Endosomal-lysosomal Cholesterol Sequestration by ...

MCB Accepted Manuscript Posted Online 12 March 2018 Mol. Cell. Biol. doi:10.1128/MCB.00529-17 Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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Endosomal-lysosomal Cholesterol Sequestration by U18666A Differentially Regulates APP Metabolism in Normal and APP Overexpressing Cells

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J. Chunga,b, G. Phukana,c, D. Vergoted, A. Mohamede, M. Maulika$, M. Stahna, R.J. Andrewf, G. Thinakaranf, E. Posse de Chavese and S. Kara,b,d#

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Centre for Prions and Protein Folding Diseases, Departments of bPsychiatry, cMedicine and Pharmacology, dFaculté Saint-Jean, University of Alberta, Edmonton, Alberta, Canada T6G 2M8; fDepartments of Neurobiology, Neurology, and Pathology, The University of Chicago, Chicago, IL 60637, USA. e

Current Address: Department of Biological Sciences, Indian Institute of Science Education and Research - Kolkata (IISER-K), Mohanpur, West Bengal 741246, India. Number of total characters (excluding spaces): 39955 Number of total words for Materials and Methods: 1594 Number of total words for Introduction, Results and Discussion sections: 5636 Number of Figures: 15 Number of Tables: 1

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E-mails: J. Chung - [email protected]; G. Phukan - [email protected]; D. Vergote [email protected]; A. Mohamed - [email protected]; M. Maulik - [email protected]; M. Stahn - [email protected]; R.J. Andrew - [email protected]; G. Thinakaran [email protected]; E. Posse de Chaves - [email protected]; S. Kar [email protected]

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Running title: Endosomal-lysosomal cholesterol and APP processing

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Key words: Alzheimer’s disease, Amyloid precursor protein, β-amyloid, β-secretase, Delipidation, Endosomal-lysosomal system, γ-secretase, Lipid-raft, Address correspondence to: Satyabrata Kar, Ph.D. Centre for Prions and Protein Folding Diseases Departments of Medicine (Neurology) and Psychiatry University of Alberta Edmonton, Alberta, Canada T6G 2M8 Tel. no: (780) 492 9357; Fax no: (780) 492 9352 E-mail: [email protected]

Downloaded from http://mcb.asm.org/ on March 14, 2018 by Univ of Chicago

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ABSTRACT

42 Amyloid β (Aβ) peptide derived from amyloid precursor protein (APP) plays a critical role in the 43 development of Alzheimer’s disease. Current evidence indicates that altered levels/subcellular

45 cholesterol sequestration within the endosomal-lysosomal (EL) system can influence APP 46 metabolism. Thus, we evaluated the effects of U18666A, which triggers cholesterol redistribution 47 within EL system, on mouse N2a cells expressing different levels of APP in the presence or absence 48 of extracellular cholesterol/lipids provided by fetal bovine serum (FBS). Our results reveal that 49 U18666A and FBS differentially increase the levels of APP and its cleaved products α/β/η-C50 terminal fragments in N2a cells expressing normal levels of mouse APP (N2awt) or higher levels of 51 human wild-type APP (APPwt) or “Swedish” mutant APP (APPsw). The cellular levels of Aβ152

40/Aβ1-42 were

markedly increased in U18666A-treated APPwt and APPsw cells. Our studies further

53 demonstrate that APP and its cleaved products are partly accumulated in the lysosomes possibly due 54 to decreased clearance. Finally, we show that autophagy inhibition plays a role in mediating 55 U18666A

effects. Collectively, these results suggest that altered levels/distribution of

56 cholesterol/lipids can differentially regulate APP metabolism depending on the nature of APP 57 expression.

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44 distribution of cholesterol can regulate Aβ production/clearance, but it remains unclear how

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INTRODUCTION

59 Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the presence 60 of tau-positive intracellular neurofibrillary tangles, extracellular β-amyloid (Aβ)-containing neuritic

62 accumulation of Aβ peptides generated from the amyloid precursor protein (APP) contributes to the 63 loss of neurons and development of AD (2). Under normal conditions, APP is proteolytically 64 processed either by non-amyloidogenic α-secretase or amyloidogenic β-secretase pathways (3). The 65 α-secretases such as, tumor necrosis factor-α converting enzyme (ADAM17), ADAM10 or 66 ADAM9, cleave APP within the Aβ domain, yielding soluble APPα (sAPPα) and a 10kD C67 terminal fragment (α-CTF) that can be further processed by γ-secretase to generate Aβ17-40/Aβ17-42 68 fragments. The β-secretase (i.e., β-site APP cleaving enzyme, BACE1), on the other hand, cleaves 69 APP to generate soluble APPβ (sAPPβ) and an Aβ-containing C-terminal fragment (β-CTF), which 70 is subsequently processed by γ-secretase to yield full-length Aβ1-40/Aβ1-42 peptides. Whereas the α71 secretase processing occurs mostly at the plasma membrane and in the late secretory pathway, the 72 endosomal-lysosomal (EL) system plays a critical role in BACE1 cleavage and the production of 73 Aβ peptides (3, 4). The autophagy pathway, which regulates degradation of long-lived proteins, has 74 also been shown to influence APP metabolism (5). More recently, APP has been reported to be 75 processed via another pathway, mediated by membrane-type 5-matrix metalloproteinases (MT576 MMP) called η-secretase, leading to the generation of η-CTF. The η-CTF is subsequently cleaved 77 by ADAM10 or BACE1 to generate Aη-α or Aη-β peptides. There is evidence that Aη-α can inhibit 78 long-term potentiation and the η-CTF fragment is enriched in dystrophic neurites in animal models 79 as well as human AD brains (6-10). Nevertheless, in contrast to the canonical APP processing via

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61 plaques and the loss of neurons in selected regions of the brain (1). Evidence suggests that the

80 the α/β-secretase pathway, very little is known about the cellular regulation of the η-secretase 81 pathway. 82 Cholesterol has long been identified as a factor that modulates APP cleavage and Aβ production 83 (11-15). Studies under in vitro conditions and in animal models indicated that, overall, an increase

