Dr Richard B Parsons
email: r.parsons@sgul.ac.uk

Gemma C Price

Joanna K Farrant

Daryl Subramaniam

Jubril Adeagbo-Sheikh

Brian M Austen

STATINS INHIBIT THE DIMERISATION OF ß-SECRETASE VIA BOTH ISOPRENOID-AND CHOLESTEROL-MEDIATED MECHANISMS

Keywords: Alzheimer's disease, amyloid-ß, dimerisation, palmitoylation, prenylation, trafficking Alzheimer's disease, APP: amyloid precursor protein, ATORVA: atorvastatin, BACE: ß-site amyloid cleaving enzyme (BACE), isoprenoid, LOVA: lovastatin, MA: mevalonate, PA: palmitic acid, PBS: phosphate-buffered saline, SIMVA: simvastatin, TGN: trans-Golgi network

We have previously reported that protein lipidation in the form of palmitoylation and farnesylation is critical for the production of ß-amyloid, the dimerisation of ßsecretase and its trafficking into cholesterol-rich microdomains. As statins influence these lipid modifications in addition to their effects upon cholesterol biosynthesis, we have investigated the effects of lovastatin and simvastatin at a range of concentrations chosen to distinguish different cellular effects upon ß-amyloid production and ßsecretase structure and its localisation in bHEK cells. We have compared the changes brought about by statins to those brought about by the palmitoylation inhibitor cerulenin and the farnesyltransferase inhibitor CVFM. The statin-mediated reduction in Aß production correlated with an inhibition of ß-secretase dimerisation into its more active form at all concentrations of statins investigated. These effects were reversed by the administration of mevalonate, showing that these effects were mediated via hydroxymethylglutaryl-CoenzymeA-dependent pathways. At low (1µM) statin concentrations, reduction in Aβ production and inhibition of ß-secretase dimerisation was mediated by inhibition of isoprenoid synthesis. At high (>10µM) concentrations of statins, inhibition of ß-secretase palmitoylation occurred, which we demonstrated to be regulated by intracellular cholesterol levels. There was also a concomitant concentration-dependent change in ß-secretase subcellular trafficking.

Significantly, ß-amyloid release from cells was markedly higher at 50µM simvastatin than at 1µM, whereas these concentrations resulted in similar reductions in total ßamyloid production, suggesting that low-dose statins may be more beneficial than high doses for the therapeutic treatment of Alzheimer's disease.

INTRODUCTION

Patients on long-term statin treatment have been shown to have a 70% lower risk of developing Alzheimer's disease (AD) [START_REF] Jick | Statins and the risk of dementia[END_REF][START_REF] Wolozin | Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3methyglutaryl coenzyme A reductase inhibitors[END_REF], and a recent study has shown that atorvastatin (ATORVA) improved the cognitive ability of patients with mild-tomoderate AD [START_REF] Sparks | Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results[END_REF]. We have previously reported that the production of ß-amyloid (Aß), whose aggregation and deposition as senile plaques is a pathological hallmark of AD [START_REF] Hardy | Amyloid deposition as the central event in the aetiology of Alzheimer's disease[END_REF], is reduced by lovastatin (LOVA) in-vitro, which can be reversed by addition of exogenous cholesterol [START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF]. Inhibition of the final step of cholesterol biosynthesis using BM15.766 reduces Aß production [START_REF] Refolo | A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease[END_REF], suggesting that statins may reduce Aβ production by lowering cholesterol levels.

However, statins have other cellular effects, the most relevant being an antiinflammatory effect mediated by in part by inhibition of the synthesis of the isoprenoids farnesyl and geranylgeranyl pyrophosphate [START_REF] Cordle | 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses[END_REF]. There is an inflammatory response in a significant number of AD cases [START_REF] Mcrae | Antibodies in the cerebrospinal fluid of some Alzheimer's disease patients recognize amoeboid microglial cells in the developing rat central nervous system[END_REF]. Non-steroidal anti-inflammatories have been shown to reduce the incidence of AD by 40-50% and improve cognitive ability [START_REF] Mcgeer | Brain inflammation in Alzheimer disease and the therapeutic implications[END_REF], however such treatments can be prohibitively toxic [START_REF] Tabet | Indomethacin for the treatment of Alzheimer's disease patients[END_REF]. Aß stimulates the release of soluble inflammatory markers from microglia [11], and statins have been shown to reduce microglia activation [START_REF] Cordle | 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses[END_REF]. Statin-mediated inhibition of isoprenoid synthesis reduces the release of Aß from cells but increases the amount remaining within the cell [START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF].

Several studies have shown that statin actions on Aβ release is likely to be mediated by ß-secretase. ß-secretase (ß-site amyloid-cleaving enzyme, BACE) is a 140kDa membrane-associated homodimer [START_REF] Sidera | Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH[END_REF]14] containing extensive N-glycosylation sites which undergo successive processing and sulphation within the ER and Golgi apparatus [START_REF] Sidera | Post-translational processing of betasecretase in Alzheimer's disease[END_REF]. BACE is palmitoylated on cysteine residues 478, 482 and 485, which promote its recruitment into cholesterol-enriched microdomains (rafts) [START_REF] Benjannet | Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro-and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production[END_REF]. Inhibiting the palmitoylation of these residues with the fatty acid synthase inhibitor cerulenin prevents BACE dimerisation and changes its intracellular trafficking, resulting in a decrease in Aß production [START_REF] Parsons | Protein lipidation of BACE[END_REF]. There is also substantial evidence that BACE associates with further proteins, for example BRI3 [START_REF] Wickham | ß-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furing[END_REF] and nicastrin [START_REF] Hattori | BACE1 interacts with nicastrin[END_REF], an integral component of the γ-secretase complex.

BACE is localised primarily to the acidic intracellular compartments such as the endosome and trans-Golgi network (TGN) [START_REF] Stephens | Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells[END_REF], however a small yet significant amount also associates with APP within cholesterol rafts within the cell and the plasma membrane [START_REF] Parkin | Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein[END_REF]. BACE does not contain a glycosylphosphatidylinositol (GPI) anchor, an important modification of proteins which associate with cholesterol-rich rafts, but BACE does associate with GPI-coupled proteins which may stabilise BACE within these cholesterol-rich rafts [START_REF] Tun | Lipid rafts play an important role in A beta biogenesis by regulating the beta-secretase pathway[END_REF].

