p2-343: peripherally overexpessed neprilysin in muscle reduces brain amyloid in a transgenic mouse...
TRANSCRIPT
Methods: PF-4360365, a humanized monoclonal antibody specific for theC-terminus of A�40, was administered to 5-month-old plaque-bearingAPPxPS1 mice (Swedish mutation of APP, �E9 mutation of PS1). Brain,CSF, and plasma were harvested 5 days following a single intraperitonealinjection of vehicle or 0.1-10 mg/kg PF-4360365 to determine the effect onA� levels. Results: A�1-X, 1-40, and 1-42 were not significantly reducedin cortex. In hippocampus, all three A� fragments were significantlyreduced to varying degrees, although no dose-response relationship wasobserved. A�1-X levels in CSF were significantly elevated at the 10 mg/kgdose; no other significant changes were detected. Assaying samples re-vealed up to 10,000% increase in plasma A�1-X but only up to 35%change in A�1-40 due to interference of the therapeutic antibody withA�1-40 detection. Samples were extracted with guanidine and antibodieswere removed using solid-phase extraction (SPE). Following this clean-upstep, A�1-40 levels correlated with A�1-X levels. A�1-42 levels in plasmaappeared to be reduced by up to 50% before SPE and increased by up to300% following SPE. However, using synthetic A� standards in solutionor spiked into plasma, it was discovered that excess A�1-40 concentrationscan increase the apparent A�1-42 signal with the polyclonal antibodiesbeing used to measure A�42. When a proprietary monoclonal A�42-selective monoclonal antibody was used to capture A�42, no change inplasma A�42 levels were observed after PF-4360365 treatment. Therapeu-tic antibody concentrations in plasma significantly correlated to A�1-X andA�1-40 in plasma, but not to any A� fragment in CSF or brain. Nointer-compartmental correlation was observed for A� levels in vehicle orantibody-treated mice. Conclusions: A single injection of PF-4360365showed selective mobilization of A� in the CNS, accompanied by robustelevation of A�40 in novel A� assays following denaturation and chro-matographic separation of the antigen-antibody complex.
P2-341 A ROBOTIC SCREEN OF A SMALL-MOLECULECHEMICAL LIBRARY FOR INHIBITORS OF A�OLIGOMER FORMATION
Harry LeVine, III1, Corinne E. Augelli-Szafran2, Randal S. Voss3,John A. Walker4, 1University of Kentucky, Lexington, KY, USA;2Harvard Medical School and Brigham and Women’s Hospital, Boston,MA, USA; 3Department of Biololgy, University of Kentucky, Lexington,KY, USA; 4Department of Biology, University of Kentucky, Lexington,KY, USA. Contact e-mail: [email protected]
Background: Soluble oligomers of �-amyloid peptide are believed to bethe proximal insult in Alzheimer’s disease. Preventing the initial assemblyof A� into stable toxic structures would short circuit the cascade ofbiological responses that lead to neuronal death and the clinical sequelae.Methods: The Apexscreen 5040 library of small organic molecules (�500m.w.) was initially screened in duplicate at 10 �g/ml and a bioA�42concentration of 10 nM on a Tecan Genesis LiHa/TeMo robotics system.Hits, defined as producing �75% inhibition of the oligomer signal in theinitial screen, were then titrated by serial dilution and an IC50 calculatedbased on their molecular weight. Results: On average between 1-2 hitswere confirmed per plate (80 cpds). Numerous compounds enhanced theoligomer signal and were not further analyzed. This effect is also seen withthe detergent SDS which has been ascribed to micelle formation. Lowmicromolar hits comprising several distinct series of compounds, each withcommon structural features, were identified. Conclusions: Confirmed hitsare currently being evaluated on their potential for future chemical devel-opment to enhance their activity and drug-like properties. Since the bio-tinylated A� single-site assay format detects �30 kDa oligomers, furthertesting is necessary to determine if certain compounds block assembly bystabilizing small oligomers of the synthetic peptide which normally revertto monomer under the assay conditions. The effects of compounds oncellular biological oligomer production, assembly and biological activityare also being evaluated. Funded by AG028816-01.