85 while a decrease in cholesterol reduces Aβ generation and increases APP cleavage by α-secretase 86 (16-21). However, one study found that cerebral Aβ levels did not change upon a decrease of 87 cholesterol (22). Moreover, in some studies an increase of cholesterol reduced A levels (23, 24), 88 and the decrease of cholesterol elevated Aβ levels (21, 25). Additionally, it has been reported that 89 enhanced levels of intracellular cholesterol can differentially regulate production/secretion of Aβ in 90 cultured neurons and non-neuronal cells (26-31). These paradoxical results could possibly be due to 91 variation in cellular levels/sites of cholesterol accumulation or the type of APP (normal vs mutant) 92 expressed in cells/animals used in different studies. Since EL system acts as a major site of APP 93 processing (3, 4) and exhibits marked changes in vulnerable neurons prior to extracellular Aβ 94 deposition in AD brains (32), it is of relevance to establish how alterations in the levels of 95 cholesterol within the endosomal/lysosomal compartments can influence APP metabolism. 96 97 Cells acquire cholesterol by de novo synthesis in the endoplasmic reticulum (ER), and by uptake 98 from an extracellular source (lipoproteins). Cholesterol esters present in lipoproteins are delivered 99 to the EL system where they are hydrolysed by lysosomal acid lipase resulting in the release of free 100 cholesterol. Subsequently, cholesterol exits the EL system via Niemann-Pick type C (NPC) 1 and 2 101 protein-dependent mechanisms and is distributed to other cellular compartments including the ER 102 and plasma membrane (33, 34). Trafficking of extracellular cholesterol back to the ER is crucial to

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84 in cellular cholesterol induces changes that favour APP amyloidogenic cleavage and Aβ production,

103 switch off the SREBP pathway, a main regulator of cholesterol homeostasis (35). The lack of 104 NPC1/2 protein or exposure to class-II amphiphile U18666A impairs trafficking of cholesterol 105 leading to its accumulation within the EL system (14, 36-40), thus providing a suitable paradigm to 106 evaluate its effects on APP metabolism. In the present study, we have demonstrated that U18666A

108 Aβ peptides depending on the levels and the nature of APP expressed. Experiments involving 109 autophagy inhibitor 3-methyladenine (3-MA) further revealed that altered autophagic pathway 110 contributes to the U18666A-induced accumulation of APP and its cleaved products. Additionally, 111 we observed that cholesterol sequestration within the EL system can markedly increase the levels of 112 η-CTF in the cells. Taken together, these results suggest that cholesterol level/accumulation, 113 depending on the levels and nature of APP expressed in the cells, can differentially influence APP 114 metabolism.

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107 can differentially regulate the cellular accumulation/secretion of APP-cleaved products including

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RESULTS

116 Effects of U18666A, extracellular lipids and APP on cellular cholesterol redistribution/levels: To 117 determine if the extracellular lipid content and cholesterol sequestration in the EL system regulate

119 used mouse Neuro2a neuroblastoma cells expressing normal mouse APP (N2awt) or cells that are 120 overexpressing either normal human APP (APPwt) or Swedish mutant human APP (APPsw). These 121 cells were maintained in media containing different concentrations (0, 5 and 10%) of fetal bovine 122 serum (FBS) and were exposed to 3µg/ml U18666A for 24hr. The concentration of U18666A used 123 in this study was based on earlier results (26, 27, 29). As demonstrated previously (29, 39), 124 U18666A triggers the sequestration of cholesterol into the EL system (Fig. 1). In untreated N2awt, 125 APPwt or APPsw cells grown with various FBS concentrations, the staining of unesterified 126 cholesterol with filipin revealed a faint labeling throughout the cytoplasm without any accumulation 127 of cholesterol. Exposure of any of the three cell lines to 3µg/ml U18666A for 24hr caused a marked 128 increase in intracellular filipin staining irrespective of the FBS concentrations (Fig. 1A-F). The 129 cellular cholesterol content was not altered following U18666A treatment in any of the three cell 130 lines grown in either 10% or 5% FBS, which is consistent with previous reports (35, 39), but 131 decreased in 0% FBS media (Fig. 1G-I), suggesting that extracellular lipids or other components of 132 FBS play a role in maintaining the cholesterol homeostasis in U18666A-treated cells. 133 134 Cholesterol synthesis is controlled by ER cholesterol (41), which is modulated by extracellular 135 cholesterol availability and by trafficking of intracellular cholesterol back to the ER. U18666A may 136 alter cholesterol homeostasis by two opposing effects. First, U18666A transiently reduces 137 cholesterol trafficking to the ER, which in turn induces cholesterol synthesis (42). On the other

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118 APP metabolism depending on the nature (wild-type vs mutant) or the level of APP expression, we

138 hand, U18666A inhibits enzymes of the cholesterol synthesis pathway (38, 43). To determine if the 139 level and nature of APP alter cholesterol homeostatic mechanisms, we evaluated the status of 140 SREBP-2, the main transcription factor regulating expression of cholesterol synthesis enzymes (44). 141 Low cholesterol levels in the ER activate SREBP-2 by cleavage of the precursor form ((P)SREBP-2)

143 in N2awt, APPwt and APPsw cells cultured under different FBS concentrations with or without 144 U18666A treatment (Fig. 2A-C). Untreated N2awt, APPwt and APPsw cells responded to the 145 decreased availability of extracellular serum with a significant increase of (M)SREBP-2 (Fig. 2A146 C). This SREBP-2 activation in response to decreased extracellular serum was also observed in 147 cells treated with U18666A. In the absence of extracellular serum and at 5% FBS, U18666A 148 significantly increased SREBP-2 activation (Fig. 2A-C). 149 150 Effects of FBS and U18666A treatment on APP metabolism: To investigate if U18666A-induced 151 cholesterol redistribution differentially regulates APP metabolism, we first measured the levels of 152 APP holoprotein in N2awt, APPwt and APPsw cells grown under different FBS concentrations (Fig. 153 3A-C). Our results revealed no significant alteration in APP levels in any of the three cell lines with 154 increasing concentrations of FBS in the absence of U18666A (Fig. 3A-C). Treatment with 155 U18666A also did not cause any alteration in APP levels in N2awt cells at any FBS concentrations, 156 whereas APPwt cells showed marked increase in holoprotein levels at 0% and 5% FBS (Fig. 3A, B). 157 The APPsw cells, on the other hand, showed an increase in APP levels at all FBS concentrations 158 (Fig. 3C). Additionally, APP levels were increased at 0% FBS compared to 10% FBS in both 159 APPwt and APPsw cell lines following U18666A treatment (Fig. 3B, C).