Though BACE does not contain the necessary isoprenylation consensus sequences, the demonstration by Cole and colleagues that reduction in isoprenoid synthesis by statins reduces Aß production [START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF] suggests that isoprenoids may still be involved in the production of Aß by BACE. We recently demonstrated that inhibiting protein farnesylation using the farnesyltransferase inhibitor CVFM resulted in a concentration-dependent decrease in Aß production, inhibition of BACE dimerisation and alteration in BACE subcellular localisation. This may involve an as-yet unidentified 53kDa isoprenylated protein which co-purifies with BACE [START_REF] Parsons | Protein lipidation of BACE[END_REF].

Statins reduce both cholesterol and isoprenoid synthesis via their inhibition of hydroxymethylglutaryl-coenzyme A (HmGCoA) reductase. HmGCoA reductase catalyses the conversion of HmGCoA to mevalonate (MA), a common intermediate of both isoprenoid and cholesterol synthesis. Concentrations in the range of 1-5µM inhibit isoprenoid biosynthesis [START_REF] Bellosta | HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages[END_REF] whereas reduction of cholesterol biosynthesis requires concentrations in the region of 20-40µM [START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF]. We have shown that 40µM LOVA inhibits the palmitoylation of the CLR sequence of the BACE C-terminal tail [START_REF] Austen | Palmitoylation of ß-secretase abolished by lovastatin[END_REF]. The marked concentration differences in statin actions may be of relevance for the therapy of AD. We have investigated the concentration-dependence of statinmediated Aß production in an in-vitro model system, and compared the effects of LOVA and simvastatin (SIMVA) to the effects of cerulenin and CVFM. The results are presented here.

MATERIALS AND METHODS

Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (Poole, Dorset, UK). Statistical analysis was performed using the InStat statistical package Copyright 2006 Biochemical Society (GraphPad, San Diego, USA) using one-way ANOVA followed by Tukey post-hoc comparison test, comparing treated cells with untreated (media only).

Cell culture incubations

We used a cell-line of HEK293 transfected with the Asp-2 gene plus a poly-histidine coding tag (bHEK) [START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF][START_REF] Sidera | Post-translational processing of betasecretase in Alzheimer's disease[END_REF] (a kind gift from GlaxoSmithkline, Harlow, UK) cultured in DMEM supplemented with 2% v/v Ultroser® G (Pall BioSepra, Cergy-Saint-Christophe, France). Ultroser® G is free of cholesterol, palmitate and isoprenoid precursors, requiring cells to rely upon their own de-novo synthesis for these components. Other workers have studied the processing of BACE in transfected human 293 cells [START_REF] Austen | Palmitoylation of ß-secretase abolished by lovastatin[END_REF]27,[START_REF] Capell | Maturation and pro-peptide cleavage of betasecretase[END_REF]. Cells expressing BACE were selected using geneticin (250µg/ml, Invitrogen, Paisley, UK).

The effects of LOVA and SIMVA were studied at 1, 10 & 50 µM. Farnesyltransferase activity in the cells was inhibited by the addition of CVFM (0.1µM, Alexis Corp, Nottingham, UK). Palmitoyltransferase activity was inhibited by the addition of cerulenin (5µg/ml). For MA reincorporation experiments, cells were co-incubated with 50µM SIMVA and 0 -400µM MA. This has been shown to maintain isoprenoid levels whilst reducing cholesterol synthesis [START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF][START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF] and to return Aß production back to those observed in untreated cells [START_REF] Famer | Rosuvastatin reduces caspase-3 activity and upregulates alpha-secretase in human neuroblastoma SH-SY5Y cells exposed to A beta[END_REF].

Enzyme-Linked Immunosorbent Assay (ELISA) detection of Aß

Aß production was measured independently in three compartments: released, remaining and total [START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF]. Released Aß consisted of Aß secreted into the culture media.

Remaining Aß was obtained by removing media from the cells, washing once with phosphate-buffered saline (PBS, Invitrogen) and lysing with RIPA buffer [START_REF] Parsons | Protein lipidation of BACE[END_REF], diluted 1:10 in PBS, pH 7.4, for 30 mins at 4 o C. Total Aß production was obtained by lysing cells into the culture media by the addition of 1xRIPA (final concentration 0.1X) to the culture media and incubating for 30 mins at 4 o C. Prior to assay, all samples were centrifuged for 10 mins at 14,000 rpm in order to pellet insoluble material.

Aß was quantitated using a double-antibody Aß ELISA [11] using a combination of rabbit-anti-NTA4 [START_REF] Stephens | Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells[END_REF][START_REF] Austen | Palmitoylation of ß-secretase abolished by lovastatin[END_REF][START_REF] Davies | Profiling of amyloid beta peptide variants using SELDI Protein Chip arrays[END_REF] (2µg/ml), monoclonal anti-6E10 (1:2000, IDLabs, Glasgow, UK) and anti-mouse IgG, horse radish peroxidase (HRP) conjugate (1:500, AmershamBiosciences, Chalfont St. Giles, UK). The ELISA detected all species of Aß monomers, and is linear in the Aß concentration range of 1-100ng/ml [START_REF] Parsons | Protein lipidation of BACE[END_REF][START_REF] Parsons | Statins reduce ß-amyloid production but increase its release from the cell[END_REF]. The specificity of the ELISA for soluble monomeric Aß over oligomeric Aß was confirmed by using an Aß oligomer ELISA [11] to measure Aß oligomers in the synthetic Aß 1-40 samples used to construct the calibration curve for the Aß monomer ELISA. The Aß oligomer ELISA utilised anti-6E10 as a capture antibody in concert with biotinylated anti-6E10 to detect oligomers within samples. No oligomers were detected in any of the Aß standards used (data not shown), demonstrating that the combination of anti-NTA4 and anti-6E10 antibodies were specific for monomeric Aß. Samples (50µl) were applied in triplicate and incubated for 1.5 hours. Colour was developed by the addition of TMB peroxidase substrate (Europa Bioscience, Ely, UK). Absorbance at 420nm was measured using a MultiSkan EX 96-well plate reader (ThermoElectron Corporation, Basingstoke, UK). Concentrations were calculated from the average of three experiments all performed in triplicate and expressed as Aß concentration in ng/ml using standard concentrations of synthetic Aβ1-40.