P2-342 VACCINATION OF AMYLOID PRECURSORPROTEIN TRANSGENIC MICE WITH ABACTERIOPHAGE-BASED SHORT AMYLOID-�PEPTIDE VACCINES WITHOUT ADJUVANTINDUCE A STRONG TH2-TYPE ANTI-AMYLOID-�IgG AND IgM RESPONSE AND REDUCEAMYLOID-� BURDEN
Qing-You Li1, Marcia N. Gordon1, Bryce Chackerian2,Jennifer Alamed1, Kenneth E. Ugen1, Dave Morgan1, 1University ofSouth Florida, Tampa, FL, USA; 2University of New Mexico,Albuquerque, NM, USA. Contact e-mail: [email protected]
Background: Previous work has shown that Q� bacteriophage -based A�
N-terminal aa1-9 short peptide vaccine, induced high antibody titers againstA� in C57BL/6 or SJL mice. The antibodies were predominately IgG2c,characteristic of a Th1-type response. Methods: In this study we vaccinatedAPP transgenic mice (Tg2576) at age of 9 months with either A� 1-9 (10�g)or C-terminal A� 28-40 (10�g) conjugated to Q� bacteriophage without usingadjuvant. Results: Our data showed that mice immunized with the A� 1-9peptide elicited high IgG antibody titers against A�1-40 (average titer � 4.37�g/ml; n � 6) compared to the A�28-40 peptide (average titer � 0.91�g/ml;n � 6) after two inoculations. However, following eight monthly boosts theanti-A� 40 IgG titers induced by the A� 1-9 were gradually decreasing,whereas the A� 28-40 domain increased the titers following each boost andreached maximal antibody levels at the fourth boost. Interestingly, immuniza-tion with A� 1-9 peptide induced strong IgG1 and IgG2b, Th2-type, immuneresponse with lower IgG2a and IgG2c in all six immunized APP transgenicmice. Vaccination with A� 28-40 peptide predominantly elicited IgG2b im-mune response and IgG1 titers which were similar to IgG2a and IgG2c.Intriguingly, the A� 1-9 domain induced a robust anti-A�1-40 IgM response.From the second immunization through the final boost, IgM titers were muchhigher than the IgG response induced by this immunogen. The A� 28-40vaccine failed to induce an IgM response. There was no anti-A� antibodyresponse in mice vaccinated with PBS vehicle control. Both vaccines reducedamyloid burden in the brain in APP transgenic mice and the A� 28-40 peptidealso significantly enhanced circulating total A� levels. Conclusions: Thus, thisnovel A� vaccination approach could be more effective with a greater safetyprofile than traditional vaccines using adjuvants in Alzheimer’s disease therapy.
P2-343 PERIPHERALLY OVEREXPESSED NEPRILYSININ MUSCLE REDUCES BRAIN AMYLOID IN ATRANSGENIC MOUSE MODEL OF AD
Yinxing Liu1, Hanjun Guan1, Ronald L. Klein2, Salvatore Oddo3,Frank M. LaFerla3, M. Paul Murphy1, Louis B. Hersh1, 1University ofKentucky, Lexington, KY, USA; 2Louisiana State University HealthSciences Center, Shreveport, LA, USA; 3University of California Irvine,Irvine, CA, USA. Contact e-mail: [email protected]
Background: Neprilysin (NEP) is a peptidase that efficiently degrades theamyloid beta peptides (A�) believed to be the causative agent of Alzheimer’sdisease (AD). Methods: We introduced adeno-associated virus (AAV) vectorcarrying the mouse NEP gene into muscle tissue of 3�Tg-AD mice to producea sustained level of NEP. Results: Here we show that this overexpression ofNEP in muscle significantly decreases brain A� with no apparent adverseeffects. Conclusions: Here we show that this overexpression of NEP in musclesignificantly decreases brain A� with no apparent adverse effects.
P2-344 NEUROPROTECTIVE EFFECTS OFACTIVATORS/AGONISTS OF THE SIGMA-1CHAPERONE PROTEIN AGAINST AMYLOIDTOXICITY IN A MOUSE MODEL
Tangui Maurice1, Vanessa Villard1, Johann Meunier1, Emeline Keller1,Fanny Malhaire1, Alexandre Vamvakides2, 1INSERM U 710,Montpellier, France; 2ANAVEX Life Sciences, Pallini, Greece. Contacte-mail: [email protected]
T473Poster Presentations P2: