S1-02-05

Symposia S1-02: Disease Mechanisms (APP and A␤)

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A␤40. This suggests that the longer A␤ species is processed at every three residues from its carboxyl portion on the ␣-helical surface of the APP transmembrane domain. Furthermore, recently established CHAPSO-solubilized assay system has clearly shown that the amounts of A␤40, A␤42, and A␤43 are equal to those of AICD, irrespective of wild-type or mutant APP and presenilin 1/2. Several species of A␤, including trace amounts of A␤48 and A␤49, were generated by this system, but only two species of AICD, AICD49-99 and AICD50-99, were detectable. These data suggest that ⑀-cleavage is a primary one and that longer A␤s (A␤48 and A␤49) generated by ⑀-cleavage are processed at every three residues, thereby producing A␤40, A␤42, and A␤43. S1-02-02

GAMMA-SECRETASE COMPLEX ASSEMBLY AND INTRAMEMBRANE PROTEOLYSIS BY G X G D PROTEASES

Christian Haass, Christoph Kaether, Harald Steiner, Regina Fluhrer, Ludwig-Maximilians-University Munich, Munich, Germany. Contact e-mail: [email protected] Background: ␥-Secretase mediates the final cleavage of APP C-terminal fragments and liberates amyloid ␤-peptide (A␤). Reconstitution experiments as well as genetic evidence demonstrate that ␥-secretase is a complex composed of presenilin (PS1/PS2), nicastrin, pen-2, and aph-1. Additional regulatory and assembly factors may exist, however, these are unlikely to be integral components of the ␥-secretase complex. The proteolytically active center of ␥-secretase is comprised of PS1 or PS2. We have shown earlier that all presenilins contain a GxGD motif in their C-terminal active site in trans-membrane domain 7. The GxGD motif is fully conserved in signal peptide peptidase (SPP). Objectives and Methods: (1) We previously proposed a mechanism of complex assembly by which unassembled subunits are retained in the ER and only the fully assembled complex is exported from the ER. (2) We compared the proteolytic mechanisms of members of the GxGD family of proteases. Conclusions: (1) We have now further proven our concept on ER based ␥-secretase complex assembly by our finding that unassembled pen-2 is selectively retained in the ER by Rer-1, a putative ER-retention/retrieval protein originally identified in yeast. Overexpression of Rer-1 apparently facilitates maturation of the ␥-secretase complex. (2) The GxGD motif is functionally conserved in the SPP-like proteins, SPPL2 and SPPL3. Differential subcellular distribution of the SPP family members suggests individual functions and substrate specificities. We will also demonstrate a surprisingly similar mechanism of intramembrane proteolyis by SPPL2 and ␥-secretase. Both apparently involve a step-wise cleavage within the transmembrane domain. However, SPP family members do not require additional co-factors and only accept type-2 oriented substrates. S1-02-03

PHARMACOLOGICAL MODULATION OF AB42 GENERATION (BY NSAIDS)

Edward Koo, University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Abstract not available. S1-02-04

ROLE OF AB OLIGOMERS IN AD

Dennis Selkoe1, Matthew Townsend1, Ganesh Shankar1, Tapan Mehta1, Marcia Podlisny1, Dominic Walsh2, 1Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 2Laboratory for Neurodegenerative Research, Conway Institute, University College, Dublin, Ireland. Contact e-mail: [email protected] Background: Converging lines of evidence suggest a central role for amyloid ␤-protein (A␤) in the genesis of Alzheimer’s disease. Studies we’ve conducted over the past decade suggest that small, soluble oligomers of A␤ rather than insoluble amyloid fibrils may be early effectors of synaptic dysfunction. We initially observed that stable CHO cell lines

expressing wt APP (7W) or FAD mutant APP (7PA2) secrete highpicomolar concentrations of SDS-stable oligomers, providing a ready source for examining the biological activity of natural oligomers of human A␤ in vitro and in vivo. Objectives: To characterize further the neurobiological properties of soluble oligomers and assess ways to block their synaptotoxicity. Methods: 7PA2 and parental CHO- cells were conditioned with or without putative inhibitors of fibrillogenesis. For additional biochemical specificity, media were fractionated by SEC and the activities of oligomer and monomer fractions compared. With our collaborators, bioactivity was assessed in rats and mice in several ways, including measuring a) hippocampal LTP, an electrophysiological correlate of learning and memory; b) performance in remembering a complex learned behavior; c) effects of certain small-molecule modulators of A␤; and d) effects of exogenous or endogenous anti-A␤ antibodies. Results: A␤ oligomers could be quantitatively separated from monomers by non-denaturing SEC. The cell-derived oligomers did not affect baseline synaptic transmission but consistently blocked hippocampal LTP, with trimers appearing more potent than dimers. Monomers had no effect. A␤ vaccination in wt rats rescued the inhibition of hippocampal LTP caused by ICV microinjection of the oligomers, and the extent of rescue correlated with the levels of anti-oligomer antibodies. We identified several small molecules that decreased oligomer levels and abrogated their block of LTP. ICV injection of oligomers in rats interfered transiently and potently with performance on a complex lever-pressing task. We are now searching for longer-term effects of the oligomers on neuronal and glial biochemistry, including tau alteration. Conclusions: Disease-relevant concentrations of naturally secreted oligomers of human A␤ alter both synaptic plasticity and cognitive behavior in vivo. Moreover, anti-A␤ antibodies and certain small molecules that retard oligomer formation can each rescue oligomer-mediated electrophysiological effects, providing a rational mechanism for therapeutic intervention in AD. S1-02-05

DEGRADATION OF AB

Takaomi Saido, Riken Brain Science Institute, Wako Shi, Japan. Contact e-mail: [email protected] Background: Amyloid beta peptide (Abeta) is a primary pathogenic agent in Alzheimer’s disease (AD) development, and its quantity in the brain is determined by the metabolic balance between anabolism and catabolism. Objective(s): We aim to determine the primary cause of Abeta deposition in sporadic AD, which accounts for more than 99% of all AD cases, and to develop new therapeutic strategies using Abeta-degrading mechanisms. Methods: We have identified the major Abeta-degrading enzyme as neprilysin and demonstrated that reduced catabolism causes elevation of Abeta levels in the brain whereas familial AD has been shown to be caused by increased anabolism. We also have succeeded in establishing an experimental gene therapy using human neprilysin cDNA, and recently discovered that a neuropeptide, somatostatin, regulates Abeta metabolism, which indicates that somatostatin receptor(s) can be a pharmacological target for prevention and treatment of AD. An attempt to produce a second-generation mouse model of AD that overproduces Abeta without overexpressing amyloid precursor protein will also be discussed. Conclusions: It is very much likely that reduced Abeta degradation may be a primary cause for sporadic AD and that activation of neuronal neprilysin using somatostatin agonists may contribute to prevention and treatment of AD. S1-02-06

CLEARANCE OF A␤

Berislav V. Zlokovic, University of Rochester, Rochester, NY, USA. Contact e-mail: [email protected] The levels of amyloid-␤ peptide (A␤) in the brain are controlled by its rates of production from a larger A␤-precursor protein (APP) and the rates of clearance. Here, we will discuss a hypothesis that late-onset Alzheimer’s disease (AD), as well as some of the related familial forms of the disease caused by genetic mutations in A␤ protein (e.g., Dutch, Iowa), can develop due to brain storage disorders and the problem of clearance. We will show that abnormal-