Increasing Membrane Cholesterol Level Increases the ...

Hindawi Publishing CorporationJournal of Neurodegenerative DiseasesVolume 2013 Article ID 407903 12 pageshttpdxdoiorg1011552013407903

Research ArticleIncreasing Membrane Cholesterol Level Increases theAmyloidogenic Peptide by Enhancing the Expression ofPhospholipase C

Yoon Sun Chun1 Hyun Geun Oh1 Myoung Kyu Park1

Tae-Wan Kim2 and Sungkwon Chung1

1 Department of Physiology Samsung Biomedical Research Institute Sungkyunkwan University School of MedicineSuwon 440-746 Republic of Korea

2Department of Pathology Columbia University Medical Center New York NY 10032 USA

Correspondence should be addressed to Tae-Wan Kim twk16columbiaedu and Sungkwon Chung schungskkuedu

Received 6 November 2012 Accepted 9 December 2012

Academic Editor Yasuji Matsuoka

Copyright copy 2013 Yoon Sun Chun et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cerebral elevation of 42-residue amyloid120573-peptide (A12057342) triggers neuronal dysfunction inAlzheimerrsquos disease (AD) Even thougha number of cholesterol modulating agents have been shown to affect A120573 generation the role of cholesterol in the pathogenesisof AD is not clear yet Recently we have shown that increased membrane cholesterol levels downregulates phosphatidylinositol45-bisphosphate (PIP

2) via activation of phospholipase C (PLC) In this study we tested whether membrane cholesterol levels may

affect the A12057342 production via changing PIP2levels Increasingmembrane cholesterol levels decreased PIP

2and increased secreted

A12057342 Supplying PIP2 by using a PIP

2-carrier system blocked the effect of cholesterol on A12057342 We also found that cholesterol

increased the expressions of 1205731 and 1205733 PLC isoforms (PLC1205731 PLC1205733) Silencing the expression of PLC1205731 prevented the effects ofcholesterol on PIP

2levels as well as on A12057342 production suggesting that increased membrane cholesterol levels increased secreted

A12057342 by downregulating PIP2via enhancing the expression of PLC1205731 Thus cholesterol metabolism may be linked to A12057342 levels

via PLC1205731 expression and subsequent changes in PIP2metabolism

1 Introduction

AD is a progressive and irreversible neurodegenerative dis-order leading to cognitive memory and behavioral impair-ments Cerebral elevation and accumulation of A120573 are nec-essary steps in the pathogenesis of AD [1ndash3] Sequentialproteolytic cleavages of amyloid precursor protein (APP) bymembrane-bound 120573-secretase and 120574-secretase produce twomajor isoforms of A120573 A12057340 and A12057342Therefore this path-way is called amyloidogenic pathway More amyloidogenicA12057342 is considered as a pathogenic agent [4 5] AlternativelyAPP can be sequentially processed by 120572-secretase and120574-secretase precluding A120573 production (nonamyloidogenicpathway) Even though advanced age serves as a major riskfactor approximately 5 of AD cases are familial (FAD)and some of them are attributable to autosomal dominantmutations in presenilin (PS) genes PS1 and PS2 PS1 and

PS2 function as catalytic subunits of 120574-secretase and FADmutations in PSs affect APP processing increasing the ratioof A12057342 to A12057340 [6ndash8]

Growing evidence indicates that dysregulation of lipidpathways have regulatory consequences for APP processingand A120573 generation [9] Especially cholesterol has beensuggested to participate in the etiology of AD by increasingthe generation of A120573 [10] Cholesterol can directly regulatethe activities of 120573-secretase or 120574-secretase to alter amyloido-genesis [11ndash13] Alternatively changes in cholesterol level mayaffect the lipid environment for APP processing and A120573generation APP is located either within or outside of lipidrafts Since BACE1 (120573-secretase) is predominantly locatedin lipid rafts APP processing occurring within lipid raftsis amyloidogenic whereas APP processing occurring out-side lipid rafts is considered nonamyloidogenic [14] Whencholesterol is depleted the association of BACE1 with lipid

2 Journal of Neurodegenerative Diseases

rafts is decreased producing less A120573 [15ndash17] In contrastincreasing cholesterol induces the co-clustering of APP andBACE1 producing more A120573 [18] From these results it couldbe hypothesized that high cholesterol levels may be respon-sible for initiating the pathogenesis of AD However it wasrecently demonstrated that lowering cholesterol levels resultsin increased 120573-amyloid production in neurons [19] Abad-Rodriguez et al reported that lowering the intracellular levelsof cholesterol could increase the rate of amyloidogenic pro-cessing of APP by placing the hydrolyzing enzyme (BACE1)and APP in close proximity within the same intracellularcompartments Therefore more experiments will be neededto clarify the conflicting results about the role of cholesterolin pathogenesis of AD

PIP2is known as one of phospholipid component of cell

membrane [20] playing important regulatory roles in a vari-ety of cell functions such as rearrangement of the cytoskele-ton and membrane trafficking [21] We have reported thatFAD-linked PS mutants down-regulate PIP

2levels and that

PIP2levels are inversely correlated to the production of

A12057342 [22] We also demonstrated that increased membranecholesterol level decreases the level of PIP

2via the activation

of PLC [23] Therefore there exists a crosstalk betweentwo plasma membrane-enriched lipids cholesterol and PIP

2

Considering the close relationship between PIP2levels and

the production of A12057342 we suspected that increased mem-brane cholesterol levels affect the A12057342 production viadown-regulating PIP

2levels In this study we found that

membrane cholesterol decreased PIP2levels and increased

secretedA12057342 Supplying PIP2by using a PIP

2-carrier system

blocked the effect of cholesterol which might indicate thatthe effect of cholesterol on A12057342 was by downregulation ofPIP2levels Enriching membrane with cholesterol increased

the expression of some PLC isoforms such as PLC1205731 andPLC1205733 Blocking the new protein synthesis prevented theeffect of cholesterol on PIP

2levels as well as on A12057342

production We found that the expression of PLC1205731 wasspecifically linked to A12057342 production These results suggestthat increased membrane cholesterol levels and FAD-linkedPS mutations may share the same molecular mechanismthat is the downregulation of PIP

2 which may serve as the

molecule linking cholesterol metabolism to the pathogenesisof AD

2 Materials and Methods

21 Cell Culture HeLa cells stably transfected with APP751

carrying the Swedish mutation (APPsw) were cultured at37∘C 5 CO

2 in Dulbeccorsquos Modified Eagle Medium

(DMEM) supplemented with 10 heat-inactivated fetalbovine serum containing 100 unitsmL penicillin 100 120583gmLstreptomycin 260120583gmL Zeocin and 400 120583gmL G418Human neuroblastoma SH-SY5Y cells were cultured inDMEM containing 10 heat-inactivated fetal bovine serum100 unitsmL penicillin and 100120583gmL streptomycin

22 Procedure To enrich the cells with cholesterol cells wereexposed to DMEM culture medium containing methyl-120573-cyclodextrin (M120573CD SigmaUSA) saturatedwith cholesterol

(water-soluble cholesterol) During the incubation cells weremaintained in a humidified CO

2incubator at 37∘C In some

experiments to avoid the use of M120573CD cells were incubatedwith cholesterol which was solubilized by sonication For thispurpose cholesterol in methanolchloroform mixture (1 1vv) was dried under nitrogen gas and sonicated for 2min inphosphate-buffered saline before use

In some experiments cells were pretreated with 10 120583MActinomycin-D (Sigma) or 50120583gmL cyclohexamide (Sigma)for 05 h before 75120583M water-soluble cholesterol was addedPIP2were delivered into the cells using the PIP

