ad is a progressive and irreversible neurodegenerative disorder leading to cognitive , memory , and behavioral impairments . cerebral elevation and accumulation of a are necessary steps in the pathogenesis of ad [ 13 ] . sequential proteolytic cleavages of amyloid precursor protein ( app ) by membrane - bound -secretase and -secretase produce two major isoforms of a , a40 , and a42 . alternatively , app can be sequentially processed by -secretase , and -secretase , precluding a production ( nonamyloidogenic pathway ) . even though advanced age serves as a major risk factor , approximately 5% of ad cases are familial ( fad ) , and some of them are attributable to autosomal dominant mutations in presenilin ( ps ) genes , ps1 and ps2 . ps1 and ps2 function as catalytic subunits of -secretase , and fad mutations in pss affect app processing increasing the ratio of a42 to a40 [ 68 ] . growing evidence indicates that dysregulation of lipid pathways have regulatory consequences for app processing and a generation . especially , cholesterol has been suggested to participate in the etiology of ad by increasing the generation of a . cholesterol can directly regulate the activities of -secretase or -secretase to alter amyloidogenesis [ 1113 ] . alternatively , changes in cholesterol level may affect the lipid environment for app processing and a generation . app is located either within or outside of lipid rafts . since bace1 ( -secretase ) is predominantly located in lipid rafts , app processing occurring within lipid rafts is amyloidogenic , whereas app processing occurring outside lipid rafts is considered nonamyloidogenic . when cholesterol is depleted , the association of bace1 with lipid rafts is decreased , producing less a [ 1517 ] . in contrast , increasing cholesterol induces the co - clustering of app and bace1 , producing more a . from these results it could be hypothesized that high cholesterol levels may be responsible for initiating the pathogenesis of ad . however , it was recently demonstrated that lowering cholesterol levels results in increased -amyloid production in neurons . abad - rodriguez et al . reported that lowering the intracellular levels of cholesterol could increase the rate of amyloidogenic processing of app by placing the hydrolyzing enzyme ( bace1 ) and app in close proximity within the same intracellular compartments . therefore , more experiments will be needed to clarify the conflicting results about the role of cholesterol in pathogenesis of ad . pip2 is known as one of phospholipid component of cell membrane , playing important regulatory roles in a variety of cell functions , such as rearrangement of the cytoskeleton and membrane trafficking . we have reported that fad - linked ps mutants down - regulate pip2 levels , and that pip2 levels are inversely correlated to the production of a42 . we also demonstrated that increased membrane cholesterol level decreases the level of pip2 via the activation of plc . therefore , there exists a crosstalk between two plasma membrane - enriched lipids , cholesterol and pip2 . considering the close relationship between pip2 levels and the production of a42 , we suspected that increased membrane cholesterol levels affect the a42 production via down - regulating pip2 levels . in this study , we found that membrane cholesterol decreased pip2 levels and increased secreted a42 . supplying pip2 by using a pip2-carrier system blocked the effect of cholesterol , which might indicate that the effect of cholesterol on a42 was by downregulation of pip2 levels . enriching membrane with cholesterol increased the expression of some plc isoforms , such as plc1 and plc3 . blocking the new protein synthesis prevented the effect of cholesterol on pip2 levels as well as on a42 production . these results suggest that increased membrane cholesterol levels , and fad - linked ps mutations may share the same molecular mechanism , that is , the downregulation of pip2 , which may serve as the molecule linking cholesterol metabolism to the pathogenesis of ad . hela cells stably transfected with app751 carrying the swedish mutation ( appsw ) were cultured at 37c , 5% co2 , in dulbecco 's modified eagle medium ( dmem ) supplemented with 10% heat - inactivated fetal bovine serum containing 100  units / ml penicillin , 100  g / ml streptomycin , 260  g / ml zeocin , and 400  g / ml g418 . human neuroblastoma sh - sy5y cells were cultured in dmem containing 10% heat - inactivated fetal bovine serum , 100  units / ml penicillin , and 100  g / ml streptomycin . to enrich the cells with cholesterol , cells were exposed to dmem culture medium containing methyl--cyclodextrin ( mcd , sigma , usa ) saturated with cholesterol ( water - soluble cholesterol ) . during the incubation , cells were maintained in a humidified co2 incubator at 37c . in some experiments , to avoid the use of mcd , cells