Cytotoxicity assay

The cytotoxicity of inhibitors was measured in the bHEK cells using the LDH Cytotoxicity Kit (Roche Diagnostics, Lewes, UK). Results were calculated from the average of three experiments all performed in triplicate and were expressed as percentage cell death.

BACE and γ-secretase activity assays

BACE and γ-secretase activities were measured using the fluorescence-based ßsecretase and γ-secretase assay kits (R&D Systems, Abingdon, UK). Results were expressed as specific activities (F.U./hour/mg protein), with protein concentrations measured using the BioRad D c Protein Assay Kit (BioRad Laboratories, Hemel Hempstead, UK).

Cellular cholesterol assay

Total cellular cholesterol levels were measured using the fluorescence-based Amplex total cholesterol assay (Molecular Probes, Invitrogen). Total cellular cholesterol content was calculated and expressed as µg total cholesterol/mg protein.

Western blotting analysis of BACE

BACE protein was detected in protein lysates using Western blotting as previously described [START_REF] Sidera | Post-translational processing of betasecretase in Alzheimer's disease[END_REF] with appropriate modifications using our own anti-BACE antibody which detects both monomeric (70kDa) and dimeric (140kDa) forms of BACE [START_REF] Sidera | Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH[END_REF][START_REF] Parsons | Protein lipidation of BACE[END_REF][START_REF] Stephens | Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells[END_REF]. As palmitoyl and farnesyl moieties are attached to proteins via reducible thioester bonds, samples were prepared by diluting samples 1:1 in both reducing (Laemmli sample buffer, buffer R) and non-reducing (buffer NR comprising Laemmli sample buffer without ß-mercaptoethanol) buffers, followed by boiling for 5 minutes.

Protein lysate samples (30µl) were electrophoresed on a 4-12% NuPage Bis/Tris gel (Invitrogen), then transferred to PVDF membrane (Waters Ltd., Elstree, UK). BACE was detected using a combination of a rabbit anti-BACE peptide antibody (1:2000), mouse-anti-rabbit IgG, HRP conjugate (1:5000) (AmershamBiosciences) and electrochemiluminescence (ECL) detection (AmershamBiosciences) using Hyperfilm M (AmershamBiosciences). Molecular weights of visualised proteins were calculated using a calibration plot constructed using standards of known molecular weight and expressed as kDa ± 9.3%, calculated as the average error between actual molecular weights of the standards used and their observed molecular weights calculated from the resultant calibration line.

Confocal microscopy

The subcellular localisation of BACE was visualised using dual-label confocal microscopy as previously described [START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF] using a combination of rabbit-anti-BACE (1:200) and a premixed secondary antibody complex consisting of biotinylated mouse-anti-rabbit IgG (1:500, Dako, Ely, UK) and streptavidin-linked Rhodamine (1:500, Dako). Cholesterol was detected using the cholesterol binding agent filipin (50µg/ml). Fluorescence was observed using a Leica 510 Confocal Microscope using a x40 objective lens, with BACE observed at 543nm (red fluorescence) and cholesterol observed at 488nm (green fluorescence).

Incorporation of palmitoyl and farnesyl moieties

Incorporation of palmitoyl using [ 3 H]-palmitate into BACE

In order to assess the effects of LOVA and cholesterol upon the incorporation of palmitoyl into BACE, the incorporation of [ 3 H] (9,10n)-palmitic acid (PA) using a method based upon that of Benjannet et al. [START_REF] Benjannet | Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro-and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production[END_REF] was employed. bHEK cells (5x10 6 cells/flask) were incubated for 16 hours with [ 3 H]-PA (AmershamBiosciences) (1mCi/ml) in the presence and absence of LOVA. Additional cells were treated with 50µM LOVA and 200µg/ml cholesterol in cyclo-ß-methyldextrin (CMD) plus [ 3 H]-PA for 16 hours. BACE was isolated using nickel affinity purification as previously described [START_REF] Sidera | Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH[END_REF]. Samples prepared in sample buffers R & NR were electrophoresed as above and stained for protein using the PlusOne silver staining kit (AmershamBiosciences). Proteins in which [ 3 H]-PA was incorporated were visualised using Amplify fluorographic reagent (AmershamBiosciences) followed by exposure to HyperFilm MP (AmershamBiosiences) for 4 days at -70 0 C.

Identification of incorporated isoprenyl using [ 3 H]-mevalonate

The nature of the isoprenylation of the 53kDa protein which co-purifies with BACE was characterised by assessing the incorporation of [ 3 H]-isoprenoid ([ 3 H]-IP) into this protein in the presence and absence of CVFM. Cells were incubated for 16 hours with 50µM LOVA, after which media was replaced with media supplemented with 50µM SIMVA plus [ 3 H]-MA (200µCi/ml, Tocris Bioscience, Bristol, UK) in the presence or absence of CVFM (0.1µM) as appropriate for a further 16 hours. Radiolabel incorporation was assessed as described above, with HyperFilm MP exposed to the gel for 5 weeks at -70 o C.

RESULTS

LOVA and SIMVA reduce the production of Aß by a mechanism independent of cholesterol

Total Aß production was significantly reduced at all concentrations of statin investigated with the exception of 50µM LOVA (Figures 1A andB), which also gave an increase in cell death (3.9 ± 1.95% vs 6 ± 2.2%); however, this was not significant.

LOVA (1µM and 10µM) reduced total Aß production by 58% and 67% respectively (p<0.001), with 50µM LOVA resulting in a 11% decrease in total Aß production (p<0.05). Aß release was not significantly affected, whereas Aß remaining within the cell decreased significantly by 81% and 95% at 1µM and 10µM respectively Copyright 2006 Biochemical Society (p<0.001). SIMVA reduced total Aß production at all three concentrations used, resulting in a 48% decrease in total Aß production at 50µM (p<0.01). In contrast to LOVA, SIMVA significantly increased the release of Aß at 10µM and 50µM, resulting in a 20% and a 66% increase in Aß release respectively (p<0.05 and p<0.01 respectively). Aß remaining within the cell decreased significantly at all three concentrations of SIMVA, decreasing by 66%, 79% and 85% respectively (p<0.01).

