S3-02-03

Symposia S3-02: Disease Mechanisms (APOE) cholesterol to sphingomyelin, correlating to disease age of onset and A␤40/A␤42 ratio. Cholesterol and sphingomyelin are main building blocks of lipid microdomains suggesting APP/A␤ involvement in the regulation of raft function. These regulatory cycles are equally effective in primary neurons, human neuroblastoma cell culture lines as well as a variety of APP expressing peripheral cells and cells of rodent, primate and human origin. Two metabolic enzymes were identified to be regulated by A␤. Remarkably, both enzymes (HMG-Co A reductase for A␤40 and SMase for A␤42) were previously shown to represent the main regulatory enzymes in their respective homeostatic pathways, already indicating that A␤ function for lipid homeostasis is essential and of critical relevance. Indeed, knock-out of presenilin or APP in mice abolishes cholesterol and sphingomyelin down-regulation. Whereas A␤-feeding of APP or PS I/II knock-out cells recovers cholesterol and sphingomyelin regulation. The strict necessity of PS I and PS II for APP cleavage in A␤ mediated lipid homeostasis shows close relation to cholesterol and sphingomyelin regulation by SREBP cleavage and signaling, indicating the additional involvement of higher level regulatory cycles. Statins (cholesterol lowering drugs) are under investigation for prevention and treatment of AD. Interestingly, statins mechanistically replace A␤40 in this regulatory feed-back cycle, resulting in reduced cholesterol and A␤ production. S3-02-02

ACAT INHIBITORS IN REDUCING ABETA GENERATION

Dora M. Kovacs, Harvard Medical School, Charlestown/Boston, MA, USA. Contact e-mail: [email protected] Increasing evidence shows that intracellular cholesterol regulates amyloid precursor protein (APP) processing and A␤ production. Consequently, therapies already developed for dyslipidemia and atherosclerosis are becoming attractive as potential strategies for reducing ADrelated amyloid pathology. Amongst compounds with systemic lipidlowering effects, only statins are relatively safe, widely available, and effective in altering cellular cholesterol metabolism. Results from clinical trials with statins have not been consistent, likely due to differences in the type of statin used, dosage, length of treatment, severity of the disease, and other factors. These studies are ongoing. We have been investigating a different class of compounds, acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, which prevent conversion of cholesterol and fatty acids into cholesteryl-esters. ACAT inhibitors are not marketed but are being actively developed for treatment of cardiovascular disease. We have previously shown that a well-characterized ACAT inhibitor, CP-113,818, reduces secreted A␤ levels in cellular models of AD by inhibiting amyloidogenic processing of APP. We have also found that CP-113,818 ameliorates AD-like pathology in the brains of transgenic mice expressing human APP751 containing the London (V717I) and Swedish (K670M/N671L) mutations. Additionally, CP113,818 improved spatial learning, which correlated with decreased A␤. In non-transgenic animals, we have demonstrated that CP-113,818 treatment regulates processing of endogenous APP resulting in decreased APP-CTF production. A similar decrease in APP-CTF levels was also observed in neuronal and non-neuronal cells treated with two different ACAT inhibitors. We have recently found that a different ACAT inhibitor, CI-1011 (avasimibe) also reduces A␤ generation in cell-based assays, while decreasing APP-CTF levels. CI-1011 has previously reached phase III trials for prevention of atherosclerosis. CI1011 was also effective in reducing brain cholesteryl-esters in mice. Currently, we are establishing whether CI-1011 can reduce A␤ generation in transgenic or non-transgenic mouse brains. In mechanistic experiments, we are elucidating the role of lipid rafts as potential sites for A␤ production in the presence of ACAT inhibitors. Both statins and ACAT inhibitors may offer hope as novel strategies for the treatment and prevention of Alzheimer’s disease.

S3-02-03

S47

DISEASE MODIFICATION: FROM ETIOLOGICAL HYPOTHESIS TO CLINICAL TESTING

Judes Poirier, McGill University, Verdun, PQ, Canada. Contact e-mail: [email protected] Background: The discovery that the apolipoprotein E4 (apoE4) allele is strongly linked to both sporadic and familial late onset Alzheimer’s disease (AD) raises the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain of AD subjects. The presence of the E4 allele has been associated with lower levels of apoE in both serum and brain tissues in AD. In an attempt to reverse the apoE deficit in AD, we identified and characterized several apoE inducer agents using a low throughput astrocyte screening assay. Positive candidates include estrogen, probucol, tacrine and heptylphysostigmine (two potent butirylcholinesterase inhibitors) and, indomethacine, a potent anti-inflammatory drug. In contrast, statins, cholesterol synthesis inhibitors, were found to inhibit apoE synthesis and secretion in vivo and in vitro. Objective(s): Probucol and statin are known to both act as potent cholesterol lowering drugs but somehow diverge in their effect toward apoE synthesis and expression. The objective of this study is thus to further characterize the effect of drugs on brain lipid metabolism and apoE neurobiology and to identify brain specific targets responsible for diverging modes of action. Methods: C57/6J mice and human astrocytomas treated with various concentrations of i) statins and ii) probucol were used to profile alterations in apoE and apoE receptor expression, cholesterol synthesis and cholesterol concentration in acute and chronic time course analyses. Conclusions: Not all statin variants were found to affect brain apoE, apoE receptor and HMG CoA reductase expression in the brain. When significant, the brain alterations matched those observed in the liver. In contrast, probucol was shown to enhance both apoE synthesis and secretion, apoE/apoB (LDL) receptor activity as well as intracellular cholesterol transport while increasing synaptic density in specific areas of the hippocampus. S3-02-04

EXPLORING THE MECHANISMS BEHIND RISK FACTORS FOR ALZHEIMER’S DISEASE

Angel Cedazo-Minguez, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: Deterministic genetic Alzheimer’s disease (AD)-causing mutations are rare and long-term multifactorial processes are in the origin of the majority of the AD cases, called ‘sporadic’. Complex interactions between both genetic and environmental risk factors are likely behind the mechanisms leading to sporadic AD. The presence of the E4 isoform of the apolipoprotein E (apoE) is the most important known genetic risk factor for AD. Additionally, several life-style factors have been described by epidemiological studies, including vascular risk factors such us high serum cholesterol. Objectives: To investigate the mechanisms by which apoE4 and high cholesterol diet increase the risk for AD. Methods: We explored the effects of hypercholesterolemia and of the apoE genotype in brain using cDNA microarrays in two models: Wild type and ApoE knockout mice on normal and high cholesterol diets; and neuroblastoma cells treated with exogenous human apoE isoforms (E3, E4). We further investigated some selected molecules with classical biochemical methods in different models, including human brain from AD sufferers. Conclusions: We have identified several molecules altered by apoE4 or / and by high cholesterol diet that can be linked to the AD pathology. We found a disruption of the endogenous antioxidants glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) in AD brains. GRX1 and TRX1 are involved in protein folding and maintenance of redox homeostasis, and are also regulators of apoptosis by modulating the apoptosis signal-regulating kinase-1 (ASK1). We also demonstrated that oxidations of GRX1 and TRX1 are key primary events in the mechanisms of Abeta toxicity. In another study, we showed that the synergistic effects between lack of apoE and high cholesterol diet invariably causes tau hyperphosphorylation in mice brain by the up-regulation of several tau kinases. Description of the mechanisms behind risk factors for AD will help develop deeper understanding of the basic neuropathology of