were incubated with cholesterol which was solubilized by sonication . for this purpose , cholesterol in methanol / chloroform mixture ( 1  :  1 v / v ) was dried under nitrogen gas and sonicated for 2  min in phosphate - buffered saline before use . in some experiments , cells were pretreated with 10  m actinomycin - d ( sigma ) or 50  g / ml cyclohexamide ( sigma ) for 0.5  h before 75  m water - soluble cholesterol was added . carrier - pip2 complex was incubated with app - transfected hela cells for 4  h in the absence or presence of 75  m water - soluble cholesterol . the antisense oligonucleotides ( idt , usa ) targeted at plc1 and plc3 were designed to be complementary to the 5 sequences and were phosphorothionated at all positions to minimize intracellular cleavage by enzymes and to enhance their stability ( 5-actccgggttgagccccggc-3 for plc1 and 5-tccaactgcagcgcgtggac-3 for plc3 ) . antisense oligonucleotide ( 5-gccccgtatgaccgcgccgg-3 ) having no target was used as a control in all of experiments . the app - transfected hela cells were plated at a density of 2  10 cells per 60  mm dish and incubated overnight and then treated with the 10  m antisense oligonucleotides for 4  h in dmem culture medium without serum . after treatment , the medium was replaced by a new medium containing 10  m antisense oligonucleotides with or without water - soluble cholesterol for 2  h. media were collected to measure levels of a , and cells were homogenized to confirm plc expression levels and pip2 levels . filipin staining of cells ( 0.05% , dmso 1% ) was performed for 1  h at room temperature after cholesterol enrichment to confirm the changes of free cholesterol levels at the plasma membrane . fluorescence images were obtained using a lsm 710 confocal microscope ( zeiss ) using laser emitting at 351  nm . images were quantified to obtain the mean fluorescence density values of plasma membrane from the edge of the cell to 500  nm inside using the imagej program . a elisa kits ( usa ) or wako -amyloid elisa kits ( high - sensitive ; japan ) . for sapp , sapp detection , app - transfected hela cells at 80% confluence in a 35  mm dish were cultured for 8  h with water - soluble cholesterol in dmem culture medium without serum . control cells were treated similarly and incubated with serum - free dmem solution without any cholesterol . after exposure to cholesterol , supernatants were collected to measure levels of a , sapp , or sapp. to detect a from sh - sy5y cells , supernatants were desalted using pd-10 desalting column ( ge healthcare , usa ) , dried , and reconstituted in water . the samples were then centrifuged at 1,000  g for 10  min at 4c to remove nuclei and debris . supernatants were separated by centrifugation at 100,000  g for 1  h at 4c into membrane ( pellet ) and cytosol ( supernatant ) fractions . whole cell lysates were prepared by homogenizing with lysis buffer ( 10  mm tris - hcl , 150  mm nacl , 1% triton x-100 , 0.25% nonidet p-40 , 2  mm edta , ph7.4 ) using a cell scraper . the lysed cells were centrifuged at 12,000  g for 10  min at 4c . the protein in the supernatant was determined by bradford assay ( bio - rad , usa ) . membranes were blocked with 5% nonfat milk powder in tris - buffered saline / tween 20 ( tbst ) for 1  h at room temperature , then incubated with rabbit polyclonal anti - plc1 ( sc-9050 ) , plc2 ( sc-206 ) , plc3 ( sc-13958 ) , plc4 ( sc-20760 ) , plc2 ( sc-9015 ) , mouse monoclonal anti - plc1 ( sc-7290 ) antibodies ( santa cruz biotechnology , usa ) , anti - app antibody ( ln27 , zymed ) , anti -actin ( a5441 , sigma ) , and rabbit anti -tubulin ( t2200 , sigma ) for overnight at 4c . dilutions were 1  :  500 for plc isozymes and 1  :  4000 for -tubulin , -actin , and app . after washing , membranes were incubated for 1  h at room temperature with horseradish peroxidase - conjugated goat anti - rabbit igg or goat anti - mouse igg antibodies ( 1  :  2000 dilution ; zymed , usa ) and washed . blots were quantified with the multi gauge software using a las-3000 system ( fugifilm , japan ) . the amount of pip2 extracted from app - transfected hela cells were measured by using pip2 mass elisa kit ( echelon biosciences inc . pip2 was extracted from the control cells or cells treated with water - soluble cholesterol according to the supplier 's instructions . cellular pip2 quantities were estimated by comparing the values from the standard curve , which showed linear relationship at the range from 0.5 to 1000  pm concentrations . statistical comparisons between the controls and treated experimental groups were performed using the student 's t - test . mcd , a water - soluble cyclic oligosaccharide , has hydrophobic cavity that is able to encapsulate insoluble compounds , thus enhances the solubility of cholesterol . mcd saturated with cholesterol ( water - soluble cholesterol ) has been used to