Active c-secretase is localized to detergent-resistant membranes in human brain ˚ Ji-Yeun Hur, Hedvig Welander, Homira Behbahani, Mikio Aoki*, Jenny Franberg, Bengt Winblad, Susanne Frykman and Lars O Tjernberg Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer’s Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden Keywords Alzheimer’s disease; detergent-resistant membranes; human brain; lipid rafts; c-secretase Correspondence L O Tjernberg, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, KASPAC, Plan 5, 141 57 Huddinge, Sweden Fax: +46 585 83610 Tel: +46 585 83620 E-mail: Lars.Tjernberg@ki.se *Present address Genomic Science Laboratories, Functional Genomics Group, Osaka, Japan (Received 29 October 2007, revised 22 December 2007, accepted January 2008) doi:10.1111/j.1742-4658.2008.06278.x Several lines of evidence suggest that polymerization of the amyloid b-peptide (Ab) into amyloid plaques is a pathogenic event in Alzheimer’s disease (AD) Ab is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by b-secretase and c-secretase, and it has been suggested that these enzymes could be targets for treatment of AD c-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins Studies in cell lines have shown that c-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids Here, we studied c-secretase in detergent-resistant membranes (DRMs) prepared from human brain DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in c-secretase components and activity The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the c-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa) Co-immunoprecipitation studies further supported the notion that the c-secretase components are associated even at high concentrations of CHAPSO Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in c-secretase activity, suggesting that DRMs from fresh rat brain may be useful for c-secretase activity studies Finally, confocal microscopy showed co-localization of c-secretase components and a lipid raft marker in thin sections of human brain We conclude that the active c-secretase complex is localized to lipid rafts in human brain The loss of synapses and neurons in Alzheimer’s disease (AD) is thought to be, at least partly, induced by toxic species formed by the amyloid b-peptide (Ab) [1] Ab is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavages mediated by b-secretase (BACE) and c-secretase [2] An initial cleavage by b-secretase produces soluble APP (b-APPs) and a membrane-bound C-terminal fragment (C99) that is cleaved by c-secretase, generating the APP intracellular domain (AICD) and Ab Two major forms of this amyloidogenic peptide are produced, Ab40 and Ab42, the latter being less abundant but more prone to aggregation [3–5] The polymerization of Ab into fibrils leads to formation Abbreviations AD, Alzheimer’s disease; AICD, APP intracellular domain; Aph-1, anterior pharynx defective-1; APP, amyloid precursor protein; Ab, amyloid b-peptide; BACE, b-site APP cleaving enzyme; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; CTF, C-terminal fragment; CT-B, cholera toxin subunit B; DRM, detergent-resistant membranes; Endo H, endo-b-N-acetylglucosaminidase; ER, endoplasmic reticulum; Nct, nicastrin; NTF, N-terminal fragment; Pen-2, presenilin enhancer-2; PNGase F, peptide N-glycosidase F; PS, presenilin; SEC, size-exclusion chromatography 1174 FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al of amyloid plaques in the brain, and several lines of evidence support the notion that oligomeric Ab species formed in this process are involved in AD pathogenesis [6,7] c-Secretase is a transmembrane protein complex, containing presenilin (PS), nicastrin, anterior pharynx defective-1 (Aph-1) and presenilin enhancer-2 (Pen-2) The stoichiometry of c-secretase components is not clear, but the lowest possible size of the c-secretase complex is approximately 220 kDa with a stoichiometry of : : : (PS : nicastrin : Aph-1 : Pen-2) c-Secretase is believed to be an aspartyl protease, as aspartate residues at positions 257 and 385 within transmembrane domains and of PS seem to constitute the active site of the protease [8] Assembly of the complex is initiated in the ER, where Aph-1 and nicastrin interact, followed by binding of PS Thereafter, Pen-2 binds to the complex and facilitates endoproteolysis of PS into N- and C-terminal fragments (PS-NTF and PS-CTF respectively), resulting in an active