Roles of β-amyloid converting enzymes BACE-1 and BACE-2 in β-amyloid secretion

s254

11160)

PRESENILIN MODULATES IN THE WNT PATHWAY

THE ACTIVITY

OF DISHEVELLED

lovastatin (SOpg/ml). The relative amount of API-42 to API-40 was increased by cholesterol, wheras the ratio of AP I-17 to Abl-40 was unchanged. Thebe changes were confirmed by measure of immunoprecipitable %-Met incorporation, and by Immunofluorescence. Raised AD-deposition in brain has been reported in cholesterolfed rabbita (Sparks DL 1996 Neurobiol. Aging 17: 291.299). Raised circulating cholesterol has been reported as a risk factor for AD in men (Notkola et al 1998 Neuroep~demiology 17; 14.2O), possibly by accelerating deposltion of neurotoxic forms of A@.

Genetic epiataais experiments have shown that Wnt proteins transduce their signal\ through dishevelled (dvl) proteins to inhibit glycogen synthase kinave 3-p (GSK3-P). Wnt signalling stabilises p-catenin and activate% TCFILEF-1 transcription. Recent reports have suggested that presemlin 1 (PSI ) plays a role in the Ftabilisation or turnover of p-catenin and that mutants of PSI. which are arwciated with familial Alzheimer’s dwxse, differentially affect p-catenm catabolicm. It has been reported that wild-type PSI forms a complex with p-catenin and GSK3-P to target it to the ubiquitin proteasome system whereas mutants of PSI do not bind GSK-3P and hence reduce p-cat&n degradation. We have mvestigated the mteractions between PSI (wild-type and several mutantsl and human dirhevelled protein. a member of the Wnt pathway. As anticipated. over-expression of dvl resulted in the cytosolic atabilisation and nuclear tranrlocatmn of p-catenin, presumably via inhibition of GSK-3P. Wild type PSI modulated thic effect by cauung a reduction in the number of cell\ showing dvl induced nuclear tran~location of p-catenin. Thw our results further demonstrate that PSI can regulate Wnt signaIling

11161(

PLAQUE-INDEPENDENT, HIBITION OF SYNAPTIC LOID @PEPTIDE.

CYCLOOXYGENASE-MEDIATED INNA+,K’ -ATPASE ACTIVITY BY AMY-

,

Mounting evidence wpports the involvement of I) senile plaque-independent action\ of soluble amylold @ (Ab)-peptide and 2)the cyclooxygenase (COX) activity of prostaglandin H yynthase in the pathogenesia of Al~heimer‘a disease. We have begun to investigate the subcellular actions of a dimethylsulfoxide-solubilized form of A@ 2.5-35, the putative neurotoxic fragment of A& in osmotically ly>ed nerve-endmg (~ynapto~omal) membrane fractions prepared from rat cerebral cortex. Thi\ fraction contain!. both synaptic placma membranes and mitochondria. We report that a low concentration (10 nM) of AP 25-35, but not the reverse sequence peptide (AP 35-25). produced n rapid (withm minute\) albeit labile inhibitjon of plasma membrane Na’.K- -ATPaw actiwty, a fundamental regulator of neuronal function and viability. by up to 48% in the relatively crude synaptowmal membranea. AP 25-35 did not Inhibit Na-,K’-ATPase activity in the more purified synaptic plasma membwtw which contained much lab mltochondrm a\ asewed by marker enzyme activity. Preincubatmn of the syneptowmal membrane\ with the COX inhibitor indomethacin completely prevented the Inhibition of Na’, K’-ATPase actiwty by AP 25-35. Indeed. AD 25-35 produced a rapid increase in indomethacin-sensitive proataglandm immunoreactivity m the $ynaptosomal membranes by up to 53% Moreover, 100 nM of the hydroperoxide product (PGG2) of COX-dependent arachidonic aad metaboli\m, but not the corresponding alcohol (PGHZ), inhibited Na+,K+-ATPahe activity by up to 64 L? in the synaptowmal membranea but not in the synaptic plasma membranes. Thex finding? suggest that a soluble form of Al3 may produce neurotoxicity. at least in part, by activating COX and, subsequently. inhibiting Na+. K ’-ATPase activity hy a mechanism that could involve perturbation of mitochondrial Cunction by PGG?. Furthermore, they provide a possible ba\l+ for the apparent efficacy of COX inhIbitor\ (i.e., NSAIDS)in the prevention/delay and. perhap\. treatment of Alrheimer‘\ Disease. Future work will be aimed at defining the mechanwn of COX activation, the site (e.g., plasma membrane or mitochondrial of A(J action. and the suggatcd role of mitoch[mdria.

/ii@

CHOLESTEROL UPREGULATES PRODUCTION AND I-42 IN TRANSFECTED CELLS.

OF

AP

l-40

ApoE 19 a susceptibility factor for AlLheimer’s disease (AD), and one possibility 1s that the risk is mediated by raising cholesterol levels. Changes in cholesterol content of intracellular membranes could alter trafficking of u, p- or y-%xretases, and their action on APP. We measured secretion of Ab varients from APP-transfected HEK cells by capture from cell media onto an antibody to the N-terminal IO residues of A!3 covalently linked on a ProteinChip array. M&s analyaea permitted detection of 12 d~xrret AP fragmentr. with l-40 being the major specie\ (see fig). The ProtemChip was illso used to chow that AP l-42 is the major variant in AD brain homogenates. A@ I-40 secreted from cells was increased 1.8?0.07 fold by preincubation with cxogenow cholearol (2oO~glml). and decreased 40% by preincubatmn with

ROLES OF P-AMYLOID CONVERTING BACE-2 IN f%-AMYLOID SECRETION

ENZYMES BACE-I AND

The aspanyl protease BACE-I has been reported to act as p-secretase, which cleaves the P-amyloid peptIde precursor, APP, before realdue Asp-l of the P-amyloid peptide (AP) sequence. The BACE-I protease domain is 80% homologous to the corraponding domain of BACE-2, a protease encoded by a gene on chromosome 21. These proteases have been conserved during evolution. A C. rlejinns protease shows 60% homology to the protease domain of BACE-2, and the yeast Bar1 protease ia 50% homologous. To confirm that BACE po\se
THE APOE ALLELE IS ASSOCIATED WITH ENHANCED NITRIC OXIDE PRODUCTION AND ENHANCED ARGININE TRANSPORT.

Although apolipoprotein E (ApoE) participates in hpid transport and regulates tiaaue cholesterol flux, ApoE alw plays a role in the immune system. Treatment of macrophaga wth ApoE enhances the production of nitric oxide (NO), a critlcal