2-carrier sys-

tem (Echelon Bioscience Inc USA) Carrier-PIP2complex

was incubated with APP-transfected HeLa cells for 4 h in theabsence or presence of 75120583Mwater-soluble cholesterol

23 Antisense Oligonucleotides Treatments The antisenseoligonucleotides (IDT USA) targeted at PLC1205731 and PLC1205733were designed to be complementary to the 51015840 sequencesand were phosphorothionated at all positions to mini-mize intracellular cleavage by enzymes and to enhancetheir stability (51015840-actccgggttgagccccggc-31015840 for PLC1205731 and51015840-tccaactgcagcgcgtggac-31015840 for PLC1205733) Antisense oligonu-cleotide (51015840-gccccgtatgaccgcgccgg-31015840) having no target wasused as a control in all of experiments The APP-transfectedHeLa cells were plated at a density of 2 times 106 cells per 60mmdish and incubated overnight and then treatedwith the 10 120583Mantisense oligonucleotides for 4 h in DMEM culture mediumwithout serum After treatment the mediumwas replaced bya new medium containing 10 120583M antisense oligonucleotideswith or without water-soluble cholesterol for 2 h Media werecollected tomeasure levels ofA120573 and cellswere homogenizedto confirm PLC expression levels and PIP

2levels

24 Cholesterol Assay Filipin staining of cells (005 DMSO1) was performed for 1 h at room temperature after choles-terol enrichment to confirm the changes of free cholesterollevels at the plasma membrane Fluorescence images wereobtained using a LSM 710 confocal microscope (Zeiss) usinglaser emitting at 351 nm Images were quantified to obtainthe mean fluorescence density values of plasma membranefrom the edge of the cell to 500 nm inside using the ImageJprogram

25 sAPP120572 sAPP120573 and A120573 Peptide Assay Levels of A120573peptides were assayed by using the Invitrogen A120573 ELISAkits (USA) or Wako 120573-amyloid ELISA kits (high-sensitiveJapan) For sAPP120572 sAPP120573 detection samples were analyzedby ELISA kit from IBL (USA) APP-transfected HeLa cellsat 80 confluence in a 35mm dish were cultured for 8 hwith water-soluble cholesterol in DMEM culture mediumwithout serum Control cells were treated similarly andincubated with serum-free DMEM solution without anycholesterol After exposure to cholesterol supernatants werecollected tomeasure levels of A120573 sAPP120572 or sAPP120573 To detectA120573 from SH-SY5Y cells supernatants were desalted usingPD-10 desalting column (GE Healthcare USA) dried andreconstituted in water The samples were analyzed by ELISAkits according to the supplierrsquos instructions

Journal of Neurodegenerative Diseases 3

26 Protein Extraction Cell fractionations were obtainedby homogenizing with hypotonic buffer using a 23-gaugeneedle The samples were then centrifuged at 1000timesg for10min at 4∘C to remove nuclei and debris Supernatantswere separated by centrifugation at 100000timesg for 1 h at 4∘Cinto membrane (pellet) and cytosol (supernatant) fractionsWhole cell lysates were prepared by homogenizing with lysisbuffer (10mM Tris-HCl 150mM NaCl 1 Triton X-100025Nonidet P-40 2mMEDTA pH74) using a cell scraperThe lysed cells were centrifuged at 12000timesg for 10min at 4∘CThe protein in the supernatant was determined by Bradfordassay (Bio-rad USA)

27 Western Blot Analysis Proteins were resolved on SDS-PAGE and transferred to nitorcelluose membrane Mem-branes were blocked with 5 nonfat milk powder in Tris-buffered salineTween 20 (TBST) for 1 h at room temperaturethen incubated with rabbit polyclonal anti-PLC1205731 (SC-9050)PLC1205732 (SC-206) PLC1205733 (SC-13958) PLC1205734 (SC-20760)PLC1205742 (SC-9015) mouse monoclonal anti-PLC1205741 (SC-7290)antibodies (Santa Cruz Biotechnology USA) anti-APP anti-body (LN27 Zymed) anti 120573-actin (A5441 Sigma) andrabbit anti 120573-tubulin (T2200 Sigma) for overnight at 4∘CDilutions were 1 500 for PLC isozymes and 1 4000 for 120573-tubulin 120573-actin and APP After washing membranes wereincubated for 1 h at room temperature with horseradishperoxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies (1 2000 dilution Zymed USA) andwashed Peroxidase activity was visualized with enhancedchemilluminescence Blots were quantified with the MultiGauge software using a LAS-3000 system (FugiFilm Japan)

28 PIP2

Assay The amount of PIP2

extracted fromAPP-transfected HeLa cells were measured by using PIP

2

Mass ELISA kit (Echelon Biosciences Inc USA) PIP2was

extracted from the control cells or cells treated with water-soluble cholesterol according to the supplierrsquos instructionsCellular PIP

2quantities were estimated by comparing the

values from the standard curve which showed linear relation-ship at the range from 05 to 1000 pM concentrations

29 Statistical Analysis Data was expressed as mean plusmn SEMStatistical comparisons between the controls and treatedexperimental groups were performed using the Studentrsquost-test 119875 lt 005 was considered statistically significant

3 Results

31 Increasing Membrane Cholesterol Levels DownregulatesPIP2and Increases Secreted A12057342 M120573CD a water-soluble

cyclic oligosaccharide has hydrophobic cavity that is able toencapsulate insoluble compounds thus enhances the solubil-ity of cholesterol M120573CD saturated with cholesterol (water-soluble cholesterol) has been used to increase membranecholesterol level since it acts as a cholesterol donor [24ndash26]We incubated APP-transfected HeLa cells with 15 75 or150120583M water-soluble cholesterol for 8 h and filipin stainingwas performed for 1 h at room temperature to monitor

the membrane cholesterol level Typical confocal imagein Figure 1(a) shows that the membrane cholesterol levelincreased by 75120583M water-soluble cholesterol The changesof cholesterol level were confirmed by quantifying the filipinfluorescent intensities from plasma membranes By incubat-ing cells with 15 and 75120583M water-soluble cholesterol thefluorescent intensities were increased by 585 plusmn 58 and833 plusmn 159 (119899 = 6) respectively (Figure 1(b)) We alsotested the time-dependent accumulation of cholesterol inthe membrane by incubating cells with 75120583M water-solublecholesterol Cholesterol levels increased after 05 h and itsteadily increased further after 15 h or 5 h (SupplementaryFigures 1(a) and 1(b) see Supplementary Material availableonline at httpdxdoiorg1011552013407903) From theseresults we concluded that the direct administration of thewater-soluble cholesterol leads to the increases in the mem-brane cholesterol levels

Recently we have reported that augmentation of mem-brane cholesterol levels downregulates PIP

2level [23] To val-

idate this observation in the current system APP-transfectedHeLa cells were incubated with 15 75 or 150120583M water-soluble cholesterol for 8 h and the steady state levels of PIP

2

were measured using a PIP2ELISA PIP

2levels in 75 and

150 120583Mcholesterol-treated cells were downregulated by 232plusmn50 and 261 plusmn 25 (119899 = 6) respectively (Figure 1(c)) Wealso tested the time-dependent effect of increased membranecholesterol on the levels of PIP

2by incubating cells with

75 120583Mwater-soluble cholesterol As shown in Figure 1(d) thesteady state levels of PIP