c-secretase complex [9] c-Secretase activity can be reconstituted in Saccaromyces cerevisiae, which lacks endogenous c-secretase activity, by co-expressing PS, nicastrin, Aph-1 and Pen2 [10] Thus, these four proteins appear to be sufficient for c-secretase activity, but it is possible that other proteins could play a regulatory role For instance, recent studies have shown that TMP21, a protein involved in protein transport and quality control in the ER and Golgi, as well as the transmembrane glycoprotein CD147, interact with c-secretase and decrease Ab production [11,12] Importantly, c-secretase has several other substrates in addition to APP, all of which are type transmembrane proteins The multitude of c-secretase substrates [13] has made development of clinically useful inhibitors for the treatment of AD difficult For instance, gastrointestinal side-effects related to decreased Notch signaling have been reported [14] Therefore, it is necessary to obtain detailed knowledge on how c-secretase activity is regulated and how the complex selects its substrate in order to design drugs that selectively modify the cleavage of APP Not only protein–protein interactions but also the lipid membrane environment can affect the activity of proteins High cholesterol levels increase Ab production, and high cholesterol levels in mid-life are correlated with the incidence of AD at older ages [15] Apolipoprotein E (ApoE) is involved in cholesterol transport, and the ApoE4 isoform is a risk factor for AD [16] Thus, cholesterol seems to have an important role in APP processing and AD pathogenesis Cholesterol and sphingolipids are the major lipid constituents of ordered microdomains in cell membranes These microdomains are called lipid rafts and are considered Human brain c-secretase in DRMs to be dynamic platforms of importance for cell signaling, membrane protein sorting and transport [17] Lipid rafts are difficult to study in living cells due to their small size, suggested to be in the range of 10–200 nm [18], and their short lifetime [19] As an alternative, the cells can be treated with detergents such as Triton X-100 at °C, resulting in partial dissolution The insoluble parts of the lipid membranes, called detergent-resistant membranes (DRMs), can be isolated by centrifugation and are thought to reflect the composition of lipid rafts However, different detergents give different results [20], and DRM preparations not capture the dynamics of lipid rafts Thus, the occurrence of a protein in DRMs indicates that it could be localized to lipid rafts, but further studies in intact cells or tissue sections are needed to confirm such localization Certain proteins are concentrated to lipid rafts, and several studies have suggested that the trafficking and processing of APP partly depends on lipid rafts [21– 25] APP, BACE and c-secretase have been shown to localize to lipid rafts, but the degree of localization differs between studies [21–27] Possible explanations for the different results include choice of cell lines, whether the cells overexpress the proteins of interest, and the various detergents used for preparation of DRMs As the majority of studies on c-secretase have been performed using cell lines (in many cases transfected cell lines), further studies in brain material are warranted Here, we show that c-secretase components, as well as c-secretase activity, are highly enriched in DRMs prepared from human brain The size of the DRMs containing c-secretase was estimated by size-exclusion chromatography (SEC) to be > 2000 kDa, indicating the presence of other proteins and lipids Preparations of DRMs from rat brain showed a similar distribution of the c-secretase components and a postmortem timedependent decline in c-secretase activity Finally we used confocal microscopy and verified the co-localization of c-secretase components and a lipid raft marker in thin sections of human brain In summary, our data indicates that the active c-secretase complex is localized to lipid rafts in human brain Results The c-secretase complex is present in DRMs Previous studies have suggested that BACE1, c-secretase and APP are located in lipid rafts in cultured cells and mouse brain [21–24] However, the association of c-secretase with DRMs in human brain has not FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1175 Human brain c-secretase in DRMs J.