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142 into a mature, transcriptionally active form ((M)SREBP-2) (45). We examined SREBP-2 activation

160 In addition to APP, we analysed the levels of α- and β-secretase cleavage products α-CTF and β161 CTF, respectively (Fig. 4A-C). In the absence of U18666A, increasing the concentration of FBS in 162 the medium from 0% to 5% and 10% causes a significant enhancement in the levels of -CTF in all 163 three cell lines and an increase in -CTF levels only in APPsw cells (Fig. 4A-C). U18666A

165 and APPsw cells compared to respective untreated controls. N2awt cells treated with U18666A 166 exhibited an increase in -CTF levels only at 5% and 10% FBS and -CTF levels at 10% FBS 167 compared to the respective control cells (Fig. 4A-C). The increased levels of β-CTF in APPsw cells 168 following U18666A treatment, observed using the C-terminal rabbit monoclonal antibody (mAb) 169 Y188, was further validated with mAb 6E10 that selectively reacts with A residues 1-16 (data not 170 shown). Interestingly, the levels of sAPPα/sAPPβ in the medium are not significantly altered in 171 N2awt cells either as a function of FBS concentrations or following U18666A treatment (Fig. 5A, 172 B). In untreated APPwt and APPsw cells, sAPPα levels are significantly increased at 10% and 5% 173 FBS compared to 0% FBS condition. Treatment with 3µg/ml U18666A also enhanced the levels of 174 sAPPα in both cell lines at all FBS conditions (Fig. 5C, E). Conversely, sAPPβ levels remained 175 unaltered with or without U18666A treatment at all FBS conditions in both APPwt and APPsw cells 176 (Fig. 5D, F). These results suggest that levels of APP and its cleaved products are differentially 177 regulated in three cell lines by the accumulation of cholesterol within the EL system as well as 178 extracellular lipid content in the culture media. 179 180 In addition to the canonical α/β-secretase pathways, APP can be processed by a novel -secretase 181 pathway to generate η-CTF (6, 10). Unlike the α/β-CTFs, η-CTF is not apparent in cell lysates 182 under normal physiological conditions unless lysosomal protease inhibitors are used to block its

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164 treatment causes a further increase of -CTF and -CTF levels at all FBS concentrations in APPwt

183 degradation (7, 8). Since intracellular accumulation of cholesterol can impair lysosomal clearance 184 (46), we wanted to examine whether variable FBS or cholesterol sequestration within the EL system 185 can influence η-CTF accumulation. Our results revealed that the steady-state levels of η-CTF, 186 detected using mAb Y188 (6), did not vary in untreated N2awt, APPwt or APPsw cells as a

188 increased η-CTF levels in all three cells at 5% and 10% FBS conditions compared to the respective 189 untreated cells (Fig. 6A-C). Moreover, the higher FBS concentration further enhanced the effect of 190 U18666A on the levels of η-CTF in all three cell lines (Fig. 6A-C). These results were validated 191 using a rabbit polyclonal NTG-449 antibody, generated against human APP ectodomain residues 192 306-600 (data not shown). In parallel, we show that the levels of η-CTF are markedly increased 193 following the inhibition of BACE1 cleavage of APP by BIV (data not shown), as reported earlier 194 (6). 195 196 Effects of FBS and U18666A treatment on Aβ1-40 and Aβ1-42 levels/secretion: To determine the 197 influence of cholesterol sequestration on Aβ levels, we measured the intracellular and secretory 198 Aβ1-40 (Fig. 7A-F) as well as Aβ1-42 (Fig. 8A-F) levels in N2awt, APPwt and APPsw cells. The 199 N2awt cells did not exhibit any alterations in intracellular Aβ1-40 (Fig. 7A) or Aβ1-42 (Fig. 8A) levels 200 either with increasing concentration of FBS or following U18666A treatment. In contrast, both 201 APPwt and APPsw cells showed an increase in intracellular Aβ1-40 (Fig. 7C, E) and Aβ1-42 (Fig. 8C, 202 E) levels as a function of FBS concentrations as well as following treatment with U18666A. In fact, 203 U18666A treatment drastically enhanced intracellular Aβ1-40 (Fig. 7C, E) and Aβ1-42 (Fig. 8C, E) 204 levels in APPwt and APPsw cells at all FBS conditions compared to respective controls. The 205 secretory levels of Aβ1-40 in the media, as observed with intracellular levels, displayed variations

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187 function of FBS concentrations (Fig. 6A-C). However, treatment with U18666A markedly

206 depending on the experimental paradigm. In untreated N2awt cells, the levels of secreted Aβ1-40 207 (Fig. 7B) or Aβ1-42 (Fig. 8B) were unaltered by FBS concentrations, whereas U18666A treatment 208 markedly decreased secretory Aβ1-40/Aβ1-42 levels compared to the respective controls. Untreated 209 APPwt and APPsw cells, on the other hand, showed a gradual increase in the secretion of both Aβ140

(Fig. 7D, F) and Aβ1-42 (Fig. 8D, F) as a function of FBS concentrations, but the exposure to

211 U18666A induced differential response. Whereas the secretory Aβ1-40 levels decreased following 212 U18666A treatment at 5% and 10% FBS conditions (Fig. 7D, F), Aβ1-42 levels in the media were 213 elevated in U18666A-treated cells regardless of the FBS conditions (Fig. 8D, F). Thus, while higher 214 FBS levels increased the levels of Aβ1-40 and Aβ1-42 in APPwt and APPsw cells, the accumulation of 215 cholesterol within the EL system had the opposite effects on their secretion. 216 217 Effects of FBS and U18666A on APP secretases levels and activity: To assess if observed changes 218 in the levels of α/β-CTFs and Aβ are due to differential APP processing by altered levels/activity of 219 secretases, we first evaluated steady-state levels of α-secretase ADAM10, β-secretase BACE1 and 220 the γ-secretase complex in N2awt, APPwt and APPsw cells treated with or without U18666A under 221 different FBS concentrations. Our results clearly showed that the levels of ADAM10 and BACE1 222 did not change in any of the cell lines either as a function of FBS concentrations or following 223 treatment with U18666A (data not shown). Similar results were obtained with all four components 224 of the γ-secretase complex i.e., presenilin 1 (PS1), anterior pharynx defective-1 (APH1), presenilin 225 enhancer 2 (Pen2) and nicastrin (data not shown) in N2awt, APPwt and APPsw cells. Since APP 226 processing can be altered in the absence of any changes in the enzyme levels (4, 47), we evaluated 227 the activities of ADAM10 and BACE1 in N2awt, APPwt and APPsw cells grown in 5% FBS with 228 or without U18666A treatment. No alteration was evident either with ADAM10 or BACE1 (data

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229 not shown) activity in any of the cell lines, thus indicating that higher levels of α/β-CTFs observed 230 in the study is unlikely due to an increase in APP secretase levels or activity. 231 232 Effects of U18666A on surface APP levels and APP stability: We considered two other

