P4-256

S592 P4-256

Poster P4:: Wednesday Posters PACAP PEPTIDES AS NEW ␣-SECRETASE ACTIVATORS

Elzbieta Kojro, Rolf Postina, Katja Gehrig-Burger, Falk Fahrenholz, Institute of Biochemistry, Johannes Gutenberg-University, Mainz, Germany. Contact e-mail: [email protected] Proteolytic cleavage of the amyloid precursor protein (APP) by ␣-secretase within the A␤ sequence precludes formation of amyloidogenic peptides and leads to a release of soluble APPs␣, which has neuroprotective, anti-apoptotic and growth promoting properties. Therefore, pharmacological up-regulation of the ␣-secretase could be a possible approach for treatment of Alzheimer disease (AD). The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic, neuroprotective as well as anti-apoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC1 is expressed in several CNS regions including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on ␣-secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPs␣). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPs␣ secretion which was completely inhibited by the PAC1 receptor specific antagonist PACAP (6-38). In HEK cells stably overexpressing functional PAC1 receptors PACAP-27 and PACAP-38 strongly stimulated ␣-secretase cleavage of APP. The PACAP-induced APPs␣ production was dose-dependent and saturable. This increase of ␣-secretase activity was completely abolished by hydroxamate-based metalloproteinase inhibitors including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors we show that the MAP-kinase pathway (including ERK1 and ERK2), and PI3-kinase mediate the PACAP-induced ␣-secretase activation. PACAP treatment had no influence on expression level of ␣-secretases (ADAM 10, ADAM 17 and ADAM 9) and on the substrate APP. According to our results, the neuroprotective effects of PACAP may be mediated by activation of the ␣-secretase pathway. PACAP neuropeptides are able to cross the blood brain barrier and might be of therapeutic value for the treatment of Alzheimer disease. Therefore, we are currently testing PACAP peptides in an APP mouse model. P4-257

ACTIVATION OF PROTEIN KINASE C SUPPRESSES THE ␤-SECRETASE ACTIVITY THROUGH REGULATION OF THE DEGRADATION AND TRANSLOCATION OF BACE1

Lizheng Wang1, Hoon Shim1, Chengsong Xie1, Huaibin Cai2, 1National Institutes of Health, Bethesda, MD, USA; 2National Institutes of Health, Bethesda, MD, USA. Contact e-mail: [email protected] Background: BACE1, the ␤-site APP cleavage enzyme1, is the ␤-secretase responsible for generation of amyloid-␤ (A␤) peptides that are involved in the pathogenesis of Alzheimer’s disease (AD). Since BACE1 is a high priority target for anti-amyloidosis therapy in AD, it is important to identify signaling pathways that regulate the expression and function of BACE1. Previous studies suggest that activation of protein kinase C (PKC)-mediated intracellular signaling transduction pathways enhances the ␣-secretase-mediated cleavage of APP and reduces the production of A␤. Objective: To address whether activation of PKC modulates the accumulation and ␤-secretase activity of BACE1. Methods: Cultured mouse fibroblasts and neurons were treated with PKC activator, phorbol myristate acetate (PMA), as well as inhibitors of PKC, proteasome, and lysosome. The accumulation of BACE1, the ␤-secretase activity of BACE1, and the translocation of BACE1 to the cell surface were quantified. Conclusion: Here we report that activation of PKC by PMA inhibited ␤-secretase activity through either suppressing the accumulation of BACE1 protein or promoting the translocation of BACE1 protein to the cell surface. We found that the accumulation of BACE1 protein in mouse fibroblasts was suppressed by PMA through enhancement of the intracellular protease-mediated protein degradation pathways. In contrast, the

accumulation of BACE1 protein in primary cultured mouse cortical neurons was not affected following PMA treatment. Instead, treatment of PMA increased the presentation of BACE1 protein at the plasma membrane of neurons. Since the optimal conditions for ␤-secretase activity exist in the lumen of intracellular organelles such as Golgi, secretory vesicles, and endosomes, the postulated diminished levels of BACE1 protein within intracellular organelles following PMA treatment may cause decreased ␤-secretase activity in these neurons. Together, these findings demonstrate that activation of PKC inhibits ␤-secretase activity in neurons and nonneuronal cells through different mechanisms and further support the notion that elevation of PKC activity may serve as a potential therapeutic strategy to ameliorate amyloidosis in AD. P4-258

HETEROLOGOUS AMYLOID VACCINES INDUCE A GENERIC ANTI-AMYLOID RESPONSE

Pritam Das, Rudi Hrncic, Todd Golde, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: By definition, amyloids regardless of primary sequence of the subunit, share common structural features. Encouraged by the previous identification of anti-amyloid mAbs that recognize multiple amyloids and data showing that anti-amyloid antibodies can enhance clearance of light chain amyloid in mice, we tested the hypothesis that immunization with heterologous amyloids (hAs) formed from peptides that lack homology to any human or mouse protein could induce a generic anti-amyloid response. We find that vaccination of mice with hAs induces a robust anti-amyloid Ab response and that the anti-amyloid Abs are capable of recognizing multiple amyloids regardless of subunit. Objective(s): Our original rationale for this approach was largely driven by trying to avoid a harmful T-cell response. In most cases, we are immunizing with amyloids composed of subunits lacking homology to human or mouse proteins; thus MHC presentation and activated T-cells would be targeting non-self peptides. Furthermore, we find that amyloids formed from a 6 amino acid peptide induce a generic anti-amyloid response. In this latter case, sequence homology becomes less of an issue; such small peptides theoretically lack the ability to bind to MHC. We had originally speculated that such an approach might avoid self-tolerance issues and induce a stronger anti-amyloid immune response than fA␤42 vaccination. However, this speculation was largely based on the idea that an anti-amyloid response was induced by a thymus dependent (TD) mechanism. Current data suggests that amyloid is not a TD antigen but a thymus independent type 2 (TI2) antigen. The rules or immune recognition of a TI2 antigen are distinct from those for TD responses, and little is known about self-tolerance with respect to TI2 responses. Methods: By definition, TI2 antigens do not require help from ␣␤ T-cells or antigen presentation by MHC class II. Therefore, to determine if fA␤42 and hAs are TI2 antigens, we are currently vaccinating TcR ␣␤ knockout mice and MHC class II knockout mice with several hAs and fA␤42. Conclusions: This novel approach enables testing of the hypothesis that immunotherapy targeting amyloid and, potentially, a preamyloid aggregate is beneficial in mouse models of AD. P4-259

INHIBITORS OF AMYLOID AGGREGATION: SYNTHESIS AND EVALUATION BY QCM

Hiroaki Okuno1, Kanae Mori1, Tomoko Okada2, 1Toho University, Funabashi, Japan; 2AIST, Tsukuba, Japan. Contact e-mail: [email protected] Background: We found1-3 that the A␤16-20 amino acid residue (KLVFF) of amyloid-beta (A␤) plays a very important role for the aggregation formation. Objective(s): To synthesize aggregation inhibitors of amyloidbeta, and evaluate the ability by quartz-crystal microbalance (QCM) as well as in vitro test. Methods: Used a peptide synthesizer for the efficient development of the target compounds. Conclusions: A series of the inhibitors consisting of a molecular recognition element and an aggregation disrupting part were prepared based largely on amino acid analogues, and