-Y Hur et al previously been reported, and there are no studies on the activity of c-secretase in DRMs from mammalian brain Here, we studied the co-localization of active csecretase with DRMs in human brain We also included preparations from rat brain in our study, because we wished to determine whether there are any significant differences between the two species regarding c-secretase activity and distribution in DRMs To investigate association of the c-secretase complex with lipid rafts in brain, we used a procedure based on centrifugation in a stepped sucrose gradient in which the DRMs float to the interface between 5% and 35% sucrose In the initial experiment, we used freshly prepared membranes (P3, 100 000 g pellet) from rat brain as well as from SH-SY5Y neuroblastoma cells We chose 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) to dissolve the membranes as it is the detergent that best preserves c-secretase activity [28–31] A concentration of around 0.4% CHAPSO gives the highest activity [31], but separation between DRMs and soluble components using 0.25–1.0% CHAPSO was poor (data not shown) The separation was improved when DRMs were prepared from membranes solubilized in 2.0% CHAPSO Western blot analysis showed that PS1-NTF and caveolin-1 (a lipid raft marker) were localized to a large extent to the interface between 5% and 35% sucrose (fraction 2), while calnexin (a non-raft marker) was found in the 45% sucrose fraction (fraction 5) (Fig 1A) Another lipid raft marker, flotillin-1, showed poor separation in rat brain (Fig 1A) In contrast, CHAPSO DRMs prepared from SH-SY5Y cells showed a distinct localization of flotillin-1 to the 5–35% interface (fraction 3, Fig 1B) The same pattern was observed for another lipid raft marker, GM1, which is labeled by the cholera toxin subunit B (Fig 1B) The pronounced separation of lipid raft markers from a non-raft marker in SH-SY5Y cells indicates that it is easier to prepare DRMs from SH-SY5Y cells than from brain tissue The c-secretase components PS1-NTF and Pen-2 were also found in the 5–35% interface in SH-SY5Y cells For comparison, we also prepared DRMs in 1% Triton X-100, a detergent that is frequently used for isolating DRMs, but no PS1NTF was found in the DRM fraction (Fig 1C,D) Thus, 2.0% CHAPSO is suitable for separation of DRMs containing c-secretase components from soluble material The 5–35% interface, which contains DRMs and c-secretase, will be referred to as the DRM fraction Using the protocol described above, we prepared DRMs from human brain Six fractions were collected from the top of the tube and subjected to western blot analysis using antibodies directed to the c-secretase 1176 components BACE, APP, APP C-terminal fragments (APP-CTFs) and raft and non-raft markers Fraction (at the 35–45% interface) and fraction (45% sucrose) were enriched in the non-lipid raft markers, calnexin (ER) and adaptin-c (trans-Golgi network) PS1-NTF, nicastrin, Aph-1aL and Pen-2 were found in the DRM fraction, while only around 10% of the total protein was found in this fraction (Fig 2A,C) Interestingly, the majority of BACE1, full-length APP and APP-CTFs were distributed to fractions and The procedure was repeated using rat brain, and the results were in line with those obtained for human brain (Fig 2B,D) However, in rat brain, the localization of flotillin-1 and caveolin-1 differed between preparations, and they were also found in fraction to a varying extent This could possibly be due to the more heterogenous and more lipid-rich starting material as the whole rat brain was used The mature form of nicastrin is found in DRMs In the active c-secretase complex, nicastrin is glycosylated [32] To determine the glycosylation status of nicastrin in DRMs and fractions and from human and rat brain, endoglycosidase H (Endo H) or N-glycosidase F (peptide-N-glycosidase F, PNGase F) were applied to deglycosylate nicastrin Endo H works on a more limited range of substrates than PNGase F Untreated DRMs contained a nicastrin species of approximately 125 kDa (Fig 2E) Endo H decreased the apparent molecular weight of nicastrin from approximately 125 kDa to approximately 100 kDa, indicating the presence of high-mannose oligosaccharides Upon treatment with PNGase F, which also removes complex oligosaccharides, the deglycosylation was more pronounced, resulting in a diffuse band at approximately 80 kDa (Fig 2E) The deglycosylation pattern of nicastrin was the same in fractions and as in