increase membrane cholesterol level , since it acts as a cholesterol donor [ 2426 ] . we incubated app - transfected hela cells with 15 , 75 , or 150  m water - soluble cholesterol for 8  h , and filipin staining was performed for 1  h at room temperature to monitor the membrane cholesterol level . typical confocal image in figure 1(a ) shows that the membrane cholesterol level increased by 75  m water - soluble cholesterol . the changes of cholesterol level were confirmed by quantifying the filipin fluorescent intensities from plasma membranes . by incubating cells with 15 and 75  m water - soluble cholesterol , the fluorescent intensities were increased by 58.5  5.8% and 83.3  15.9% we also tested the time - dependent accumulation of cholesterol in the membrane by incubating cells with 75  m water - soluble cholesterol . cholesterol levels increased after 0.5  h , and it steadily increased further after 1.5  h or 5  h ( supplementary figures  1(a ) and 1(b ) , see supplementary material available online at http://dx.doi.org/10.1155/2013/407903 ) . from these results we concluded that the direct administration of the water - soluble cholesterol leads to the increases in the membrane cholesterol levels . recently , we have reported that augmentation of membrane cholesterol levels downregulates pip2 level . to validate this observation in the current system , app - transfected hela cells were incubated with 15 , 75 , or 150  m water - soluble cholesterol for 8  h , and the steady state levels of pip2 were measured using a pip2 elisa . pip2 levels in 75 and 150  m cholesterol - treated cells were downregulated by 23.2  5.0% and 26.1  2.5% ( n = 6 ) , respectively ( figure 1(c ) ) . we also tested the time - dependent effect of increased membrane cholesterol on the levels of pip2 by incubating cells with 75  m water - soluble cholesterol . as shown in figure 1(d ) , the steady state levels of pip2 after 1.5  h and 5  h incubation time were downregulated by 20.3  5.1% and 26.3  5.3% ( n = 6 ) , respectively . since we have reported that cellular pip2 levels are closely correlated with the a42 levels , we tested the effect of increased membrane cholesterol levels on secreted a. for this purpose , app - transfected hela cells were incubated for 8  h with 15 , 75 , or 150  m water - soluble cholesterol , and a levels were measured from the conditioned media by using an elisa kits specific for a40 or a42 . the secreted a40 levels were not changed by increased membrane cholesterol levels ( open bars in figure 1(e ) ) . however , a42 levels were increased by 28.1  8.4% , and 36.2  8.1% ( n = 6 ) when cells were incubated with 75 , and 150  m water - soluble cholesterol , respectively ( closed bars in figure 1(e ) ) . we also tested the effect of increased membrane cholesterol levels on the levels of secreted a from neuroblastoma sh - sy5y cells . the level of endogenous a42 increased significantly ( closed bars in figure 1(f ) ) , while the endogenous a40 level was not changed ( open bars in figure 1(f ) ) . thus , these results suggest that the effect of cholesterol enrichment is specific to a42 levels , and is not cell - type specific . alternatively , app can be cleaved sequentially by -secretase followed by -secretase precluding the production of a. thus , the effect of increased membrane cholesterol levels on a levels can occur in any of those processes . to begin to investigate the effects of membrane cholesterol on app processing increased membrane cholesterol levels led to a moderate increase in the full - length app levels ( figure 2(a ) ) . then , we tested the effects of increased membrane cholesterol levels on the activities of -secretase and -secretase . for this purpose , we measured the levels of sapp and sapp from the conditioned media using specific elisa kits , since they are produced via the activities of -secretase and -secretase , respectively . in this experiment , we used sapp elisa kit for swedish mutant . as shown in figure 2(b ) , the levels of both sapp and sapp were also slightly increased by increased membrane cholesterol levels , which might be due to the increased level of their precursor , app . however , the amount of increased sapp level was not robust to explain the a42-selective changes associated with increased membrane cholesterol levels . since membrane cholesterol levels affect a42 but not a40 , it is conceivable that the effects of cholesterol may influence the specificity of -secretase - mediated cleavage of amyloidogenic app c - terminal fragments ( e.g. , c99 ) . in order to elucidate the role of pip2 for the effect of increased membrane cholesterol levels on secreted a42 , we used a pip2-carrier system for the intracellular delivery of pip2 . because carrier compounds are  charge - neutralization  species after the carriers were added at a one - to - one molar ratio with pip2 at room