234 with U18666A. First, we examined whether the U18666A treatment increases secretory trafficking 235 of APP. For this, we assessed the levels of surface APP by cell surface biotinylation of APPsw cells 236 treated with U18666A. The results revealed that the U18666A treatment caused a dramatic increase 237 in surface APP levels at all FBS concentrations (Fig. 9A). Next, we asked whether the U18666A 238 treatment reduces the rate of clearance of APP CTFs. To this end, APPsw cells grown in 5% FBS 239 were treated with or without 3µg/ml U18666A and then exposed to protein synthesis inhibitor 240 cycloheximide for different periods of time in the presence of U18666A (48, 49). Our results 241 showed that U18666A treatment differentially decreased the turnover of APP and its metabolites 242 over the course of 5hr (Fig. 9B-F). Thus, the higher levels of APP cleaved products in U18666A243 treated cells could partly be due to an increase in the pool of APP that has reached the surface and 244 available for processing by the secretases as well as a decrease in the turnover of these molecules. 245 246 Effects of U18666A on lysosomal accumulation of APP, APP-CTFs and Aβ: To establish if 247 cholesterol sequestration triggered an accumulation of APP and its cleaved products in the 248 lysosomes (50-52), we assessed the intracellular localization of APP, APP-CTFs, and Aβ in control 249 and U18666A-treated APPsw cells using confocal microscopy (Figs. 10, 11). Full-length APP was 250 localized using the N-terminal APP mAb 22C11 and the localization of APP/APP-CTFs was 251 determined with mAb Y188. Previously, we showed that Y188 immunoreactivity overlaps

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233 possibilities that could account for the marked increase in the levels of APP CTFs in cells treated

252 completely with that of a C-terminal APP antibody CTM1 but only partially with the N-terminal 253 APP mAb 22C11 (27). The mAb 4G8 (epitope Aβ17-24), which recognizes Aβ without reacting with 254 APP or APP-CTFs (52-54) was used to label A. Immunoreactivity detected using 22C11, Y188 255 and 4G8 antibodies exhibited perinuclar localization and punctate staining throughout the

257 following U18666A treatment (Figs. 10D, J, 11D). Moreover, the perinuclar accumulation of 258 APP/APP-CTFs and Aβ ensue in lysosomal-associated membrane protein 1 (LAMP1)-positive 259 organelles following U18666A treatment (Figs. 10E, F, K, L, 11E, F) compared to untreated APPsw 260 cells (Figs. 10B, C, H, I, 11B, C). Thus, it is likely that enhanced levels of APP and its cleaved 261 products following U18666A treatment represent an accumulation of these molecules in the 262 lysosomal pathway. 263 264 Effects of U18666A on lipid raft distribution: Lipid rafts are membrane domains rich in cholesterol 265 and sphingolipids. It is reported that ADAM10 resides mostly in the low-cholesterol content non266 raft domains (56), whereas a subset of BACE1 and γ-secretase components are associated with 267 cholesterol-rich raft microdomains of the plasma membrane and intracellular organelles (57). APP, 268 however, exists in both the raft and non-raft domains of the membranes. These observations raised 269 the possibility that amyloidogenic vs non-amyloidogenic processing of APP occurs in different 270 membrane microdomains and an alteration in their distribution may influence APP processing (58, 271 59). A direct interaction of APP with cholesterol might play a role in APP localization to lipid rafts 272 (60). Changes in membrane cholesterol content significantly affect the lipid raft integrity, and it is 273 reported that cholesterol accumulation upon the loss of NPC1 expression triggers a shift in APP 274 localization to lipid rafts (61). To determine if U18666A treatment alters the distribution of APP in

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256 cytoplasm in untreated APPsw cells (Figs. 10A, G, 11A); the perinuclear staining intensified

275 raft vs non-raft domains, we analyzed untreated and U18666A-treated APPsw cells cultured with 5% 276 FBS by lipid raft fractionation. The presence of raft marker prion protein predominantly in fractions 277 3-5 and non-raft marker transferrin receptor in fractions 8-12 validated our fractionation protocol 278 (Fig. 12A). As reported earlier (56, 62), APP was present in both raft and non-raft fractions (Fig.

280 non-rafts fractions, nor changed the distribution profile of prion protein and transferrin receptor (Fig. 281 12B-D). 282 283 Effect of delipidated FBS on U18666A-induced APP metabolism: FBS contains numerous 284 components including cholesterol and other lipids such as sphingolipids, which have previously 285 been linked to APP processing (63-66). To determine if the lipid component of the serum is 286 responsible for the effect of FBS on APP metabolism, we cultured APPsw cells with normal FBS 287 and delipidated FBS (>97% reduction in cholesterol levels compared to normal FBS) and then 288 treated the cells with or without 3µg/ml U18666A for 24hr (Fig. 13). Our results showed no 289 difference in APP levels between delipidated and normal FBS following U18666A treatment (Fig. 290 13A, B). Similarly, increasing the delipidated FBS concentration did not change the levels of α/β291 CTFs. U18666A treatment, however, was able to enhance the levels of both CTFs in delipidated 292 and lipidated FBS conditions compared to the untreated controls. The progressive increase in the 293 levels of CTFs observed as a function of FBS following U18666A treatment was also not evident 294 under delipidated conditions (Fig. 13A, C, D). These results confirm that lipids in the FBS are 295 indeed responsible for enhancing the levels of CTFs under normal conditions as well as following 296 treatment with U18666A. 297

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279 12A). U18666A treatment neither altered significantly the proportion of APP localized to rafts vs

298 Effects of cholesterol supplementation on APP metabolism: Since FBS contains numerous lipids 299 including cholesterol (64, 66-68), we wanted to determine whether the cholesterol component of the 300 serum is partly responsible for the observed effects on APP metabolism. Thus, APPsw cells 301 cultured with 0% FBS were exposed to either 25 or 50µM cholesterol and then processed to

303 APP, APP-CTFs (i.e., α-/β-/η-CTFs) and Aβ1-40 (Fig. 14A-F) were enhanced following cholesterol 304 supplementation. These results indicate that extracellular cholesterol present in the FBS are largely 305 responsible for enhancing the levels of APP and its cleaved products. 306 307 Effects of 3-MA on U18666A-regulated APP metabolism: Earlier studies indicated that the 308 autophagic pathway modulates APP metabolism (28, 69-72). Thus, to determine whether APP 309 metabolism regulated by U18666A treatment is partly mediated by the autophagic pathway, we 310 treated APPsw cells with U18666A in the presence or absence of the autophagy inhibitor 3-MA. 311 Consistent with earlier data (31, 73-76), the treatment with U18666A increased the levels of 312 autophagy marker LC3-II, which is accompanied by enhanced levels of APP, APP-CTFs and Aβ1-40 313 (Fig. 15A-H). Interestingly, the combined treatment of cells with U18666A and 3-MA did not 314 produce an additive effect (Fig. 15A-H) suggesting that the inhibition of the autophagic pathway, as 315 demonstrated in earlier studies (28, 77-79), may have a role in U18666A-induced accumulation of 316 APP and its cleaved products in cells.