2after 15 h and 5 h incubation time

were downregulated by 203 plusmn 51 and 263 plusmn 53 (119899 = 6)respectively

Since we have reported that cellular PIP2levels are closely

correlated with the A12057342 levels [22] we tested the effect ofincreased membrane cholesterol levels on secreted A120573 Forthis purpose APP-transfected HeLa cells were incubated for8 h with 15 75 or 150 120583M water-soluble cholesterol and A120573levels were measured from the conditioned media by usingan ELISA kits specific for A12057340 or A12057342 The secreted A12057340levels were not changed by increased membrane cholesterollevels (open bars in Figure 1(e)) However A12057342 levelswere increased by 281 plusmn 84 and 362 plusmn 81 (119899 = 6)when cells were incubated with 75 and 150120583Mwater-solublecholesterol respectively (closed bars in Figure 1(e)) We alsotested the effect of increased membrane cholesterol levels onthe levels of secreted A120573 from neuroblastoma SH-SY5Y cellsThe level of endogenous A12057342 increased significantly (closedbars in Figure 1(f)) while the endogenous A12057340 level was notchanged (open bars in Figure 1(f))Thus these results suggestthat the effect of cholesterol enrichment is specific to A12057342levels and is not cell-type specific

A120573 is produced by the sequential cleavages of APP by120573-secretase followed by 120574-secretase Alternatively APP canbe cleaved sequentially by 120572-secretase followed by 120574-secretaseprecluding the production of A120573Thus the effect of increasedmembrane cholesterol levels on A120573 levels can occur in anyof those processes To begin to investigate the effects ofmembrane cholesterol on APP processing we first examinedthe levels of full-length APP Increasedmembrane cholesterollevels led to a moderate increase in the full-length APP


CholesterolControl

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Figure 1 Augmentation of membrane cholesterol levels downregulated PIP2levels and increased A12057342 (a b) Incubating APP-transfected

HeLa cells with water-soluble cholesterol increased cholesterol levels in the plasmamembrane Cells were incubated with 0 15 75 and 150120583Mwater-soluble cholesterol for 8 h at 37∘C Filipin staining was performed for 1 h at room temperature after cholesterol enrichment (a) A typicalfluorescence image with 75 120583M water-soluble cholesterol is shown in (b) Incubating the cells with water-soluble cholesterol increased thecholesterol contents in a concentration-dependent manner Fluorescent intensities from plasma membranes were quantified as described inSection 2 (119899 = 6) (c) Incubating cells with water-soluble cholesterol downregulated PIP

2levels APP-transfected HeLa cells were incubated

for 8 h with 0 15 75 and 150120583M water-soluble cholesterol PIP2levels in the membrane fractions were measured by using a PIP

2ELISA kit

as described in Section 2 (119899 = 6) (d) Incubating cells with water-soluble cholesterol downregulated PIP2levels in time-dependent manner

Cells were incubated with 75 120583M water-soluble cholesterol for 05 15 and 5 h (119899 = 6) (e) Incubating cells with water-soluble cholesterolselectively increased secreted A12057342 levels (closed bars 119899 = 6) In contrast the levels of A12057340 were not changed by cholesterol enrichment(open bars 119899 = 6) APP-transfected HeLa cells were incubated with 0 15 75 and 150120583M water-soluble cholesterol for 8 h A12057340 and A12057342levels were measured from the conditioned media by using ELISA method as described in Section 2 (f) Incubating cells with water-solublecholesterol increased secreted A12057342 levels (closed bars 119899 = 6) but not A12057340 levels (open bars 119899 = 4) from neuroblastoma SH-SY5Y cellsTheendogenous A12057340 andA12057342 levels weremeasured from the conditionedmedia by using ELISAmethod lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001


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Figure 2 Elevation of PIP2level prevented the effect of cholesterol on A12057342 production (a) Incubating APP-transfected HeLa cells with

water-soluble cholesterol did not significantly change the full-length APP level Cells were incubated with 75 120583M water-soluble cholesterolfor 8 h Similar Western blotting results were obtained from 4 different experiments (b) Both sAPP120572 and sAPP120573 were slightly increased byincubating cells with 75120583M water-soluble cholesterol for 8 h (119899 = 4) The levels of sAPP120572 and sAPP120573 were measured from the conditionedmedia as described in Section 2 (c) Supplying PIP

2decreased A12057342 production and prevented the effect of cholesterol APP-transfectedHeLa

cells were incubated with 0 2 and 5120583M carrier-PIP2complex in the absence and the presence of 75 120583M water-soluble cholesterol for 4 h

A12057342 levels were measured from the conditioned media by using ELISAmethod Without treating cells with water-soluble cholesterol A12057342production decreased as PIP

2concentration increased (119899 = 6 open bars) As expected treating cells with water-soluble cholesterol increased

the production A12057342 in the absence of PIP2(the first closed bar) However the effects of cholesterol on A12057342 were prevented by the presence

of 2120583M and 5 120583M PIP2(the second and the third close bars 119899 = 6) (d) Elevation of PIP

2level did not change A12057340 production (119899 = 5)

lowastlowastlowast119875 lt 0001

levels (Figure 2(a)) However the increase was less than10 (119899 = 4) Then we tested the effects of increasedmembrane cholesterol levels on the activities of 120572-secretaseand 120573-secretase For this purpose we measured the levels ofsAPP120572 and sAPP120573 from the conditionedmedia using specific

ELISA kits since they are produced via the activities of 120572-secretase and 120573-secretase respectively In this experimentwe used sAPP120573 ELISA kit for Swedish mutant As shownin Figure 2(b) the levels of both sAPP120572 and sAPP120573 werealso slightly increased by increased membrane cholesterol


levels which might be due to the increased level of theirprecursor APP However the amount of increased sAPP120573level was not robust to explain the A12057342-selective changesassociated with increased membrane cholesterol levels Sincemembrane cholesterol levels affect A12057342 but not A12057340 it isconceivable that the effects of cholesterol may influence thespecificity of 120574-secretase-mediated cleavage of amyloidogenicAPP C-terminal fragments (eg C99)

32 Intracellular Delivery of PIP2Prevents the Effect of

Increased Membrane Cholesterol Levels on Secreted A12057342In order to elucidate the role of PIP

2for the effect of

increased membrane cholesterol levels on secreted A12057342 weused a PIP

2-carrier system for the intracellular delivery of

PIP2 Because carrier compounds are ldquocharge-neutralizationrdquo

species it could deliver the anionic PIP2into the cells [27]

After the carriers were added at a one-to-one molar ratiowith PIP

2at room temperature the complex was diluted

to the desired final concentration Then the carrier-PIP2

complexwas incubatedwith cells for 4 h beforeA120573 levels weremeasured from the conditioned media

The presence of 2120583M and 5 120583M carrier-PIP2complex

decreased secreted A12057342 levels by 128 plusmn 121 and 415 plusmn41 (119899 = 6) respectively (open bars in Figure 2(c)) Thisresult is consistent with our previous result showing theclose correlation between PIP

2levels and A12057342 production

[22] In the absence of carrier-PIP2complex incubating cells

with 75 120583M water-soluble cholesterol for 4 h increased A12057342levels by 177 plusmn 29 (119899 = 6) which is consistent with theresult in Figure 1(e) However the presence of either 2120583M or5 120583M carrier-PIP

2complex completely prevented the effect

of water-soluble cholesterol on A12057342 levels (closed bars inFigure 2(c)) These results suggest that the relative levels ofcholesterol and PIP

2correlate closely with secreted A12057342

levels in a positive or negative manner respectively UnlikeA12057342 the A12057340 levels were not affected by the presenceof carrier-PIP

2complex (open bars in Figure 2(d)) Also

the A12057340 levels were not affected by increased membranecholesterol levels in the presence of carrier-PIP

2complex

(closed bars in Figure 2(d)) which was consistent with thespecific effect of cholesterol on A12057342 level