temperature , the complex was diluted to the desired final concentration . then , the carrier - pip2 complex was incubated with cells for 4  h before a levels were measured from the conditioned media . the presence of 2  m and 5  m carrier - pip2 complex decreased secreted a42 levels by 12.8  12.1% and 41.5  4.1% ( n = 6 ) , respectively ( open bars in figure 2(c ) ) . this result is consistent with our previous result showing the close correlation between pip2 levels and a42 production . in the absence of carrier - pip2 complex , incubating cells with 75  m water - soluble cholesterol for 4  h increased a42 levels by 17.7  2.9% ( n = 6 ) , which is consistent with the result in figure 1(e ) . however , the presence of either 2  m or 5  m carrier - pip2 complex completely prevented the effect of water - soluble cholesterol on a42 levels ( closed bars in figure 2(c ) ) . these results suggest that the relative levels of cholesterol and pip2 correlate closely with secreted a42 levels in a positive or negative manner , respectively . unlike a42 , the a40 levels were not affected by the presence of carrier - pip2 complex ( open bars in figure 2(d ) ) . also , the a40 levels were not affected by increased membrane cholesterol levels in the presence of carrier - pip2 complex ( closed bars in figure 2(d ) ) , which was consistent with the specific effect of cholesterol on a42 level . since we suspected that the effect of increased membrane cholesterol levels on the secreted a42 levels is due to the downregulation of pip2 , we tested whether increased membrane cholesterol levels affect the activity of plc . we first examined the expression levels of plc isoforms by monitoring them using western blot analysis from cytosol and membrane fractions in app - transfected hela cells . the expression levels of plc1 were significantly increased by cholesterol only in membrane fractions as shown in figure 3(a ) . densitometry analysis of the bands corresponding to plc1 clearly supports this conclusion ( figure 3(b ) ; n = 5 ) . after 0.5  h incubation time with 75  m water - soluble cholesterol , expression levels of plc1 were increased by two folds in membrane fractions . the effect of cholesterol on plc1 expression lasted as long as 5  h. the expression level of plc3 was also increased in membrane fractions by cholesterol ( figures 3(a ) and 3(c ) ) . however , the use of mcd may cause nonspecific effects in addition to enrichment of membrane cholesterol levels . to avoid the use of mcd , free cholesterol when cells were incubated with 75  m solubilized cholesterol for 1  h , the expression of plc1 in the membrane fraction was increased ( supplementary figure  2 ) . at 1  h incubation time , the expression of plc3 was not changed . these results indicated that the effect of cholesterol on plc1 expression was not due to the nonspecific effect of mcd . cholesterol - induced increases in the levels of plc1 and plc3 were observed almost exclusively in the membrane fraction . also , increased membrane cholesterol levels did not change the expression levels of plc2 as shown in figure 3(a ) from a typical experiment . the expression levels of other plc isoforms ( plc2 , plc4 , and plc1 ) were not changed either ( supplementary figure  3(a ) ) . we also observed specific increase of plc1 and plc3 expressions from sh - sy5y cells by enriching membrane cholesterol levels ( supplementary figure  3(b ) ) .   to determine whether the effect of cholesterol on plc expression was due to increased transcription , cells were preincubated for 10  min with the transcription inhibitor , actinomycin - d ( act - d , 10  m ) , or with the translation inhibitor , cyclohexamide ( chx , 50  g / ml ) . then , cells were incubated further for 0.5  h with 75  m water - soluble cholesterol . representative western blots for plc1 and plc3 from membrane fractions are shown in figure 4(a ) . the effect of cholesterol on plc1 and plc3 expressions was completely prevented either by act - d or by chx , indicating that the effect of cholesterol on plc1 and plc3 expression was via the up - regulation of transcription . then , we tested the effect of act - d on the steady state level of pip2 . as we expected , pip2 levels were downregulated by 18.9  0.3% ( n = 6 ) when cells were treated with 75  m water - soluble cholesterol for 1  h in the absence of act - d ( figure 4(b ) ) . however , the effect of cholesterol on pip2 levels was completely prevented when cells were pretreated with act - d ( n = 6 ) . these results confirmed that increased membrane cholesterol levels increases the expression of plc1 and plc3 , leading to the downregulation of pip2 levels . next , we tested the effect of act - d on the level of secreted a42 . for this purpose , cells were incubated for 4  h with 75  m water - soluble cholesterol with or without act - d . as expected , cholesterol increased