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302 measure the levels of APP and its cleaved products. The results clearly show that cellular levels of

317

DISCUSSION

318 The present study shows that cholesterol sequestration within the EL system can differentially alter 319 the levels of APP and its cleavage products in mouse neuroblastoma cells expressing endogenous

321 concentrations in the culture media can, to some extent, influence APP metabolism. This is 322 supported by data which showed that i) the treatment of cells with U18666A, but not the presence 323 of FBS in the media, triggers the intracellular accumulation of cholesterol in N2awt, APPwt and 324 APPsw cells; ii) the steady-state APP levels are not influenced by the FBS concentrations but 325 increased in APPwt and APPsw cells following U18666A treatment, whereas levels of APP-CTFs 326 are differentially elevated as a function of FBS as well as U18666A treatment in all three cell lines; 327 iii) intracellular Aβ1-40 and Aβ1-42 levels are increased with FBS concentrations in U18666A-treated 328 APPwt and APPsw cells; iv) U18666A treatment results in the accretion of APP, APP-CTFs and 329 Aβ peptides in lysosomal compartments, v) delipidation of the FBS in the culture media attenuates 330 the levels of APP-CTFs, but not APP holoprotein, in U18666A-treated APPsw cells and vi) 331 autophagy inhibition did not alter the effects of U18666A on the levels of APP or its cleaved 332 products. Collectively, these results suggest that cholesterol sequestration within the EL system and 333 the extracellular lipids can significantly influence APP metabolism, especially in cells 334 overexpressing human APP. 335 336 The influence of cellular cholesterol on APP metabolism has long been studied in view of the 337 evidence that intracellular trafficking, localization and processing of APP are regulated by the levels 338 of cholesterol within the cells. Many studies, however, reported contradictory results raising the 339 possibility that variation in levels/sites of cholesterol accumulation or the type of APP (normal vs

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320 APP or those overexpressing wild-type or familial AD-linked mutant APP. Additionally, FBS

340 mutant) expressed in cells/animals may underlie the cause of discrepancy (17-30). Supporting the 341 notion, our results showed that extracellular lipids in combination with intracellular accretion of 342 cholesterol can differentially regulate APP metabolism depending on the nature/levels of APP 343 expression. Furthermore, our study reveals how variation in the levels/types of APP can

345 existing discrepancy with regards to the cause of variations in APP metabolism observed following 346 treatments that affect cholesterol levels. 347 348 U18666A is one of the most well characterized class-2 amphiphilic compounds known to trigger 349 accumulation of cholesterol within the EL system by reducing cholesterol movement from the 350 plasma membrane to ER and from the late-endosomes/lysosomes to the plasma membrane. Recent 351 studies indicated that U18666A binds and inhibits NPC1 protein, which plays a crucial role in the 352 efflux of cholesterol out of the lysosomes, leading to its accumulation in high levels within the EL 353 system (80-82). Cellular cholesterol levels may also partly be modified by extracellular lipoproteins, 354 which are provided by FBS to the cultured cells (83, 84). In the present study, we used U18666A to 355 define the effects of EL cholesterol accumulation on APP metabolism in mouse N2a cell lines 356 expressing different levels of APP. Also, we have determined how different FBS levels can 357 influence APP metabolism in the absence or presence of U18666A. 358 359 As reported in earlier studies (29, 30, 39), the treatment with U18666A triggered EL sequestration 360 of cholesterol in all three cell lines. However, U18666A did not alter the cellular levels of total and 361 free cholesterol when provided in the presence of 5% or 10% FBS, but caused a substantial 362 decrease of cellular cholesterol in culture media lacking FBS. The reduced levels of cholesterol in

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344 differentially influence cellular cholesterol metabolism. These results provide some clarity to the

363 cells grown in 0% FBS likely results from the inhibition of cholesterol synthesis by U18666A (37, 364 38, 43, 80), which is compensated by lipoprotein-derived cholesterol when FBS is present in the 365 medium. Our data provide strong evidence on the importance of extracellular components present in 366 the serum (FBS). The increase of FBS in the medium per se did not trigger cholesterol

368 cell lines. These effects cannot be explained by variations in basic cholesterol homeostatic 369 mechanisms since all three cell lines responded similarly to the reduction of FBS concentration by 370 activating SREBP-2. 371 372 The role of APP in the regulation of cholesterol homeostasis is unclear (60). Our work provides 373 new insights on this respect. First, we found no difference in cholesterol levels among the three cell 374 types in either the absence or presence of U18666A at any FBS concentration. Second, cells 375 overexpressing (APPwt and APPsw) or not overexpressing (N2awt) APP are equally able to 376 activate SREBP-2 cleavage in response to serum deprivation. SREBP-2 cleavage and maturation 377 can be used as a reporter of intracellular cholesterol trafficking to the ER (42). Thus, our results 378 imply that overexpression of APP does not alter the movement of cholesterol to the ER. Previous 379 studies indicated that APP overexpression in cortical neurons inhibits cholesterol synthesis possibly 380 by impairing the maturation of SREBP in the Golgi (85). Our data demonstrate that in cells 381 overexpressing APP, SREBP-2 is cleaved normally in the absence of serum or at low serum 382 concentration (5%), at least during the 24hr window of our experiments. Our experiments, however 383 do not exclude the possible inhibition of cholesterol synthesis in APPwt or APPsw by mechanisms 384 independent of SREBP-2. Cholesterol sequestration by U18666A caused additional SREBP-2 385 activation in absence of serum or at 5% FBS but did not increase SREBP-2 cleavage when provided

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367 accumulation or altered APP levels, but it did cause differential increase of APP-CTFs in the three

386 together with 10% FBS. This is consistent with previous reports that when intracellular cholesterol 387 trafficking was inhibited by U18666A-treatment or NPC1 deficiency, the ability of lipoproteins to 388 suppress SREBP-2 processing is transiently abolished (4 and 8hr time points) but returns to normal 389 by 24hr and beyond (42). Overtime, in the presence of U18666A and FBS, sufficient cholesterol