Cerulenin and CVFM resulted in decreases in total Aß production of 57% and 54% respectively (Figure 1C) (p<0.001).

Reduction of total Aβ production occurred at concentrations (1µM) of statins which did not change intracellular cholesterol levels as well as at concentrations at which intracellular cholesterol was significantly reduced (Table 1), suggesting that at low (1µM) concentrations, statins were exerting their effects upon Aß production via cholesterol-independent pathways. At higher (>10µM) concentrations, the cholesterol-dependent pathways may become more important, as intracellular cholesterol was significantly reduced at this concentration for both statins. Cholesterol reduction was concomitant with a statin concentration-dependent change in BACE trafficking (Figure 2). In untreated cells, BACE was co-localised with cholesterol within the Golgi apparatus and associated vesicles. Staining was punctate, with discrete areas of intense staining concurrent with cholesterol raft localisation. There was very little staining associated with other compartments within the cell. With increasing LOVA concentration, the punctate staining associated with cholesterol raft localisation was replaced with diffuse staining throughout the membrane. Staining associated with BACE became more pronounced in intracellular locations with increasing LOVA concentration. Co-localisation of BACE with non-raft associated cholesterol increased with increasing LOVA concentration, resulting in an increase in orange/yellow staining in merged immunofluorescence images (Figure 2B &C).

Staining associated with cholesterol decreased with increasing LOVA concentration, which coincided with the measured decrease in total cellular cholesterol (Table 1). This decrease in green fluorescence associated with cholesterol staining resulted in the staining associated with BACE within membranes and intracellular regions becoming more orange in appearance at 50µM LOVA (Figure 2D). Treatment of the cells with SIMVA, cerulenin or CVFM (data not shown) produced similar effects.

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655

There was no correlation between Aß production and the in-vitro activities against synthetic substrates of either BACE or γ-secretase released from cells with detergent after incubation with statins (Table 2), suggesting that statins are not competitive inhibitors of BACE. There was also no correlation between Aß production and cell death, as there was no significant increase in cell death at any of the concentrations of statin investigated (data not shown).

LOVA and SIMVA inhibit the dimerisation of BACE

As statins are also able to inhibit both palmitoyl transfer to BACE [START_REF] Austen | Palmitoylation of ß-secretase abolished by lovastatin[END_REF] and farnesyl biosynthesis, their effects upon BACE dimerisation were assessed using Western blotting (Figure 3). In cells grown in Ultroser® G media alone, BACE consisted of two proteins of 145.5kDa and 73.7kDa, corresponding to the predicted molecular weights of the dimeric and monomeric forms of glycosylated and lipidated BACE as reported previously [START_REF] Sidera | Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH[END_REF]14,[START_REF] Parsons | Protein lipidation of BACE[END_REF]. Treatment of the cells with either LOVA or SIMVA resulted in a single monomeric BACE protein of 73.7kDa (samples prepared with reducing agent, buffer R) or 65.5kDa (samples prepared in the absence of reducing agent, buffer NR) at 1, 10 and 50µM. Treatment of cells with cerulenin (73.7 and 67.5kDa for buffers R and NR respectively) and CVFM (73.7 and 69.3kDa) gave similar results. The differences in mobilities produced by reducing agent in the sample buffer may be caused by release of covalent modifications linked via thioester bonds or the reduction of internal disulphide bonds.

Mevalonate addition reverses statin-induced reduction of Aß levels and promotes the dimerisation of BACE

The inhibition of Aβ production by 50µM SIMVA was overcome by the addition of MA in a dose-dependent manner (Figure 4), with almost 100% Aß production restored at 400µM MA. This was concomitant with the reappearance of the dimeric form of BACE (Figure 4). The addition of MA did not return total cellular cholesterol levels back to those observed in untreated cells, nor did they differ from those observed in cells incubated with 50µM SIMVA alone (Table 3). Therefore, the inhibition of Aβ production and BACE dimerisation by statins was mediated via inhibition of HmGCoA-reductase-dependent isoprenoid synthesis.

BACE palmitoylation is regulated by intracellular cholesterol levels

Our previous studies have shown that palmitoylation is not dependent upon prior protein farnesylation [START_REF] Parsons | Protein lipidation of BACE[END_REF], and it is possible that palmitoylation is mediated by alterations in cellular cholesterol levels. In cells grown in Ultroser® G media alone, [ 3 H]-PA was incorporated into both dimeric and monomeric forms of BACE (Figure 5A), resulting in labelled proteins of 141kDa and 75kDa respectively. Boiling in the presence of the reducing agent ß-mercaptoethanol removed all trace of radiolabelled protein, showing that [ 3 H]-PA was attached via a reducible thioester bond.

Incubation of cells with LOVA reduced the incorporation of [ 3 H]-PA in a dosedependent manner. [ 3 H]-PA incorporation occurred into a single protein of 73kDa at 1µM LOVA (Figure 5A). As BACE dimerisation was inhibited at this concentration (Figure 3), this suggests that BACE palmitoylation is not inhibited at this concentration and does not require prior BACE dimerisation.

Statins inhibit farnesylation of a 53kDa protein

In order to determine whether the isoprenylation present on the 53kDa protein is farnesyl or geranylgeranyl, the incorporation of [ 3 H]-MA in the presence and absence of CVFM was investigated. As previously observed, there was significant isoprenyl incorporation into many bHEK proteins (Figure 6). Boiling protein samples in buffer R removed all [ 3 H]-IP label, showing that the prenylation modifications were attached to proteins via thioester bonds. The isoprenylation of the 53kDa protein was inhibited by CVFM, showing that this protein is farnesylated. The farnesylated 53kDa protein co-purified with BACE on a nickel affinity column, suggesting that the two proteins may be part of the same complex.

DISCUSSION

As β-amyloid is implicated by the amyloid hypothesis as a causative agent in AD, BACE and γ-secretase offer potential targets for drugs for the disease. Recent evidence suggesting that statin use reduces the risk of AD makes it important to fully elucidate the mechanisms by which statins influence Aß production. The present study demonstrates that statins inhibit the dimerisation of BACE via HmGCoAreductase-dependent pathways, involving both isoprenoid synthesis and intracellular cholesterol levels, which in turn regulates the palmitoylation of BACE. These data provide further evidence that physiologically active BACE exists as a complex of proteins, whose assembly is reliant upon protein lipidation reactions.