the levels of secreted a42 by 20.1  2.8% ( n = 4 ) in the absence of act - d ( figure 4(c ) ) . however , the presence of act - d completely prevented the effect of increased membrane cholesterol levels on a42 ( n = 4 ) . these results suggest that the increase of plc transcription by cholesterol induced the downregulation of pip2 levels , which increased secreted a42 levels . we tested whether plc1 or plc3 is required for the observed effects of cholesterol on a42 . to avoid any further transfection , cells were pretreated with 10  m antisense oligonucleotides against plc isoforms for 4  h. then the media were replaced by fresh media containing the same antisense oligonucleotides with or without 75  m water - soluble cholesterol , followed by additional incubation for 2  h. antisense oligonucleotides having no specific target were used for controls in all of experiments . a typical western blot for plc1 is shown in figure 5(a ) , and the band densities are expressed relative to -tubulin density in figure 5(c ) ( n = 6 ) . however , the presence of antisense oligonucleotides against plc1 ( anti 1 ) blocked the effect of cholesterol . similarly , an increase of plc3 expression by cholesterol was blocked by antisense oligonucleotides against plc3 ( anti 3 ; figures 5(b ) and 5(d ) ) . these results demonstrated that these antisense oligonucleotides were very effective to block the effect of increased membrane cholesterol levels on the expression of plc1 and plc3 . then , we tested the effect of these antisense oligonucleotides on the pip2 levels . in the presence of control antisense oligonucleotides ( anti 1 ) , cholesterol decreased pip2 levels by 12.1    2.8% ( figure 6(a ) ; n = 6 ) . in the presence of antisense oligonucleotide against plc1 ( + anti 1 ) , cholesterol decreased pip2 levels only by 2.5    1.0% ( n = 6 ) . in contrast , cholesterol decreased pip2 levels by 10    2.8% ( n = 6 ) even in the presence of antisense oligonucleotide against plc3 ( + anti 3 ; figure 6(b ) ) . thus , inhibiting plc1 expression , but not inhibiting plc3 expression , prevented the effect of increased membrane cholesterol levels on pip2 levels . these results may suggest that the downregulation of pip2 levels by cholesterol enrichment is specifically via the increased expression of plc1 . next , we tested the effect of increased membrane cholesterol levels on the levels of secreted a42 in the presence of these antisense oligonucleotides . as we expected , cholesterol increased the levels of secreted a42 by 25.7  5.5% ( n = 8) in the presence of control antisense oligonucleotides ( figure 6(c ) ) . in the presence of antisense oligonucleotide against plc1 , cholesterol increased the secreted a42 level only by 2.0  2.1% ( n = 8) . thus , plc1 antisense oligonucleotide almost completely prevented the effect of cholesterol on the secreted a42 levels . in contrast , the presence of plc3 antisense oligonucleotides failed to prevent the effect of cholesterol . the a42 production was still increased by 16.7    3.4% ( figure 6(d ) ; n = 6 ) . together , these results strongly suggest that increased membrane cholesterol levels increased the levels of secreted a42 by down - regulating pip2 levels via specific enhancement of plc1 expression . we have previously reported that fad - linked ps mutants down - regulate pip2 levels , which are closely related to increased a42 level . in addition , up , or downregulation of pip2 levels by pharmacological means decreases or increases the production of a42 , respectively . in this study , we showed that cholesterol enrichment increases the secreted a42 levels by down - regulating pip2 levels . thus , there exists a close relationship between pip2 levels and a42 levels , consistent with our previous results . recently , we demonstrated that enrichment of cholesterol decreases the levels of pip2 via the activation plc . therefore , it was suggested that there exists a crosstalk between two plasma membrane - enriched lipids , cholesterol , and pip2 . in this paper , we confirmed that cholesterol decreases the level of pip2 via the activation plc . in addition , we showed that cholesterol specifically increases the expression levels of plc1 and plc3 . consistent with this conclusion , inhibiting transcription prevented the effects of cholesterol not only on pip2 levels but also on the production of a42 . also , the inhibition of plc1 expression , but not that of plc3 , prevented the effects of cholesterol , indicating a close link between plc1 and regulation of pip2 levels . although pip2 is a minor component in the plasma membrane , it plays important regulatory roles in a variety of cell functions , such as rearrangement of the cytoskeleton and membrane trafficking . the concept of spatially confined pip2 pools was proposed to explain the multiple roles of pip2 . cholesterol- and sphingolipid - rich rafts