33 Increasing Membrane Cholesterol Level Increases PLC1205731and PLC1205733 Expressions The major catabolic pathway forPIP2is the hydrolysis by membranous PLC Since we sus-

pected that the effect of increased membrane cholesterol lev-els on the secreted A12057342 levels is due to the downregulationof PIP

2 we tested whether increased membrane cholesterol

levels affect the activity of PLC We first examined theexpression levels of PLC isoforms by monitoring them usingWestern blot analysis from cytosol and membrane fractionsinAPP-transfectedHeLa cellsThe expression levels of PLC1205731were significantly increased by cholesterol only in membranefractions as shown in Figure 3(a) Densitometry analysisof the bands corresponding to PLC1205731 clearly supports thisconclusion (Figure 3(b) 119899 = 5) After 05 h incubation timewith 75 120583M water-soluble cholesterol expression levels ofPLC1205731 were increased by two folds in membrane fractions

The effect of cholesterol on PLC1205731 expression lasted as longas 5 h The expression level of PLC1205733 was also increased inmembrane fractions by cholesterol (Figures 3(a) and 3(c))However the effect was significant only after 5 h incubationtime

We used M120573CD to increase cholesterol levels Howeverthe use of M120573CDmay cause nonspecific effects in addition toenrichment of membrane cholesterol levels [28] To avoid theuse of M120573CD free cholesterol was solubilized in phosphate-buffered saline using sonication When cells were incubatedwith 75 120583M solubilized cholesterol for 1 h the expression ofPLC1205731 in the membrane fraction was increased (Supple-mentary Figure 2) At 1 h incubation time the expressionof PLC1205733 was not changed These results indicated that theeffect of cholesterol on PLC1205731 expression was not due to thenonspecific effect of M120573CD

Cholesterol-induced increases in the levels of PLC1205731 andPLC1205733 were observed almost exclusively in the membranefraction Also increased membrane cholesterol levels didnot change the expression levels of PLC1205742 as shown inFigure 3(a) from a typical experiment The expression levelsof other PLC isoforms (PLC1205732 PLC1205734 and PLC1205741) werenot changed either (Supplementary Figure 3(a)) We alsoobserved specific increase of PLC1205731 and PLC1205733 expressionsfrom SH-SY5Y cells by enriching membrane cholesterollevels (Supplementary Figure 3(b))

To determine whether the effect of cholesterol on PLCexpression was due to increased transcription cells werepreincubated for 10min with the transcription inhibitoractinomycin-D (Act-D 10 120583M) or with the translationinhibitor cyclohexamide (CHX 50120583gmL) Then cells wereincubated further for 05 h with 75120583M water-soluble choles-terol Representative Western blots for PLC1205731 and PLC1205733from membrane fractions are shown in Figure 4(a) Theeffect of cholesterol on PLC1205731 and PLC1205733 expressions wascompletely prevented either by Act-D or by CHX indicatingthat the effect of cholesterol on PLC1205731 and PLC1205733 expressionwas via the up-regulation of transcription Similar resultswere obtained from 4 different experiments Then we testedthe effect of Act-D on the steady state level of PIP

2 As we

expected PIP2levels were downregulated by 189 plusmn 03

(119899 = 6) when cells were treated with 75120583M water-solublecholesterol for 1 h in the absence ofAct-D (Figure 4(b)) How-ever the effect of cholesterol on PIP

2levels was completely

prevented when cells were pretreated with Act-D (119899 = 6)These results confirmed that increasedmembrane cholesterollevels increases the expression of PLC1205731 and PLC1205733 leadingto the downregulation of PIP

2levels Next we tested the effect

of Act-D on the level of secreted A12057342 For this purpose cellswere incubated for 4 h with 75 120583M water-soluble cholesterolwith or without Act-D As expected cholesterol increasedthe levels of secreted A12057342 by 201 plusmn 28 (119899 = 4) in theabsence of Act-D (Figure 4(c)) However the presence of Act-D completely prevented the effect of increased membranecholesterol levels on A12057342 (119899 = 4) These results suggestthat the increase of PLC transcription by cholesterol inducedthe downregulation of PIP

2levels which increased secreted

A12057342 levels


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Figure 3 Augmentation of membrane cholesterol levels increased PLC1205731 and PLC1205733 expressions (a) Representative western blotting resultsshowed specific increase of PLC1205731 and PLC1205733 expressions from APP-transfected HeLa cells Cells were incubated with 75120583Mwater-solublecholesterol for the indicated timesMembrane and cytosol fractions were obtained as described inMaterials andMethods Similar results wereobtained from 5 different experiments Note that PLC1205731 and PLC1205733 expressions were increased in time-dependent manner by cholesterolfrommembrane fraction but not from cytosol fraction In contrast PLC1205742 expression was not changed by cholesterol 120573-tubulin was used toconfirm the amount of proteins loaded (b c) Bars correspond to the densitometric analysis of PLC1205731 and PLC1205733 expressions frommembraneand cytosol fractions (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001

34 Inhibition of PLC1205731 Expression Prevents the Effect ofIncreased Membrane Cholesterol Levels on Secreted A12057342We tested whether PLC1205731 or PLC1205733 is required for theobserved effects of cholesterol on A12057342 To avoid any fur-ther transfection cells were pretreated with 10 120583M antisenseoligonucleotides against PLC isoforms for 4 h Then themedia were replaced by fresh media containing the sameantisense oligonucleotides with or without 75120583M water-soluble cholesterol followed by additional incubation for 2 hAntisense oligonucleotides having no specific target wereused for controls in all of experiments A typical western blotfor PLC1205731 is shown in Figure 5(a) and the band densities areexpressed relative to 120573-tubulin density in Figure 5(c) (119899 =6) As we expected cholesterol increased PLC1205731 expression

However the presence of antisense oligonucleotides againstPLC1205731 (anti 1205731) blocked the effect of cholesterol Similarlyan increase of PLC1205733 expression by cholesterol was blockedby antisense oligonucleotides against PLC1205733 (anti 1205733 Figures5(b) and 5(d)) These results demonstrated that these anti-sense oligonucleotides were very effective to block the effectof increased membrane cholesterol levels on the expressionof PLC1205731 and PLC1205733

Then we tested the effect of these antisense oligonu-cleotides on the PIP

2levels In the presence of control

antisense oligonucleotides (minusanti 1205731) cholesterol decreasedPIP2levels by 121 plusmn 28 (Figure 6(a) 119899 = 6) In the pres-

ence of antisense oligonucleotide against PLC1205731 (+anti 1205731)cholesterol decreased PIP

2levels only by 25plusmn10 (119899 = 6) In


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l)

(c)

Figure 4 Transcription inhibitor prevented the effects of augmentation of membrane cholesterol levels (a) Transcription and translationinhibitors prevented the effect of cholesterol on PLC1205731 and PLC1205733 expressions APP-transfected HeLa cells were pretreated with thetranscription inhibitor actinomycin-D (Act-D 10 120583M) or translation inhibitor and cyclohexamide (CHX 50 120583gmL) for 10min which wasfollowed by the additional 05 h incubation with 75 120583Mwater-soluble cholesterol In the presence of Act-D or CHX the effect of cholesterol onPLC1205731 and PLC1205733 expressions in membrane fractions was prevented Similar results were obtained from 3 different experiments 120573-tubulinwas used to confirm the amount of proteins loaded (b) Transcription inhibitor prevented the effect of cholesterol on PIP

2levels APP-

transfected HeLa cells were incubated in the presence or absence of 10120583M Act-D with 75 120583M water-soluble cholesterol for 1 h PIP2levels

in membrane fractions were measured by using a PIP2ELISA kit as described in Section 2 In the absence of Act-D (minusAct-D) cholesterol

decreased PIP2levels (119899 = 6) However the presence of Act-D (+Act-D) prevented the effect of cholesterol on PIP

