Previous studies suggest that NEP might rescue the pathological phenotypein mutant amyloid precursor protein (APP) transgenic (tg) mice by reduc-ing the levels of A� monomers and oligomers. Objective(s): Neprilysinappears to display neurotrophic effects and previous studies have shownthat this peptidase might cleave substrates other than A�. Methods: Weinvestigated the potential mechanisms involved in tg mice expressing APPunder the thy-1 promoter and NEP under the PDGF-b promoter. Results:Consistent with the neuroprotective effects, NEP reduced the performancedeficits and neurodegenerative alterations in APP tg mice. Biochemical

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