There is a dose-dependent effect of statins upon cellular cholesterol and isoprenoid levels

The differences in the concentrations of statins required to inhibit both isoprenoid and cholesterol biosyntheses [START_REF] Stephens | Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells[END_REF][START_REF] Davies | Profiling of amyloid beta peptide variants using SELDI Protein Chip arrays[END_REF][START_REF] Famer | Rosuvastatin reduces caspase-3 activity and upregulates alpha-secretase in human neuroblastoma SH-SY5Y cells exposed to A beta[END_REF], in which we observed significant reduction in cellular cholesterol levels at LOVA and SIMVA concentrations greater than 10µM (Table 1), may reflect the differences in the way cellular levels of isoprenoids and cholesterol are maintained. There are two pools of cholesterol, free cholesterol and esterified cholesterol. Esterified cholesterol acts as a metabolic reserve of cholesterol which can be converted back to cholesterol by cholesterol esterase in times of low cellular cholesterol levels. This reduces the reliance of the cell upon de-novo cholesterol synthesis in order to maintain cellular cholesterol levels. In contrast, isoprenoids have no such metabolic reserve, and rely solely upon de-novo synthesis in order to maintain cellular isoprenoid levels. This would make cellular isoprenoid levels much more sensitive to HmGCoA reductase inhibition than cellular cholesterol levels, also resulting in preferential isoprenoid synthesis over cholesterol synthesis in the presence of MA, resulting in isoprenoid synthesis utilising the majority of the MA passing down this shared metabolic pathway. This is in accord with our results which show no effect of MA upon cellular cholesterol levels after 50µM LOVA incubation, and is also in agreement with studies by other laboratories which have shown that MA reincorporation after statin inhibition of HmGCoA reductase preferentially increases isoprenoid synthesis over cholesterol synthesis [START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF][START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF].

Statins inhibit the dimerisation of BACE by inhibiting isoprenoid synthesis

Our study is the first demonstration that BACE dimerisation can be inhibited by statins via HmG-CoA reductase-dependent isoprenoid biosynthesis. Blocking the dimerisation of BACE is an attractive therapeutic target, as the dimer is 30-fold more active at cleaving APP than the monomer [14] and so inhibiting BACE dimerisation would reduce Aß production markedly.

BACE palmitoylation is mediated via cholesterol

LOVA treatment yielded dose-dependant concomitant changes in BACE subcellular trafficking and inhibition of palmitoylation, complete at 50µM LOVA, which were reversed by the addition of exogenous cholesterol (Figure 5), showing that both are connected to the cholesterol gradient in much the same way as the maturation of the glycosylation of BACE [START_REF] Austen | The regulation of ß-secretase by cholesterol and statins in Alzheimer's disease[END_REF]. The palmitoylation of BACE does not require prior dimerisation, as monomeric BACE was palmitoylated in the presence of both 1µM LOVA and 0.1µM CVFM. It is also clear that palmitoylation is required, though not sufficient in itself, for dimerisation to occur, as inhibiting palmitoylation using cerulenin inhibits the dimerisation of BACE (figure 3) [START_REF] Parsons | Protein lipidation of BACE[END_REF]. It is therefore likely that the cholesterol gradient may play a part in the dimerisation process, as reintroducing cholesterol after 50µM LOVA treatment results in the dimerisation of BACE and reincorporation of [ 3 H]-PA into both dimeric and monomeric forms of BACE.

Palmitoylation is usually required by proteins to stabilise their association with membranes [START_REF] Smotrys | Palmitoylation of intracellular signaling proteins: regulation and function[END_REF], an association that BACE requires for dimerisation to occur [14].

Therefore, in the temporal sequence of processing of BACE, palmitoylation occurs prior to BACE dimerisation and prosequence cleavage, which are driven by the cholesterol gradient. Inhibiting cellular cholesterol biosynthesis by LOVA lowers the intracellular cholesterol gradient [START_REF] Kirsch | Statin effects on cholesterol micro-domains in brain plasma membranes[END_REF][START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF], therefore reducing the cholesterol-driven palmitoylation and dimerisation of BACE.

BACE undergoes palmitoylation and associates with a farnesylated 53kDa protein in a sequential manner

It is common for protein palmitoylation to require prior farnesylation [START_REF] Smotrys | Palmitoylation of intracellular signaling proteins: regulation and function[END_REF], however BACE is not farnesylated [START_REF] Parsons | Protein lipidation of BACE[END_REF]. There is mounting evidence that BACE exists as a complex within cholesterol-rich rafts [START_REF] Tun | Lipid rafts play an important role in A beta biogenesis by regulating the beta-secretase pathway[END_REF], and though many associations with other proteins may be transitory in nature, some associations may be permanent. The association of BACE with a 53kDa farnesylated protein [START_REF] Parsons | Protein lipidation of BACE[END_REF] would indirectly provide BACE with a farnesyl group necessary to stabilise BACE within its lipid raft localisation. We propose that BACE undergoes a timed sequence series of maturation steps prior to entry into the cholesterol-rich rafts, as summarised in Figure 7. After production within the endoplasmic reticulum and during its cholesterol gradientdriven processing through the TGN, BACE undergoes palmitoylation which is followed by dimerisation and association with a 53kDa farnesylated protein. This in turn stabilises BACE within the membrane, enabling it to undergo prosequence cleavage and subsequent recruitment into cholesterol-rich microdomains.

The model suggests several potential therapeutic targets which could be exploited in order to disrupt the assembly of the BACE complex. Inhibition of any of these steps would prevent the dimerisation of BACE and its association with cholesterol-rich rafts, all of which we have demonstrated experimentally. Such a strategy would channel APP processing via the non-amyloidogenic α-secretase pathway. Transgenic BACE knock-out animal models show no pathological or behavioural abnormalities [35], which suggests that BACE is not necessary for survival. In contrast, BACE activity and expression increases as a consequence of natural ageing [START_REF] Fukumoto | Beta-secretase activity increases with aging in human, monkey, and mouse brain[END_REF] and studies have shown a small but significant increase in BACE activity in patients with AD compared to non-disease controls [START_REF] Li | Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer's disease patients[END_REF]. As increased Aß load is associated with an increase in AD pathology [START_REF] Hardy | Amyloid deposition as the central event in the aetiology of Alzheimer's disease[END_REF], targeting the assembly of the BACE complex may be an effective therapeutic strategy.