391 SCAP system (42). At low FBS concentration or in the absence of FBS, cholesterol accumulation 392 and spill out of the late-endosomes may take longer than at 10% FBS. 393 394 The levels of APP are mostly increased in APPwt and APPsw cells, but not in N2awt cells, 395 following treatment with U18666A. Earlier studies have in fact shown that U18666A treatment can 396 lead to increased levels of APP in APPwt and APPsw cells (30) but not in primary neurons (29), 397 suggesting a rather selective effect of the drug in cells expressing higher levels of APP. 398 Interestingly, U18666A treatment enhanced α/β-CTFs levels in APPwt and APPsw cells in all FBS 399 concentrations, whereas in N2awt cells the levels of α-CTF are increased at 5% and 10% and β-CTF 400 are increased only at 10% FBS conditions. Since FBS concentration can independently influence 401 α/β-CTFs levels, the effects of U18666A on these APP metabolites may depend not only on the cell 402 type but also on the concentration of FBS in the culture media. Previous studies have shown that 403 U18666A treatment can lead to either an increase in α/β-CTFs (29, 31) or an increase in α-CTF and 404 decrease in β-CTF (27, 30) levels. In contrast to α/β-CTFs, the levels of η-CTF were not influenced 405 by the FBS concentration but found to be enhanced in all three cell lines following U18666A 406 treatment. Although this suggests a role for cellular cholesterol in the regulation of η-secretase 407 processing of APP, the underlying mechanism remains unclear. Since η-CTF is rapidly degraded by 408 lysosomal enzyme cathepsin L under normal conditions (7), it is likely that impaired enzyme

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390 may accumulate in the late-endosomes so that some spills over to become accessible to the SREBP-

409 activity and/or clearance mechanism triggered by EL sequestration of cholesterol (28, 46) may 410 underlie its accumulation in the cells. 411 Accompanying APP-CTFs, the intracellular levels of Aβ1-40 are markedly increased in APPwt and 412 APPsw cells, while the secretory levels of the peptide are decreased in all three cell lines. With

414 its levels in the conditioned media either decreased or increased depending on the cell types. It is, 415 however, important to note that FBS was able to influence both intracellular and secretory levels of 416 Aβ peptides in APPwt and APPsw cells emphasising that the effects of U18666A can partly be 417 modulated by the cultured conditions and endogenous levels of APP expressed in the cells. This is 418 supported by the evidence that U18666A treatment has been reported to either reduce cellular 419 and/or secretory levels of Aβ1-40/Aβ1-42 in neurons, APP-transfected SH-SY5Y, CHO or mouse N2a 420 cells (27, 30, 74, 86) or increased intracellular Aβ1-42 levels without affecting its secretion in CHO 421 cells (26). Additionally, U18666A treatment can lead to an accumulation of AβX-42 in mouse 422 cortical neurons expressing endogenous APP (86) or transfected with human APP (29). 423 Notwithstanding these results, we did not observe any alteration in the levels of either ADAM-10, 424 BACE1 or the γ-secretase complex following treatment with U18666A in any of the three cell lines. 425 Although it is reported that U18666A treatment can trigger transcriptional up-regulation of PS1 (85) 426 without any alteration in protein levels, there is also evidence of PS1 redistribution to late427 endosomes following EL cholesterol sequestration (27). At present, it is unclear if U18666A 428 treatment can induce redistribution of PS1 in late-endosomes in N2awt, APPwt or APPsw cells 429 without any change in its levels. Accompanying ADAM-10 and BACE1 levels, no alteration was 430 observed in either ADAM-10 or BACE1 activity in U18666A-treated cells. Interestingly, an earlier 431 study using SH-SY5Y cells stably transfected with APP SP-C99, which acts as a direct substrate for

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413 regards to Aβ1-42, while the intracellular levels of the peptide increased in APPwt and APPsw cells,

432 γ-secretase, demonstrated that U18666A treatment can enhance γ-secretase activity leading to 433 increased production of Aβ peptides (27), suggesting an additional basis for the increased levels of 434 Aβ peptides observed in our experiments. 435 APP metabolism is known to be regulated by multiple factors that can influence not only the

437 increase the levels of APP, APP-CTFs and intracellular Aβ1-40/Aβ1-42 in APPwt and APPsw cells in 438 all FBS concentrations. Notwithstanding the unaltered levels/activity of ADAM10 and BACE1, 439 results of our biotinylation assay reveal an increase in the levels of APP at the cell surface that can 440 undergo processing, thus contributing to the increase in the levels of APP-CTFs. Concomitantly, a 441 decreased turnover of the APP-CTFs and Aβ, as a consequence of EL cholesterol sequestration, 442 may also contribute to the enhanced intracellular levels of APP-CTFs and Aβ1-40/Aβ1-42. This is 443 supported by two lines of evidence i) the levels of APP and APP-CTFs were higher in 444 cycloheximide exposed U18666A-treated cells than control cells and ii) immunoreactive APP, 445 APP-CTFs and Aβ are accumulated more in lysosomal compartments following U18666A 446 treatment than untreated cells. Additionally, we and others have reported that cholesterol 447 sequestration within the EL system resulting from NPC1 deficiency can trigger the intracellular 448 accumulation of APP-CTFs and Aβ by impairing their lysosomal clearance pathway (29, 89, 90). 449 Nevertheless, future studies are needed to define the contribution of the increased production vs 450 decreased clearance that leads to the accumulation of APP metabolites in U18666A-treated cells. 451 452 Our study further reveals that in a manner similar to treatment with U18666A, cholesterol 453 supplementation of culture media, as depicted in a recent study (67), increases the intracellular 454 levels of APP, APP-CTFs and Aβ in APPsw cells, possibly by enhancing cellular accumulation of

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436 generation but also the clearance of the peptides (3, 14, 15, 88). U18666A treatment can markedly

455 cholesterol. We also showed that U18666A treatment, as reported earlier (31, 73, 74, 76), can 456 enhance the levels of LC3-II suggesting an impaired autophagic pathway following EL cholesterol 457 sequestration. Interestingly, the levels of APP and its metabolites are not found to be altered when 458 cells were co-treated with U18666A and 3-MA compared with those treated with U18666A alone.