2levels (119899 = 6) (c)

Transcription inhibitor prevented the effects of cholesterol on A12057342 production APP-transfected HeLa cells were incubated in the presenceor absence of 10 120583M Act-D with 75120583M water-soluble cholesterol for 4 h A12057342 levels were measured from the conditioned media by usingELISA method In the absence of Act-D (minusAct-D) cholesterol increased A12057342 level (119899 = 4) However the presence of Act-D (+Act-D)prevented the increase of A12057342 production induced by cholesterol (119899 = 4) lowast119875 lt 005 lowastlowast119875 lt 001

contrast cholesterol decreasedPIP2levels by 10plusmn28(119899 = 6)

even in the presence of antisense oligonucleotide againstPLC1205733 (+anti 1205733 Figure 6(b)) Thus inhibiting PLC1205731expression but not inhibiting PLC1205733 expression preventedthe effect of increased membrane cholesterol levels on PIP

2

levels These results may suggest that the downregulation ofPIP2levels by cholesterol enrichment is specifically via the

increased expression of PLC1205731Next we tested the effect of increased membrane choles-

terol levels on the levels of secreted A12057342 in the presence of

these antisense oligonucleotides As we expected cholesterolincreased the levels of secreted A12057342 by 257 plusmn 55 (119899 = 8)in the presence of control antisense oligonucleotides (Fig-ure 6(c)) In the presence of antisense oligonucleotide againstPLC1205731 cholesterol increased the secreted A12057342 level only by20 plusmn 21 (119899 = 8) Thus PLC1205731 antisense oligonucleotidealmost completely prevented the effect of cholesterol on thesecreted A12057342 levels In contrast the presence of PLC1205733antisense oligonucleotides failed to prevent the effect ofcholesterolTheA12057342 productionwas still increased by 167plusmn


(a) (b)

(c) (d)

Figure 5 The effect of augmentation of membrane cholesterol levels on PLC1205731 and PLC1205733 expressions were blocked by the presence ofantisense oligonucleotides (a b) The effect of cholesterol on PLC1205731 and PLC1205733 expressions were prevented by the presence of antisenseoligonucleotides APP-transfectedHeLa cells were incubatedwith or without 75 120583Mwater-soluble cholesterol for 2 h and the expression levelsof PLC1205731 and PLC1205733 were tested using western blotting In some cells 10120583M antisense oligonucleotides directed against PLC1205731 (anti 1205731) orPLC1205733 (anti1205733) were pretreated for 4 h before the cholesterol treatment Similar results were obtained from4 different experiments Antisenseoligonucleotides having no specific target were used for controls in all of experiments (c d) Bars correspond to the densitometric analysis ofthe expression levels of PLC1205731 and PLC1205733 respectively (119899 = 6) lowast119875 lt 005

34 (Figure 6(d) 119899 = 6) Together these results stronglysuggest that increased membrane cholesterol levels increasedthe levels of secretedA12057342 by down-regulating PIP

2levels via

specific enhancement of PLC1205731 expression

4 Discussion

We have previously reported that FAD-linked PS mutantsdown-regulate PIP

2levels which are closely related to

increased A12057342 level [22] In addition up or downregu-lation of PIP

2levels by pharmacological means decreases

or increases the production of A12057342 respectively In thisstudy we showed that cholesterol enrichment increases thesecreted A12057342 levels by down-regulating PIP

2levels Thus

there exists a close relationship between PIP2levels and

A12057342 levels consistent with our previous results Recentlywe demonstrated that enrichment of cholesterol decreasesthe levels of PIP

2via the activation PLC [23] Therefore

it was suggested that there exists a crosstalk between twoplasma membrane-enriched lipids cholesterol and PIP

2 In

this paper we confirmed that cholesterol decreases the levelof PIP

2via the activation PLC In addition we showed

that cholesterol specifically increases the expression levelsof PLC1205731 and PLC1205733 Consistent with this conclusioninhibiting transcription prevented the effects of cholesterolnot only on PIP

2levels but also on the production of A12057342

Also the inhibition of PLC1205731 expression but not that ofPLC1205733 prevented the effects of cholesterol indicating a closelink between PLC1205731 and regulation of PIP

2levels

Although PIP2is a minor component in the plasma

membrane it plays important regulatory roles in a varietyof cell functions such as rearrangement of the cytoskeletonand membrane trafficking [21] The concept of spatiallyconfined PIP

2pools was proposed to explain the multiple

roles of PIP2[29] Cholesterol- and sphingolipid-rich rafts

may serve to confine PIP2within the plasma membrane


(a)

100

80

60

100

80

60Control Cholesterol Control Cholesterol

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast

minusAnti-1205733 +Anti-1205733

(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)

lowastlowastlowast lowast

120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)

Figure 6 Suppression of PLC1205731 expression prevented the effects of augmentation of membrane cholesterol levels on PIP2levels and A12057342

production (a b) The effect of cholesterol on PIP2levels was blocked in the presence of antisense oligonucleotides directed against PLC1205731

APP-transfectedHeLa cells were incubated with or without 75120583Mwater-soluble cholesterol for 2 h and PIP2levels in themembrane fractions

were measured by using a PIP2ELISA kit In some cells antisense oligonucleotides directed against PLC1205731 or PLC1205733 (10120583M)were pretreated

for 4 h before the cholesterol treatmentThe presence of PLC1205731 antisense oligonucleotides (119899 = 6) but not that of PLC1205733 prevented the effectsof cholesterol (119899 = 6) (c d) The effect of cholesterol on A12057342 production was blocked in the presence of antisense oligonucleotides directedagainst PLC1205731 After treating cells as in (a) and (b) A12057342 levels were measured from the conditioned media by using ELISA method Thepresence of PLC1205731 antisense oligonucleotides (119899 = 8) but not that of PLC1205733 blocked the effects of cholesterol enrichment (119899 = 6) lowast119875 lt 005lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

allowing PIP2hydrolysis to occur locally and restrict sig-

naling mechanisms to the site of activation [30 31] Inaddition to this confined regulation of PIP

2 it is possible

that the steady-state level of PIP2is dynamically determined

by the concerted action of phosphoinositide kinases andphosphatases In this study we showed another way of reg-ulating PIP

2levels within specific microdomains cholesterol

content in a specific microdomain may regulate PIP2levels

via PLC activity Interestingly PLC1205731 is shown to localize indetergent-resistant membrane microdomains prepared fromthe synaptic plasma membrane fraction of rat brain [32]For this reason increase of PLC1205731 expression by cholesterol

enrichment may directly induce downregulation of PIP2in

the confined microdomainThe effect of cholesterol enrichment on A120573 secretion was

recently demonstrated [33] It was shown that cholesterolincreases clathrin-dependent APP endocytosis and that it islikely the direct cause of the increased A12057342 secretion SincePIP2is a key regulator for the rearrangement of the cytoskele-

ton and membrane trafficking [21] it is possible that thedownregulation of PIP

2may be the underlying mechanism

for the increased clathrin-dependent APP endocytosis bycholesterol enrichment Alternatively the downregulation ofPIP2may directly activate 120574-secretase since PIP

2is shown to


inhibit 120574-secretase activity by suppressing its association withthe substrate [13] It is also possible that PIP

2induces changes

in 120574-secretase conformation to alter the generation of A12057342Recent reports show that the changes in PS1 conformationby various manipulations of PS1 itself Pen2 Aph1 APP andpharmacological agents known as 120574-secretasemodulators canallosterically modify 120574-secretase catalytic specificity leadingto increase of A12057342A12057340 ratio [34 35] Further studies willbe needed to clarify the role of PIP