SIMVA increases the release of Aß from bHEK cells

The decrease in Aß intracellular accumulation and production by low concentrations (<10µM) of statins was not unexpected. Previous studies have reported reductions in total Aß production and accumulation of upto 50% in HEK cells by concentrations of LOVA and SIMVA below 10µM [START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF][START_REF] Buxbaum | Pharmacological concentrations of the HMG-CoA reductase inhibitor lovastatin decrease the formation of the Alzheimer beta-amyloid peptide in vitro and in patients[END_REF], and upto a 40% reduction in intracellular accumulation of Aß by 4µM LOVA and SIMVA in isolated rat hippocampal neurones [START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF]39]. In contrast, Cole and colleagues have recently demonstrated that 10µM LOVA increases the intracellular accumulation of Aß 1-40 by 400% in both HEK and SH-SY5Y cells [START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF]. It is unclear as to why this is the case, but it may represent methodological differences between studies.

However, we are the first to demonstrate an increase in Aß release in response to incubation with SIMVA. SIMVA at 10µM and 50µM decreased cellular cholesterol levels (Table 1) and increased Aß release from bHEK (Figure 1). This is in contrast with previous studies which have reported a 50-90% reduction in Aß release from cells exposed to 10µM SIMVA [START_REF] Sparks | Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results[END_REF][START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF][START_REF] Buxbaum | Pharmacological concentrations of the HMG-CoA reductase inhibitor lovastatin decrease the formation of the Alzheimer beta-amyloid peptide in vitro and in patients[END_REF]39], however it is an effect we have previously observed [START_REF] Parsons | Statins reduce ß-amyloid production but increase its release from the cell[END_REF]. This leakage of Aß that we observe most likely arises from the reduction in plasma membrane cholesterol [START_REF] Kirsch | Statin effects on cholesterol micro-domains in brain plasma membranes[END_REF][START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF], which would make it more permeable to small molecules such as Aß and thus result in the statin concentrationdependent increase in Aß that we observe.

LOVA and SIMVA have different effects upon Aß production

It is clear from our data that LOVA and SIMVA, though they both have the same effect upon Aß production, differ in the degree to which they have their effects. The percentage reductions in remaining and total Aß was less for SIMVA than for LOVA, suggesting that SIMVA is less effective at reducing Aß production than LOVA. Such a difference is not unexpected as different statins exhibit different abilities in inhibiting microglia activation [START_REF] Jans | Processing of amyloid precursor protein as a biochemical link between atherosclerosis and Alzheimer's disease[END_REF]. Such differences between statins are not limited to in-vitro studies. Wolozin and colleagues reported that the prevalence of AD in patients receiving LOVA therapy was significantly reduced compared to the total patient population within their study whereas the prevalence of AD in patients receiving SIMVA did not differ from the total patient population [START_REF] Wolozin | Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3methyglutaryl coenzyme A reductase inhibitors[END_REF].

The reasons for these differences are unclear. LOVA and SIMVA, which differ structurally by an additional methyl group, are transported into the cell via passive diffusion across the membrane, and have correspondingly similar IC 50 values for the inhibition of HMGCoA reductase [START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF]. Additionally, both statins are metabolised by CYP 3A4 [START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF].

Recently, it has become possible to obtain SIMVA over-the-counter without the need for a prescription. Taking into consideration both our data showing a greater effect of Copyright 2006 Biochemical Society LOVA upon Aß production compared to SIMVA and the reduction in the prevalence of AD by LOVA as reported by Wolozin and colleagues [START_REF] Wolozin | Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3methyglutaryl coenzyme A reductase inhibitors[END_REF], it is likely that this change in prescription status for SIMVA will have little or no affect upon the incidence of AD in the general population, and that the use of LOVA would be more beneficial.

Low dose statin therapy may be beneficial for the treatment of AD

Since the original retrospective reports showing a reduction in the incidence of AD in patients taking long-term statins, there has been much discussion regarding their use as an anti-AD treatment. Initial promising results have not been replicated in largescale studies, with little effect seen on cognitive decline. A recent study has demonstrated a potentially beneficial effect of ATORVA upon the cognitive decline in patients with mild AD, however the difference observed was small [START_REF] Sparks | Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results[END_REF]. The 80mg/day dosage of ATORVA used in these clinical studies is the maximal recommended dose for patients [START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF], and therefore represents statin dosage at the highest level. We have previously shown that statins at concentrations which reduce cholesterol production (20µM) reduce the total production of Aß in bHEK cells, yet increase its release from the cell [START_REF] Parsons | Statins reduce ß-amyloid production but increase its release from the cell[END_REF]. Our present data confirm this finding whilst showing that lower doses may be just as effective at reducing total Aß production. At 1µM SIMVA, we observed a 42% decrease in total Aß produced, which differed from 50µM by a mere 5%, suggesting that low doses may be just as effective as high doses.