may serve to confine pip2 within the plasma membrane , allowing pip2 hydrolysis to occur locally and restrict signaling mechanisms to the site of activation [ 30 , 31 ] . in addition to this confined regulation of pip2 , it is possible that the steady - state level of pip2 is dynamically determined by the concerted action of phosphoinositide kinases and phosphatases . in this study , we showed another way of regulating pip2 levels within specific microdomains : cholesterol content in a specific microdomain may regulate pip2 levels via plc activity . interestingly , plc1 is shown to localize in detergent - resistant membrane microdomains prepared from the synaptic plasma membrane fraction of rat brain . for this reason , increase of plc1 expression by cholesterol enrichment may directly induce downregulation of pip2 in the confined microdomain . it was shown that cholesterol increases clathrin - dependent app endocytosis , and that it is likely the direct cause of the increased a42 secretion . since pip2 is a key regulator for the rearrangement of the cytoskeleton and membrane trafficking , it is possible that the downregulation of pip2 may be the underlying mechanism for the increased clathrin - dependent app endocytosis by cholesterol enrichment . alternatively , the downregulation of pip2 may directly activate -secretase since pip2 is shown to inhibit -secretase activity by suppressing its association with the substrate . it is also possible that pip2 induces changes in -secretase conformation to alter the generation of a42 . recent reports show that the changes in ps1 conformation by various manipulations of ps1 itself , pen2 , aph1 , app , and pharmacological agents known as -secretase modulators can allosterically modify -secretase catalytic specificity , leading to increase of a42/a40 ratio [ 34 , 35 ] . further studies will be needed to clarify the role of pip2 in the production of a.   the physiological relevance of our finding is not clear since a large increase of cholesterol in the membrane was required to induce meaningful downregulation of pip2 and up - regulation of a42 in hela cells ( figures 1(c ) and 1(e ) ) . in sh - sy5y cells , however , a42 level was significantly increased even by 15  m water - soluble cholesterol ( figure 1(f ) ) . we also observed a significant increase of plc1 expression and downregulation of pip2 level by 15  m water - soluble cholesterol when we used hek cells ( data not shown ) . thus , it seems that the effective amount of cholesterol to induce changes in pip2 and a42 levels is cell type dependent . however , even the minimal increase of a42 by the mild cholesterol enrichment will have profound cytotoxic effects since the cytotoxicity of a42 is caused by its small molecular aggregates , such as dimer form of a42 , and the produced a42 will accumulate . it has been demonstrated that cholesterol levels in the brains of ad patients are increased [ 36 , 37 ] . the levels of cholesterol increase in the brain even during normal aging . in an ad brain , cholesterol homeostasis is impaired , and cholesterol retention likely enhances a production . these results may suggest that high cholesterol levels in the brain participate in the etiology of ad by increasing the generation of a. however , further work is needed to understand how cholesterol is implicated in ad pathogenesis . in this study , we showed that membrane cholesterol levels may share the same molecular mechanism with fad ps mutations , that is , downregulation of pip2 , for the increased generation of a42 . therefore , pip2 may serve as the molecule linking cholesterol metabolism to the pathogenesis of ad .
cerebral elevation of 42-residue amyloid -peptide ( a42 ) triggers neuronal dysfunction in alzheimer 's disease ( ad ) . 
 even though a number of cholesterol modulating agents have been shown to affect a generation , the role of cholesterol in the pathogenesis of ad is not clear yet . 
 recently , we have shown that increased membrane cholesterol levels downregulates phosphatidylinositol 4,5-bisphosphate ( pip2 ) via activation of phospholipase c ( plc ) . in this study , we tested whether membrane cholesterol levels may affect the a42 production via changing pip2 levels . increasing membrane cholesterol levels decreased pip2 and increased secreted a42 . supplying pip2 , by using a pip2-carrier system , blocked the effect of cholesterol on a42 . 
 we also found that cholesterol increased the expressions of 1 and 3 plc isoforms ( plc1 , plc3 ) . silencing the expression of plc1 prevented the effects of cholesterol on pip2 levels as well as on a42 production , suggesting that increased membrane cholesterol levels increased secreted a42 by downregulating pip2 via enhancing the expression of plc1 . 
 thus , cholesterol metabolism may be linked to a42 levels via plc1 expression and subsequent changes in pip2 metabolism .