2in the production of A120573

The physiological relevance of our finding is not clearsince a large increase of cholesterol in the membrane wasrequired to induce meaningful downregulation of PIP

2and

up-regulation of A12057342 in HeLa cells (Figures 1(c) and1(e)) In SH-SY5Y cells however A12057342 level was signif-icantly increased even by 15120583M water-soluble cholesterol(Figure 1(f)) We also observed a significant increase ofPLC1205731 expression and downregulation of PIP

2level by 15 120583M

water-soluble cholesterol when we used HEK cells (datanot shown) Thus it seems that the effective amount ofcholesterol to induce changes in PIP

2and A12057342 levels is

cell type dependent However even the minimal increase ofA12057342 by the mild cholesterol enrichment will have profoundcytotoxic effects since the cytotoxicity of A12057342 is causedby its small molecular aggregates such as dimer form ofA12057342 [9] and the produced A12057342 will accumulate It hasbeen demonstrated that cholesterol levels in the brains ofAD patients are increased [36 37] The levels of cholesterolincrease in the brain even during normal aging [36] In anAD brain cholesterol homeostasis is impaired and choles-terol retention likely enhances A120573 production [37] Theseresults may suggest that high cholesterol levels in the brainparticipate in the etiology of AD by increasing the generationof A120573 However further work is needed to understand howcholesterol is implicated in AD pathogenesis In this studywe showed that membrane cholesterol levels may share thesame molecular mechanism with FAD PS mutations thatis downregulation of PIP

2 for the increased generation of

A12057342 Therefore PIP2may serve as the molecule linking

cholesterol metabolism to the pathogenesis of AD

Conflict of Interests

Theauthors of the paper do not have a direct financial relationwith the commercial identity mentioned in the paper thatmight lead to a conflict of interests for any of the authors

Acknowledgments

This work was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF)and funded by the Ministry of Education Science and Tech-nology (2009-0072220) Samsung Biomedical Research Insti-tute (B-B1-003) to S Chung and by NIH Grant (NS074536)to T-W Kim

References

[1] J A Hardy andG A Higgins ldquoAlzheimerrsquos disease the amyloidcascade hypothesisrdquo Science vol 256 no 5054 pp 184ndash1851992

[2] J Hardy and D J Selkoe ldquoThe amyloid hypothesis ofAlzheimerrsquos disease progress and problems on the road totherapeuticsrdquo Science vol 297 no 5580 pp 353ndash356 2002

[3] D M Walsh and D J Selkoe ldquoA120573 oligomersmdasha decade ofdiscoveryrdquo Journal of Neurochemistry vol 101 no 5 pp 1172ndash1184 2007

[4] T Iwatsubo A Odaka N Suzuki HMizusawa N Nukina andY Ihara ldquoVisualization of A12057342(43) and A12057340 in senile plaqueswith end-specific A120573 monoclonals Evidence that an initiallydeposited species is A12057342(43)rdquoNeuron vol 13 no 1 pp 45ndash531994

[5] D Scheuner C Eckman M Jensen et al ldquoSecreted amyloid120573-protein similar to that in the senile plaques of Alzheimerrsquosdisease is increased in vivo by the presenilin 1 and 2 andAPP mutations linked to familial Alzheimerrsquos diseaserdquo NatureMedicine vol 2 no 8 pp 864ndash870 1996

[6] B De Strooper ldquoAph-1 Pen-2 and nicastrin with presenilingenerate an active 120574-secretase complexrdquo Neuron vol 38 no 1pp 9ndash12 2003

[7] M S Wolfe ldquoThe 120574-secretase complex membrane-embeddedproteolytic ensemblerdquo Biochemistry vol 45 no 26 pp 7931ndash7939 2006

[8] M Bentahir O Nyabi J Verhamme et al ldquoPresenilin clinicalmutations can affect 120574-secretase activity by different mecha-nismsrdquo Journal of Neurochemistry vol 96 no 3 pp 732ndash7422006

[9] G Di Paolo and T W Kim ldquoErratum linking lipids toAlzheimerrsquos disease cholesterol and beyondrdquo Nature ReviewsNeuroscience vol 12 no 8 p 484 2011

[10] L M Refolo M A Pappolla B Malester et al ldquoHypercholes-terolemia accelerates the Alzheimerrsquos amyloid pathology in atransgenic mouse modelrdquo Neurobiology of Disease vol 7 no 4pp 321ndash331 2000

[11] L Kalvodova N Kahya P Schwille et al ldquoLipids as modulatorsof proteolytic activity of BACE involvement of cholesterolglycosphingolipids and anionic phospholipids in vitrordquo Journalof Biological Chemistry vol 280 no 44 pp 36815ndash36823 2005

[12] P Osenkowski W Ye R Wang M S Wolfe and D JSelkoe ldquoDirect and potent regulation of 120574-secretase by its lipidmicroenvironmentrdquo Journal of Biological Chemistry vol 283no 33 pp 22529ndash22540 2008

[13] S Osawa S Funamoto M Nobuhara et al ldquoPhosphoinosi-tides suppress 120574-secretase in both the detergent-soluble and -insoluble statesrdquo Journal of Biological Chemistry vol 283 no28 pp 19283ndash19292 2008

[14] K S Vetrivel and G Thinakaran ldquoMembrane rafts inAlzheimerrsquos disease 120573-amyloid productionrdquo Biochimica et Bio-physica Acta vol 1801 no 8 pp 860ndash867 2010

[15] D R Riddell G Christie I Hussain and C DingwallldquoCompartmentalization of120573-secretase (Asp2) into low-buoyantdensity noncaveolar lipid raftsrdquo Current Biology vol 11 no 16pp 1288ndash1293 2001

[16] R Ehehalt P Keller C Haass C Thiele and K Simons ldquoAmy-loidogenic processing of the Alzheimer 120573-amyloid precursorprotein depends on lipid raftsrdquo Journal of Cell Biology vol 160no 1 pp 113ndash123 2003

[17] C Hattori M Asai H Onishi et al ldquoBACE1 interacts with lipidraft proteinsrdquo Journal of Neuroscience Research vol 84 no 4pp 912ndash917 2006

[18] C Marquer V Devauges J C Cossec et al ldquoLocal cholesterolincrease triggers amyloid precursor protein-bace1 clustering in


lipid rafts and rapid endocytosisrdquo FASEB Journal vol 25 no 4pp 1295ndash1305 2011

[19] J Abad-Rodriguez M D Ledesma K Craessaerts et al ldquoNeu-ronal membrane cholesterol loss enhances amyloid peptidegenerationrdquo Journal of Cell Biology vol 167 no 5 pp 953ndash9602004

[20] S McLaughlin J Wang A Gambhir and D Murray ldquoPIP2and proteins interactions organization and information flowrdquoAnnual Review of Biophysics and Biomolecular Structure vol 31pp 151ndash175 2002

[21] G Di Paolo and P De Camilli ldquoPhosphoinositides in cellregulation and membrane dynamicsrdquoNature vol 443 no 7112pp 651ndash657 2006

[22] N Landman S Y Jeong S Y Shin et al ldquoPresenilin mutationslinked to familial Alzheimerrsquos disease cause an imbalance inphosphatidylinositol 45-bisphosphate metabolismrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 103 no 51 pp 19524ndash19529 2006

[23] Y S Chun S Shin Y Kim et al ldquoCholesterol modulatesion channels via down-regulation of phosphatidylinositol 45-bisphosphaterdquo Journal of Neurochemistry vol 112 no 5 pp1286ndash1294 2010