This benefit may not be solely limited to the low dose required, however. The amount of Aß released from cells increased by 66% at 50µM SIMVA, compared to a 20% increase at 1µM. The observed increase in release has ramifications for the use of statins in AD treatment. If this were to occur in the in-vivo human brain, this increase in Aß release at high statin concentrations may have detrimental consequences. An increase in Aß production under these conditions may accelerate the oligomersiation of Aß into toxic aggregates, which may therefore be masking the beneficial effects of statins in previous clinical studies. The bioavailability of both LOVA and SIMVA is below 5%, whilst the bioavailability of ATORVA is 12% [START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF]. With the standard dose of ATORVA (40mg/day), this would result in a plasma concentration of approximately 1-5nM. Statins have a very high affinity for HMGCoA-reductase, in the region of 1.2 -55nM [START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF], which places the maximal ATORVA concentration The concentrations used in our study are in line with others who have used statins in their studies [START_REF] Sparks | Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results[END_REF][START_REF] Frears | The role of cholesterol in the biosynthesis of beta-amyloid[END_REF][START_REF] Cole | Statins cause intracellular accumulation of amyloid precursor protein, betasecretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism[END_REF][START_REF] Bellosta | HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages[END_REF][START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF][START_REF] Famer | Rosuvastatin reduces caspase-3 activity and upregulates alpha-secretase in human neuroblastoma SH-SY5Y cells exposed to A beta[END_REF][START_REF] Buxbaum | Pharmacological concentrations of the HMG-CoA reductase inhibitor lovastatin decrease the formation of the Alzheimer beta-amyloid peptide in vitro and in patients[END_REF]39]. However, the differences in concentrations required to reduce cholesterol synthesis in-vivo and in-vitro may reflect differences in statin metabolism. In-vitro, alterations in plasma cholesterol levels require a number of weeks administration of statins. In contrast, the decreases in cellular cholesterol levels occur within a very short time frame. Both SIMVA and LOVA are prodrugs, administered in their lactone form which undergo subsequent reversible conversion within the body to their active open acid forms [START_REF] Shitara | Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: Drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions[END_REF]41]. It is probable that these interconversions, though they may occur in-vitro, do not occur to a significant degree in comparison, and therefore much higher concentrations of statins are required in order to achieve physiological effects. It is therefore probable that the effective concentration of statin open acid forms used in-vitro may well be within the low nM range. It is also common to administer high doses of statin in in-vivo models in order to obtain a rapid decrease in cellular cholesterol levels [START_REF] Fassbender | Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo[END_REF]. Irrespective of these considerations however, the concentrations used in our study were chosen to allow the investigation of the cholesterol-and isoprenoid-effects of statins, and we have demonstrated that each of these lipid effects mediate the production of Aß via BACE.

TABLES

  [ 3 H]-PA incorporation was reduced in monomeric BACE of 73kDa by 10µM LOVA, whereas [ 3 H]-PA incorporation was totally abolished by 50µM LOVA. The addition of 200µg/ml cholesterol to cells treated with 50µM LOVA resulted in the incorporation of [ 3 H]-PA into two proteins of 141kDa and 75kDa (Figures 5A), which corresponded to the appearance of both dimeric and monomeric BACE on Western blot (Figure 5B). The farnesyltransferase inhibitor CVFM had no effect upon palmitoylation of monomeric BACE, further supporting the hypothesis that statins inhibit BACE palmitoylation via their influence upon intracellular cholesterol levels and does not require prior BACE dimerisation. Cerulenin inhibited the palmitoylation of both forms of BACE.

Biochemical

  Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 Copyright 2006 Biochemical Society within the upper end of this range. Therefore, use of low-dose statins may demonstrate a more beneficial effect in such trials.

  calculated as the average of triplicates and expressed as percentage of total cellular cholesterol concentration in untreated cells, which was taken as 100%. In all incubations, cells were co-incubated with 50µM SIMVA and 0 -400µM MA, with the exception of untreated cells, which were incubated in Ultroser® G media alone. a Cellular cholesterol levels in cells co-incubated with 50µM SIMVA and increasing concentrations of MA were compared with cholesterol levels in cells grown in Ultroser® G media only to determine whether increasing concentrations of MA were able to reverse the reduction of total cellular cholesterol levels by 50µM back to those levels observed in untreated cells. b Cellular cholesterol levels in cells pretreated with 50µM SIMVA and increasing concentrations of MA were compared to cellular cholesterol levels in cells pretreated with 50µM SIMVA alone to determine whether total cellular cholesterol levels differed at any of the concentrations of MA investigated. n/s = not significant; *** = p<0.001. Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 Copyright 2006 Biochemical Society

Figure 1 .

 1 Figure1. LOVA and SIMVA reduce total Aß production. Cells were incubated with 0, 1, 10 and 50µM statin, 5µg/ml cerulenin or 0.1µM CVFM for 16 hours prior to assay. Aß was measured in the removed media (released), lysed cells with media (total), and lysed cells (remaining) using an Aß ELISA assay and expressed as ng/ml ± S.D., with statistical analysis of untreated-vs. statin-treated cells performed using one-way ANOVA with Tukey post-hoc comparisons. Results were calculated as the average of three independent experiments, with each experiment assayed in triplicate. (A) Effect of LOVA upon Aß release (top panel), Aß remaining within the cell (middle panel) and total Aß production (bottom panel). * = p<0.05; *** = p<0.001. (B) Effect of SIMVA upon Aß release (top panel), Aß remaining within the cell (middle panel) and total Aß production (bottom panel). * = p<0.05; ** = p<0.01; *** = p<0.001. (C) Effect of CVFM and cerulenin upon Aß release, Aß remaining within the cell and total Aß production. *** = p<0.001. Black bars: untreated cells; white bars: 0.1µM CVFM; hashed bars: cerulenin.

Figure 2 .

 2 Figure 2. LOVA changes subcellular trafficking of BACE. Cells were incubated with 0, 1, 10 & 50µM LOVA, 5µg/ml cerulenin or 0.1µM CVFM, after which BACE expression was detected using confocal microscopy using a combination of anti-BACE (1:200), biotinylated mouse-anti-rabbit IgG (1:500) and streptavidin-linked rhodamine (1:500). Cholesterol localisation was determined using the cholesterol marker filipin (50µg/ml). Fluorescent staining was observed with a Leica 510 confocal microscope using an excitation wavelength of 543nm for BACE (red fluorescence) and an excitation wavelength of 488nm for cholesterol (green fluorescence). BACE and cholesterol co-localisation was observed as orange/yellow fluorescence. In untreated cells (panel A), BACE was co-localised with cholesterol within the Golgi apparatus and associated vesicles (arrows). Staining was punctate, corresponding to cholesterol raft localisation. There was no staining within other intracellular areas of the cell (*). With increasing concentrations of LOVA (panels B and C), BACE localisation became much more general throughout the cell (*), with staining within the membrane becoming much more diffuse (arrows). This resulted in an increase in co-localisation with cholesterol (orange/yellow staining). At 50µM LOVA, BACE localisation became much more diffuse throughout the membrane, with staining present in intracellular areas (*). However, due to the decrease in