460 trigger an accretion of APP-CTFs and Aβ in EL compartments as well as LC3-positive autophagic 461 vesicles (28). These results, taken together, suggest that inhibition of the autophagic pathway may 462 have a role on the accumulation of APP and its cleaved products in U18666A-treated cells. 463 464 Neurons are considered to be the major source of Aβ in normal and AD brains (91, 92). Earlier 465 studies have shown that an increasing cellular concentration of cholesterol can enhance the level 466 and processing of APP in neurons (11, 14, 15). Our results further revealed that cholesterol 467 sequestration within the EL system together with extracellular cholesterol/lipids can have an 468 alternate effect on APP levels/processing as well as intracellular accumulation of Aβ, depending on 469 the levels and nature of cellular APP expression. Since cholesterol levels are increased in AD brains 470 (93, 94) and the cholesterol transporter Apolipoprotein E4 allele can augment the risk of developing 471 AD (14, 15, 95), it is likely that enhanced levels or altered subcellular distribution of cholesterol can 472 directly influence APP metabolism in AD brains. Additionally, some recent studies have shown that 473 intracellular Aβ peptides may possibly underlie the cause of toxicity associated with the increased 474 accumulation of cholesterol in the cells (28, 85, 96). This is supported by the finding that the 475 induction of autophagy and lysosomal biogenesis by rapamycin (97, 98), which reduces levels of 476 APP-CTFs and Aβ (79, 99), protected the cells from toxicity (28). These mechanistic features 477 provide a basis to suggest that cholesterol dysfunction associated with AD pathology (15, 40, 100,

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459 In fact, some earlier studies have shown that cholesterol sequestration within the EL system can

478 101) can regulate not only APP metabolism and generation of Aβ peptides, but also can lead to the 479 loss of neurons in the brain.


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480

MATERIALS AND METHODS

481 Materials: NuPage 4-12% Bis-Tris gels, Opti-MEM® I reduced-serum medium, Dulbecco's 482 modified eagle medium (DMEM), penicillin-streptomycin, FBS, Geneticin® selective antibiotic

484 kit, Enzyme-linked immunosorbent assay (ELISA) kits for human Aβ1-40, human Aβ1-42, mouse 485 Aβ1-40 and Aβ1-42, Alexa fluor conjugated secondary antibodies were purchased from Life 486 Technologies Corp. (Burlington, ON, Canada). ADAM10 α-secretase activity assay kit was 487 obtained from AnaSpec (Fremont, CA, USA), BACE1 β-secretase activity assay kit was from 488 Abcam (Cambridge, United Kingdom) and β-secretase inhibitor BIV was from Calbiochem 489 (Etobicoke, ON, Canada). U18666A was purchased from Enzo Life Sciences (Ann Arbor, MI, 490 USA), whereas bicinchoninic acid (BCA) protein assay kit and enhanced chemiluminescence (ECL) 491 kit were from Thermo Fisher Scientific (Montreal, QC, Canada). Sources of all primary antibodies 492 used in the study are listed in Table 1. All HRP-conjugated secondary antibodies were from Santa 493 Cruz Biotechnology (Paso Robles, CA). Filipin, cycloheximide, 3-MA and water-soluble 494 cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used in 495 our study were either from Sigma-Aldrich or Thermo Fisher Scientific. 496 497 Cell Culture and treatments: Mouse Neuro2a (N2awt) neuroblastoma cells stably overexpressing 498 the human wild-type APP (APPwt) and the “Swedish” mutant (K670N, M671L) APP (APPsw) 499 were maintained in N2a growth medium (102). N2awt cells were cultured in DMEM/OptiMem I 500 (50:50) supplemented with 5% FBS and 1% penicillin/streptomycin. APPwt and APPsw cells were 501 cultured in DMEM/OptiMem I (1:1) supplemented with 5% FBS, 1% penicillin/streptomycin and o

502 0.4% Geneticin. All cells were maintained at 37 C with 5% CO2 and split every 3-4 days. Media 503 were changed every second day and experiments were performed on day 2 after plating of cells. All

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483 (G418), phosphatase inhibitor cocktail, prolong gold antifade reagent, Amplex red cholesterol assay

504 cells were treated with or without 3µg/ml U18666A in media containing 0%, 5% or 10% FBS to 505 trigger the intracellular accumulation of cholesterol. After 24hr treatment, cells were collected in 506 phosphate-buffered saline (PBS, pH 7.4) and then centrifuged at 400g for 5min. The supernatant 507 and the pellets were used either immediately or stored at -80°C until further processing. For some

509 with 30μg/ml cycloheximide along with U18666A for 0.5, 2 or 5hr over a 24hr experimental 510 paradigm (103). In parallel, APPsw cells grown in 10% FBS were pre-treated with BACE1 inhibitor 511 BIV (10µM) or autophagy inhibitor 3-MA (250µM) for 24hr prior to the addition of 3µg/ml 512 U18666A for an additional 24hr. Untreated and treated cells were then harvested for further 513 processing as described earlier (28). In a separate series of experiments, FBS media was delipidated 514 using EDTA and butanol:diisopropyl ether solution (104) and then sterilized by vacuum filtration 515 through 2μm filters. Subsequently, cultured APPsw cells were exposed to 3µg/ml U18666A for 516 24hr with normal or delipidated serum and cells were harvested for further processing. In some 517 experiments, cultured APPsw cells following maintenance in complete medium were exposed to 0% 518 FBS in the presence or absence of 25µM or 50µM of cholesterol as described earlier (67, 68). After 519 36hr of incubation, control and treated cells were harvested for further processing. 520 521 Filipin Staining: Filipin, which labels unesterified cholesterol (105), was used to determine the 522 intracellular accumulation of cholesterol in untreated and U18666A-treated cells. In brief, cells 523 from various experimental conditions were washed in PBS and fixed with 4% paraformaldehyde 524 before incubation with 25µg/ml filipin for 1hr as described earlier (28). Stained coverslips were 525 mounted and then viewed and photographed with a Zeiss Axioskop-2 microscope (Carl Zeiss, 526 Germany). 527

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508 experiments, APPsw cells grown in 5% FBS were exposed to 3µg/ml U18666A and then treated

528 Cholesterol assay: Cultured N2awt, APPwt and APPsw cells after 24hr incubation in media 529 containing 0%, 5% or 10% FBS with or without 3µg/ml U18666A were harvested as described 530 above. Subsequently, cells were sonicated in PBS containing protease inhibitors and centrifuged at 531 10,000g for 10min. Only the supernatants were collected and their protein concentrations were