[24] A E ChristianM PHaynesM C Phillips andGH RothblatldquoUse of cyclodextrins for manipulating cellular cholesterolcontentrdquo Journal of Lipid Research vol 38 no 11 pp 2264ndash22721997

[25] V G Romanenko G H Rothblat and I Levitan ldquoModulationof endothelial inward-rectifier K+ current by optical isomers ofcholesterolrdquo Biophysical Journal vol 83 no 6 pp 3211ndash32222002

[26] M Toselli G Biella V Taglietti E Cazzaniga and M Par-enti ldquoCaveolin-1 expression and membrane cholesterol contentmodulate N-type calcium channel activity in NG108-15 cellsrdquoBiophysical Journal vol 89 no 4 pp 2443ndash2457 2005

[27] S Ozaki D B DeWald J C Shope J Chen and G DPrestwich ldquoIntracellular delivery of phosphoinositides andinositol phosphates using polyamine carriersrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol97 no 21 pp 11286ndash11291 2000

[28] R Zidovetzki and I Levitan ldquoUse of cyclodextrins to manip-ulate plasma membrane cholesterol content evidence miscon-ceptions and control strategiesrdquo Biochimica et Biophysica Actavol 1768 no 6 pp 1311ndash1324 2007

[29] P A Janmey and U Lindberg ldquoCytoskeletal regulation rich inlipidsrdquo Nature Reviews Molecular Cell Biology vol 5 no 8 pp658ndash666 2004

[30] L J Pike and J M Miller ldquoCholesterol depletion delocal-izes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnoverrdquo Journal of BiologicalChemistry vol 273 no 35 pp 22298ndash22304 1998

[31] E M Hur Y S Park B D Lee et al ldquoSensitization of epidermalgrowth factor-induced signaling by bradykinin is mediated byc-Src implications for a role of lipid microdomainsrdquo Journal ofBiological Chemistry vol 279 no 7 pp 5852ndash5860 2004

[32] K Taguchi H Kumanogoh S Nakamura and S MaekawaldquoLocalization of phospholipase C1205731 on the detergent-resistantmembrane microdomain prepared from the synaptic plasmamembrane fraction of rat brainrdquo Journal of NeuroscienceResearch vol 85 no 6 pp 1364ndash1371 2007

[33] J C Cossec A Simon C Marquer et al ldquoClathrin-dependentAPP endocytosis and A120573 secretion are highly sensitive to the

level of plasmamembrane cholesterolrdquo Biochimica et BiophysicaActa vol 1801 no 8 pp 846ndash852 2010

[34] K Uemura C M Lill X Li et al ldquoAllosteric modulation ofPS1120574-secretase conformation correlates with amyloid 1205734240ratiordquo PLoS ONE vol 4 no 11 Article ID e7893 2009

[35] A Ebke T Luebbers A Fukumori et al ldquoNovel 120574-secretaseenzyme modulators directly target presenilin proteinrdquo Journalof Biological Chemistry vol 286 no 43 pp 37181ndash37186 2011

[36] R G Cutler J Kelly K Storie et al ldquoInvolvement of oxida-tive stress-induced abnormalities in ceramide and cholesterolmetabolism in brain aging andAlzheimerrsquos diseaserdquoProceedingsof the National Academy of Sciences of the United States ofAmerica vol 101 no 7 pp 2070ndash2075 2004

[37] H Xiong D Callaghan A Jones et al ldquoCholesterol retentionin Alzheimerrsquos brain is responsible for high 120573- and 120574-secretaseactivities and A120573 productionrdquo Neurobiology of Disease vol 29no 3 pp 422ndash437 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


rafts is decreased producing less A120573 [15ndash17] In contrastincreasing cholesterol induces the co-clustering of APP andBACE1 producing more A120573 [18] From these results it couldbe hypothesized that high cholesterol levels may be respon-sible for initiating the pathogenesis of AD However it wasrecently demonstrated that lowering cholesterol levels resultsin increased 120573-amyloid production in neurons [19] Abad-Rodriguez et al reported that lowering the intracellular levelsof cholesterol could increase the rate of amyloidogenic pro-cessing of APP by placing the hydrolyzing enzyme (BACE1)and APP in close proximity within the same intracellularcompartments Therefore more experiments will be neededto clarify the conflicting results about the role of cholesterolin pathogenesis of AD

PIP2is known as one of phospholipid component of cell

membrane [20] playing important regulatory roles in a vari-ety of cell functions such as rearrangement of the cytoskele-ton and membrane trafficking [21] We have reported thatFAD-linked PS mutants down-regulate PIP

2levels and that

PIP2levels are inversely correlated to the production of

A12057342 [22] We also demonstrated that increased membranecholesterol level decreases the level of PIP

2via the activation

of PLC [23] Therefore there exists a crosstalk betweentwo plasma membrane-enriched lipids cholesterol and PIP

2

Considering the close relationship between PIP2levels and

the production of A12057342 we suspected that increased mem-brane cholesterol levels affect the A12057342 production viadown-regulating PIP

2levels In this study we found that

membrane cholesterol decreased PIP2levels and increased

secretedA12057342 Supplying PIP2by using a PIP

2-carrier system

blocked the effect of cholesterol which might indicate thatthe effect of cholesterol on A12057342 was by downregulation ofPIP2levels Enriching membrane with cholesterol increased

the expression of some PLC isoforms such as PLC1205731 andPLC1205733 Blocking the new protein synthesis prevented theeffect of cholesterol on PIP

2levels as well as on A12057342

production We found that the expression of PLC1205731 wasspecifically linked to A12057342 production These results suggestthat increased membrane cholesterol levels and FAD-linkedPS mutations may share the same molecular mechanismthat is the downregulation of PIP

2 which may serve as the

molecule linking cholesterol metabolism to the pathogenesisof AD

2 Materials and Methods

21 Cell Culture HeLa cells stably transfected with APP751

carrying the Swedish mutation (APPsw) were cultured at37∘C 5 CO

2 in Dulbeccorsquos Modified Eagle Medium

(DMEM) supplemented with 10 heat-inactivated fetalbovine serum containing 100 unitsmL penicillin 100 120583gmLstreptomycin 260120583gmL Zeocin and 400 120583gmL G418Human neuroblastoma SH-SY5Y cells were cultured inDMEM containing 10 heat-inactivated fetal bovine serum100 unitsmL penicillin and 100120583gmL streptomycin

22 Procedure To enrich the cells with cholesterol cells wereexposed to DMEM culture medium containing methyl-120573-cyclodextrin (M120573CD SigmaUSA) saturatedwith cholesterol

(water-soluble cholesterol) During the incubation cells weremaintained in a humidified CO

2incubator at 37∘C In some

experiments to avoid the use of M120573CD cells were incubatedwith cholesterol which was solubilized by sonication For thispurpose cholesterol in methanolchloroform mixture (1 1vv) was dried under nitrogen gas and sonicated for 2min inphosphate-buffered saline before use

In some experiments cells were pretreated with 10 120583MActinomycin-D (Sigma) or 50120583gmL cyclohexamide (Sigma)for 05 h before 75120583M water-soluble cholesterol was addedPIP2were delivered into the cells using the PIP

2-carrier sys-

tem (Echelon Bioscience Inc USA) Carrier-PIP2complex

was incubated with APP-transfected HeLa cells for 4 h in theabsence or presence of 75120583Mwater-soluble cholesterol