Figure 3 .Figure 1 ,

 31 Figure 3. LOVA and SIMVA inhibit BACE dimerisation. Cells were incubated as per Figure 1, and BACE was detected using Western blotting with the following antibody combination: anti-BACE (1:2000), overnight 4 o C; mouse-anti-rabbit IgG Horseradish peroxidase conjugate (1:5000), 1 hour RT. Molecular weights of detected proteins were calculated using a calibration line constructed using standards of known molecular weight and expressed as kDa. Samples were loaded in couplets, the first lane of each comprising sample boiled for 5 minutes in reducing sample buffer, and the second lane of each couplet boiled for 5 minutes in non-reducing sample buffer. Lanes 1&2: untreated cells; 3&4: 1µM LOVA; 5&6: 10µM LOVA; 7&8: 50µM LOVA; 9&10: 1µM SIMVA; 11&12: 10µM SIMVA; 13&14: 50µM SIMVA; 15&16: 5µg/ml cerulenin; 17&18: 0.1µM CVFM.

Figure 4 .Figure 3 ,

 43 Figure 4. MA reverses the effects of statins upon Aß production and BACE dimerisation. Cells were incubated with 50µM SIMVA and 0 -400µM MA for 16 hours prior to ELISA and Western blot. Total Aß production was measured using an Aß ELISA assay and expressed as ng/ml ± S.D., with statistical analysis of untreatedvs. MA-treated-cells performed using one-way ANOVA with Tukey post-hoc comparisons. Results were calculated as the average of three independent experiments, with each experiment assayed in triplicate. * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant. BACE was detected using Western blotting as per Figure 3, using samples prepared by boiling for 5 minutes in non-reducing buffer only. Molecular weights were calculated using a calibration line produced from standards of known molecular weight and expressed as kDa. Upper arrow = 140kDa; lower arrow = 70kDa.

Figure 5 .

 5 Figure 5. BACE palmitoylation is mediated by cholesterol. Cells were incubated for 16 hours in the presence of [ 3 H]-PA (1mCi/ml) in normal media or media

Figure 6 .

 6 Figure 6. A 53kDa protein which co-purifies with BACE is farnesylated. Cells were incubated for 16 hours with 50µM LOVA, followed by further overnight incubation with 50µM LOVA plus [ 3 H]-MA (200µCi/ml) in the presence and absence of CVFM (0.1µM). Cells were lysed and BACE purified using nickel affinity purification.Samples were prepared in non-reducing sample buffer only in order to ensure that any thioester-linked radiolabelled isoprenyl moieties were not removed. Samples were electrophoresed and radiolabel incorporation was determined as described in Figure5, with gel exposure at -70 0 C for 5 weeks. Molecular weights of detected proteins were calculated and expressed as kDa. The figure is a typical result constructed from three experiments and is representative of results obtained. Lanes 1&2: all radiolabelled proteins from cells incubated in the absence of CVFM prepared in non-reducing and reducing buffer respectively; 3: purified BACE sample from cells incubated in the absence of CVFM; 4: purified BACE sample from cells incubated in the presence of 0.1µM CVFM.

Figure 7 .

 7 Figure 7. Schematic representation of the palmitoylation of BACE, its association with farnesylated 53kDa protein and its stabilisation into its dimeric physiological form.Upon synthesis, BACE associates with the endoplasmic reticular membrane[START_REF] Jick | Statins and the risk of dementia[END_REF] where it undergoes cholesterol-driven palmitoylation (2), stabilising its association with the membrane. BACE then dimerises (3) upon association with farnesylated 53kDa protein (4), stabilising its dimeric structure within the membrane. BACE's prosequence is then cleaved (5), forming the physiological BACE complex, which then enters the cholesterol-rich rafts. Palmitoylation of BACE's C-terminal tail (closed circles) and association with the farnesylated 53kDa protein (open circles) provides the dual-lipidation common in lipid-raft-associated proteins.

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655

Table 1 .

 1 Effect of LOVA and SIMVA upon total cellular cholesterol concentration.

	Cholesterol concentration

Biochemical Journal

Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 Copyright 2006 Biochemical SocietyTable 2 .

 2 Effect of statins, cerulenin and CVFM upon BACE and γ-secretase activity.

			BACE activity (S.A. ±	γ-secretase activity (S.A. ±
			S.D.)	S.D.)
	Untreated		34288 ± 1824	759.8 ± 39.6
	1µM		22707 ± 1143 ***	729.6 ± 8 ns
	10µM	Lovastatin	47685 ± 1571 ***	925.5 ± 26.8 **
	50µM		27508 ± 1394 ***	812.4 ± 10.1 ns
	1µM		30186 ± 171 **	1192.1 ± 54.5 ***
	10µM	Simvastatin	29702 ± 1437 **	629.2 ± 2.58 **
	50µM		40326 ± 1632 ***	1231.9 ± 35.1 ***
	5µg/ml	Cerulenin	30329 ± 1901 ns	1377.8 ± 85.6 ***
	0.1µM	CVFM	31692 ± 678 ns	738.6 ± 10.8 ns
	Specific activity, calculated from three independent experiments in which each
	experiment was performed in triplicate, is expressed as fluorescent units/hour/mg
	protein. ns = not significant; ** = p<0.01; ** = p<0.001.

Table 3 .

 3 Effect of co-incubation of 50µM SIMVA and 0-400µM MA upon total cellular cholesterol levels.

	MA concentration	Cholesterol	vs untreated a	vs 50µM SIMVA b
	(µM)	concentration
		(percentage of
		untreated ± S.D.)
	Untreated	100 ± 5.63	-	-
	0	72.5 ± 2.97	p<0.001	-
	50	55.6 ± 2.84	p<0.001	n/s
	100	75.6 ± 2.00	p<0.001	n/s
	250	53.5 ± 1.85	p<0.001	n/s
	400	78.2 ± 6.82	p<0.001	n/s
	Total cholesterol concentration, measured as free and esterified cholesterol, was

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 FIGURE 1 FIGURE 2 Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 Copyright 2006 Biochemical Society FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 28 Jun 2006 as manuscript BJ20060655

ACKNOWLEDGEMENTS

The authors would like to thank the Wellcome Trust and the Alzheimer's Society for their financial support.