533 red cholesterol assay kit (106). Fluorescence was measured using a SpectraMax M5 plate reader 534 (Sunnyvale, CA, USA) at excitation/emission wavelengths of 560/590nm. All samples were 535 assayed in duplicate and results were from 3 independent experiments. 536 537 Western blotting: Control and treated cells from different experimental paradigms were 538 homogenized in radioimmunoprecipitation assay (RIPA) buffer and then processed for Western blot 539 analysis (106). In brief, equal amounts of protein (10-15µg) were separated on 7-17% 540 polyacrylamide or 4-12% NuPAGE Bis-Tris gels, transferred onto polyvinylidene difluoride (PVDF) °

541 membranes, blocked with 10% skimmed milk and then incubated overnight at 4 C with anti-APP, 542 anti-ADAM10, anti-BACE1, anti-nicastrin, anti-PS1, anti-PEN2, anti-APH1, 6E10 or anti-LC3 543 antibodies at dilutions listed in Table 1. After Incubation, membranes were washed and exposed to 544 HRP-conjugated secondary antibodies (1:5000) and immunoreactive proteins were detected using 545 an ECL detection kit. All blots were re-probed with anti-β-actin antibody and quantified using an 546 MCID image analyzer (Imaging Research, Inc., St Catherines, ON, Canada) (107). All experiments 547 were repeated at least three to four times. 548 549 Analysis of SREBP-2 activation: Control and U18666A-treated cells from different experimental 550 paradigms were homogenized in RIPA buffer and then equal amounts of protein from each sample

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532 determined using BCA kit. The amount of total and free cholesterol was determined using Amplex

551 were separated by SDS-PAGE as described earlier (108). Subsequently, proteins were transferred to 552 PVDF membranes, incubated with anti-SREBP-2 and anti-β-actin antibodies and then visualized 553 using by ECL Prime Western Blotting detection system. UN-SCAN-IT gel 5.3 software was used 554 for quantification of the bands and the results are expressed as the ratio of pixels SREBP-2/actin

556 (P)SREBP2 depend on (M)SREBP-2, thus, ratios (M)SREBP2/(P)SREBP-2 or (M)SREBP2/Total 557 SREBP2 cannot be used to express the results as (109). To confirm the band corresponding to 558 (M)SREBP-2 in immunoblots, we generated a control by overexpression of a construct 559 corresponding to the mature form of human SREBP-2 (kindly provided by Dr. S. Sipione, 560 University of Alberta) in St14A cells. 561 562 Enzyme activity: Control and U18666A-treated N2awt, APPwt and APPsw cells were processed to 563 measure ADAM10 and BACE1 activity using respective assay kits as reported earlier (110). For 564 ADAM10 activity, the fluorescence was measured at excitation wavelength of 490nm and emission 565 wavelength of 520nm, whereas the fluorescence for BACE1 activity was monitored at 566 excitation/emission wavelengths of 355/495nm. The specific activity of each enzyme was 567 determined by incubating parallel sets of samples with the respective inhibitors provided with the 568 kits. All samples were assayed in duplicate and the data were obtained from three independent 569 experiments. 570 571 ELISA for human and mouse Aβ1-40 and Aβ1-42: Control and treated N2awt, APPwt and APPsw 572 cells from various experiments were homogenized in ice-cold RIPA buffer and then cellular levels 573 of human and mouse Aβ1-40 and Aβ1-42 were measured using respective ELISA kits as reported

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555 referred to the untreated control. Since SREBP-2 regulates its own synthesis the levels of

574 earlier (28, 110). For secretory Aβ1-40 and Aβ1-42 peptides, cultured cells after treatments with 575 U18666A for 24hr were washed, incubated with phenol-red free OptiMem for 45min and then the 576 media were collected. Subsequently, the media were concentrated using spin columns and 577 centrifugation at 4000g for 6-9hrs and then processed to measure human or mouse Aβ1-40 and Aβ1-42

579 to a standard curve. All samples were assayed in duplicate and each experiment was repeated 3-4 580 times. 581 582 Surface protein biotinylation: APPsw cells were treated with or without U18666A for 24hrs at 583 different FBS conditions, washed with PBS containing 2.5mM CaCl2 and 1mM MgCl2 (PBS-S) and o

584 then exposed to 0.25mg/ml membrane-impermeant sulfo-NHS-biotin in PBS-S for 30min at 4 C. o

585 After biotinylation, cells were rinsed with glycine (50mM) for 5min at 4 C, harvested and 586 centrifuged at 500g for 5min and rinsed with oxidized glutathione (1mM in PBS) 3 times, each time 587 followed by a centrifugation at 500g for 5min. Cells were then lysed in PBS containing 1% Triton 588 X-100 in a cocktail of proteases and phosphatases inhibitors. Equal amounts of proteins were 589 extracted with streptavidin-agarose beads (1ml of beads is used for 1-3mg protein), whereas an 590 equivalent sample of each cell lysate not extracted with streptavidine-agarose served as the input. 591 The samples were separated by SDS-PAGE and analyzed by Western blot as above. 592 593 Immunocytochemistry: For subcellular localization of APP and its cleaved products, APPsw cells 594 grown on coverslips were treated 24hr with or without 3µg/ml U18666A in media containing 5% o

595 FBS, fixed with 4% paraformaldehyde and then incubated overnight at 4 C with anti-APP (N- and 596 C-terminal) or anti-Aβ antibody in combination with anti-LAMP1 antibody at dilutions listed in

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578 using respective ELISA kits. The OD value was converted to Aβ1-40/Aβ1-42 pg/mg protein according

597 Table 1. The coverslips were then exposed to Alexa Fluor 488/594-conjugated secondary antibodies 598 (1:1000), washed and mounted with prolong gold anti-fade medium. Immunostained cells were 599 visualized using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany) (28). 600 Lipid raft Isolation: For lipid raft isolation, we used APPsw cells maintained with 5% FBS in the

602 Tris-HCl, 150mM NaCl, 5mM EDTA, pH 7.4) containing protease inhibitors. Cells were lysed in 1% o

603 Triton X-100 using 22G and 25G needles at 4 C and incubated for 30min in the cold. After 604 normalizing the protein with BCA kit, the lysate was centrifuged for 5min at 10,000g and then 605 supernatant was used to build a discontinued OptiPrep (Sigma Aldrich, MO, USA) gradient (5%, 606 30%, 40%) and centrifuged at 100,000g for 24hr. The resulting gradient was divided into 13 607 fractions of 380µl each as described earlier (110). 608 609 Data analysis: Data are expressed as means ± SEM. Statistical significance was determined by two610 way ANOVA followed by Fisher’s LSD, Bonferroni’s post-hoc analysis for multiple comparisons 611 or unpaired two-tailed Student’s t-test for single comparison with a significance set at p