23 Antisense Oligonucleotides Treatments The antisenseoligonucleotides (IDT USA) targeted at PLC1205731 and PLC1205733were designed to be complementary to the 51015840 sequencesand were phosphorothionated at all positions to mini-mize intracellular cleavage by enzymes and to enhancetheir stability (51015840-actccgggttgagccccggc-31015840 for PLC1205731 and51015840-tccaactgcagcgcgtggac-31015840 for PLC1205733) Antisense oligonu-cleotide (51015840-gccccgtatgaccgcgccgg-31015840) having no target wasused as a control in all of experiments The APP-transfectedHeLa cells were plated at a density of 2 times 106 cells per 60mmdish and incubated overnight and then treatedwith the 10 120583Mantisense oligonucleotides for 4 h in DMEM culture mediumwithout serum After treatment the mediumwas replaced bya new medium containing 10 120583M antisense oligonucleotideswith or without water-soluble cholesterol for 2 h Media werecollected tomeasure levels ofA120573 and cellswere homogenizedto confirm PLC expression levels and PIP

2levels

24 Cholesterol Assay Filipin staining of cells (005 DMSO1) was performed for 1 h at room temperature after choles-terol enrichment to confirm the changes of free cholesterollevels at the plasma membrane Fluorescence images wereobtained using a LSM 710 confocal microscope (Zeiss) usinglaser emitting at 351 nm Images were quantified to obtainthe mean fluorescence density values of plasma membranefrom the edge of the cell to 500 nm inside using the ImageJprogram

25 sAPP120572 sAPP120573 and A120573 Peptide Assay Levels of A120573peptides were assayed by using the Invitrogen A120573 ELISAkits (USA) or Wako 120573-amyloid ELISA kits (high-sensitiveJapan) For sAPP120572 sAPP120573 detection samples were analyzedby ELISA kit from IBL (USA) APP-transfected HeLa cellsat 80 confluence in a 35mm dish were cultured for 8 hwith water-soluble cholesterol in DMEM culture mediumwithout serum Control cells were treated similarly andincubated with serum-free DMEM solution without anycholesterol After exposure to cholesterol supernatants werecollected tomeasure levels of A120573 sAPP120572 or sAPP120573 To detectA120573 from SH-SY5Y cells supernatants were desalted usingPD-10 desalting column (GE Healthcare USA) dried andreconstituted in water The samples were analyzed by ELISAkits according to the supplierrsquos instructions




28 PIP2



2






3 Results





2level [23] To val-


2


2levels in 75 and










CholesterolControl

(a)

50

100

150

200

250

0

Chol

este

rol (

o

f con

trol

)

lowastlowastlowast



(b)

20

40

60

80

100

120

0



PIP 2

( o

f con

trol)

(c)

Time (h)

20

40

60

80

100

120

0

lowastlowastlowast

PIP 2

( o

f con

trol)

0 05 15 5

(d)

40

80

120

lowastlowast

0

160

( o

f con

trol)

0 15 75 150

A 40A 42

(e)

0

50

100

150

200

lowast


( o

f con

trol)

0 15 75 150

A 40A 42

(f)






2ELISA kit




FL-APP

CholesterolControl

120573-Actin

(a)

0

40

80

120

160

sAPPsAPP


120572

120573(

of c

ontro

l)

(b)

0 2

20

40

60

80

100

120

140

05

A42

(of

cont

rol)

CholesterolControl


120573

lowastlowastlowast

(c)

20

40

60

80

100

120

140

0

A40

(of

cont

rol)


CholesterolControl

120573

120583

(d)

















2for the effect of




















2complex










2 As we








A12057342 levels


(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















28 PIP2



2






3 Results





2level [23] To val-


2


2levels in 75 and










CholesterolControl

(a)

50

100

150

200

250

0

Chol

este

rol (

o

f con

trol

)

lowastlowastlowast



(b)

20

40

60

80

100

120

0



PIP 2

( o

f con

trol)

(c)

Time (h)

20

40

60

80

100

120

0

lowastlowastlowast

PIP 2

( o

f con

trol)

0 05 15 5

(d)

40

80

120

lowastlowast

0

160

( o

f con

trol)

0 15 75 150

A 40A 42

(e)

0

50

100

150

200

lowast


( o

f con

trol)

0 15 75 150

A 40A 42

(f)






2ELISA kit




FL-APP

CholesterolControl

120573-Actin

(a)

0

40

80

120

160

sAPPsAPP


120572

120573(

of c

ontro

l)

(b)

0 2

20

40

60

80

100

120

140

05

A42

(of

cont

rol)

CholesterolControl


120573

lowastlowastlowast

(c)

20

40

60

80

100

120

140

0

A40

(of

cont

rol)


CholesterolControl

120573

120583

(d)

















2for the effect of




















2complex










2 As we








A12057342 levels


(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CholesterolControl

(a)

50

100

150

200

250

0

Chol

este

rol (

o

f con

trol

)

lowastlowastlowast



(b)

20

40

60

80

100

120

0



PIP 2

( o

f con

trol)

(c)

Time (h)

20

40

60

80

100

120

0

lowastlowastlowast

PIP 2

( o

f con

trol)

0 05 15 5

(d)

40

80

120

lowastlowast

0

160

( o

f con

trol)

0 15 75 150

A 40A 42

(e)

0

50

100

150

200

lowast


( o

f con

trol)

0 15 75 150

A 40A 42

(f)






2ELISA kit




FL-APP

CholesterolControl

120573-Actin

(a)

0

40

80

120

160

sAPPsAPP


120572

120573(

of c

ontro

l)

(b)

0 2

20

40

60

80

100

120

140

05

A42

(of

cont

rol)

CholesterolControl


120573

lowastlowastlowast

(c)

20

40

60

80

100

120

140

0

A40

(of

cont

rol)


CholesterolControl

120573

120583

(d)

















2for the effect of




















2complex










2 As we








A12057342 levels


(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FL-APP

CholesterolControl

120573-Actin

(a)

0

40

80

120

160

sAPPsAPP


120572

120573(

of c

ontro

l)

(b)

0 2

20

40

60

80

100

120

140

05

A42

(of

cont

rol)

CholesterolControl


120573

lowastlowastlowast

(c)

20

40

60

80

100

120

140

0

A40

(of

cont

rol)


CholesterolControl

120573

120583

(d)

















2for the effect of




















2complex










2 As we








A12057342 levels


(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2for the effect of




















2complex










2 As we








A12057342 levels


(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

0 51505

Rela

tive d

ensit

y

15

1

0

05

2

25

CytosolMembrane

Time (h)

PLC 1


120573

(b)

Rela

tive d

ensit

y

15

1

0

05

2

25

0 51505

CytosolMembrane

Time (h)

PLC1205733

lowast

(c)










(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)


100

80

60

40

20

0

100

80

60

40

20

0

lowastlowast

minusAct-D +Act-D

PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

(b)


100

120

80

60

40

20

0

100

120

80

60

40

20

0


A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

(c)


2levels APP-




2levels (119899 = 6) (c)




2






(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)



2levels via


4 Discussion






2levels Thus





2 In






2levels







(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

100

80

60

100

80


PIP 2

( o

f con

trol)

PIP 2

( o

f con

trol)

lowastlowast


(b)

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0Cholesterol

lowastlowast

A120573

42 (

of c

ontro

l)

A120573

42 (

of c

ontro

l)

minus minus

minus minus

+

+

+

+Anti-1205731

(c)

Cholesterol

20

40

60

80

100

120

140

0

A42

(of

cont

rol)

20

40

60

80

100

120

140

0A

42(

ofco

ntro

l)


120573120573

minusminus

minus minus

+

+ +

+

Anti-1205733

(d)








2 it is possible












2is shown to



2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2induces changes




2and











Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Increasing Membrane Cholesterol Level Increases the ...

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