DRMs, and no differences between human and rat brain were observed The above results suggest that the nicastrin that is present in DRMs (fraction 2) as well as in fractions and is highly glycosylated, including high-mannose oligosaccharides and complex oligosaccharides The results were confirmed using another nicastrin antibody (BD Biosciences, San Jose, CA, USA, data not shown) DRMs containing c-secretase elute in a high-molecular-weight SEC fraction To further purify and investigate the approximate molecular weight of DRMs containing the c-secretase complex, we injected the DRM fraction from human FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al Human brain c-secretase in DRMs Fig DRMs prepared in 2% CHAPSO are enriched in c-secretase components DRMs from (A) rat brain and (B) SH-SY5Y cells were isolated by sucrose gradient centrifugation after treatment with 2.0% CHAPSO In rat brain, six fractions were collected from the top of the tube: fraction 1, fraction (DRM fraction, interface between 5% and 35% sucrose), fraction 3, fraction (interface between 35% and 45% sucrose), fraction and fraction (pellet) In SH-SY5Y cells, 12 fractions were collected from the top of the tube: fraction 1–2, fraction (DRM fraction, interface between 5% and 35% sucrose), fraction 4–9, fraction 10 (interface between 35% and 45% sucrose), fraction 11 and fraction 12 The fractions were subjected to western blot analysis using flotillin-1 and caveolin-1 (lipid raft markers), calnexin (a non-raft marker), and PS1-NTF In SH-SY5Y cells, the ganglioside GM1 (a lipid raft marker) was detected by binding of cholera toxin subunit B using a dot-blot assay In (C) and (D), the DRMs were isolated after treatment with 1% Triton X-100 of (C) rat brain and (D) SH-SY5Y cells brain onto a Superose SEC column, collected fractions and analyzed them by western blotting using antibodies directed to all the known c-secretase complex components When using 0.25% CHAPSO as the mobile phase, the c-secretase components, APP and flotillin-1 eluted with the void volume (> 2000 kDa) (Fig 3A,B) DRMs from rat brain gave similar results (Fig 3C) The relatively narrow peak indicates that the complex is stable during separation When using 2% CHAPSO as the mobile phase, most of the nicastrin and PS1-NTF eluted in the void volume, although a second peak around 230 kDa could be observed (corresponding to fractions 17–21, Fig 3F) Thus, the c-secretase complexes are mainly present in large DRMs (> 2000 kDa) The c-secretase complex components can be co-immunoprecipitated from DRMs The stability of the complex was further evaluated by co-immunopreciptation The starting material for DRM preparation (P3), fraction (DRMs) and fraction (soluble fraction) from the rat brain DRM preparation were immunoprecipitated using an antibody against nicastrin (Fig 4) Western blotting showed that PS1-CTF, Aph-1aL and Pen-2 co-immunoprecipitated with nicastrin in P3 and the DRM fraction, and, to a lower degree, in the soluble fraction Flotillin-1 did not co-immunoprecipitate with nicastrin, indicating that flotillin-1 and c-secretase are present in different lipid rafts FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1177 Human brain c-secretase in DRMs J.-Y Hur et al Fig DRMs containing the c-secretase complex can be isolated from human and rat brain The protein concentration was analyzed by BCA assay in (A) human brain and (B) rat brain (C) Human brain membranes were treated with 2.0% CHAPSO, fractionated on a sucrose gradient, and subjected to western blot analysis using antibodies directed to the c-secretase complex components’ BACE1, APP, APP-CTFs, flotillin-1 and caveolin-1 (lipid raft markers) and calnexin and adaptin-c (non-raft markers) (D) The experiment was repeated using rat brain The higher-molecular-weight form of nicastrin (> 125 kDa, labeled with an asterisk) was only detected by one antibody, and this was due to non-specific binding (E) Fractions (DRMs), and for human brain and rat brain were denatured and incubated overnight at 37 °C with glycosidases (Endo H and PNGase F) The samples were analyzed by western blot using anti-nicastrin serum The control was incubated overnight at °C DRMs contain active c-secretase complex To investigate whether the c-secretase complex present in DRMs is active, we incubated fractions (DRMs), and from rat brain in the absence or presence of lm 1178 of the c-secretase inhibitor L-685,458 (Fig 5A), and found that AICD was produced in DRMs only in the absence of L-685,458 Although there were higher amounts of full-length APP and APP-CTFs in fractions and 5, the highest amount of AICD was clearly FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al Human brain c-secretase in DRMs Fig DRM-associated c-secretase is present in a high-molecular-weight complex, as shown by size-exclusion chromatography (SEC) The DRM fraction was injected onto a Superose 6HR column and fractions were collected from 10–50 at a flow rate of 0.5 mLỈmin)1 Solubilization buffer with 0.25% CHAPSO was used as the mobile phase (A) The absorbance at 254 nm was monitored The DRM chromatogram was normalized to the standard chromatogram (B,C) Every second fraction was analyzed by western blot for (B) human brain and (C) rat brain The rat DRM fraction was further analyzed by SEC using solubilization buffer with 0.25% or 2.0% CHAPSO as the mobile phase and the absorbance at 254 nm was monitored (D) (E,F) Every second fraction was analyzed by western blot for (E) 0.25% CHAPSO and (F) 2.0% CHAPSO Fig The c-secretase complex immunoprecipitates in DRMs Rat membranes (P3), the DRM fraction (fraction 2) and fraction were co-immunoprecipitated with anti-nicastrin serum or control rabbit IgG PS1-CTF, Aph-1aL, Pen-2 and flotillin-1 were identified by western blotting generated in DRMs The immunoreactive band comigrated with a 50-residue synthetic AICD peptide and was detected by several antibodies (data not shown) Using an exogenous substrate, C99-FLAG, and a sensitive sandwich ELISA method, we were also able to detect Ab production, which was inhibited by L-685,458 in the DRM fraction from rat brain (Fig 5B) In the next step, we determined whether c-secretase activity could be detected in a human brain sample with a postmortem time of 22 h, and were able to detect AICD production that was inhibited by L-685,458 (Fig 5C) Thus, DRMs isolated from postmortem human brain tissue contain active c-secretase that cleaves endogenous APP-CTFs FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1179 Human brain c-secretase in DRMs J.-Y Hur et al membranes (postmortem time h), the c-secretase activity, measured as AICD production, was decreased by more than 80% at a postmortem time of h After this time, the activity continued to decrease but was still detectable at 24 h postmortem (Fig 5D) and remained even after 48 h (data not shown) Thus, c-secretase activity decreases most rapidly after short postmortem times, but can be observed in brain tissue at all time points studied c-Secretase co-localizes with lipid rafts in human brain sections The presence of a protein in DRMs suggests that it is associated with lipid rafts To further investigate whether the c-secretase components are associated with lipid rafts, we performed immunofluorescence labeling on human brain sections Multiple fluorescent staining was used to study co-localization of PS1, nicastrin and APP with the lipid raft marker GM1 Confocal microscopy revealed that GM1 immunoreactivity was most pronounced in the plasma membrane of the cells PS1 and nicastrin immunoreactivity overlapped extensively with the lipid raft marker (Fig 6A,B), but the overlap of APP and GM1 was limited (Fig 6C) Thus, confocal microscopy supports the view that c-secretase is localized to lipid rafts in human brain Discussion Fig c-Secretase activity was observed in DRMs by monitoring AICD and Ab production (A) The production of AICD was assayed in fractions (DRMs), and by incubation of 100 lg of protein for 16 h at 37 °C in the absence or presence of the c-secretase inhibitor L-685,458 The supernatant was subjected to western blot using the antibody C1 ⁄ 6.1 (B) The DRM fraction (approximately 12 lg protein) was incubated for 16 h at 37 °C in the absence or presence of the c-secretase inhibitor L-685,458 Twenty nanograms of C99-FLAG were added to the samples Ab40 levels were analyzed by sandwich ELISA (C) The production of AICD from human brain was measured and detected as in (A) (D) Solubilized membranes from rat brain obtained at various postmortem times were incubated as in (A) and AICD production was measured as in (A) To investigate the effect of postmortem time on c-secretase activity, we collected rat brains after 0, 6, 12 or 24 h postmortem time (see Experimental procedures), prepared P3 and analyzed this fraction for c-secretase activity Compared to freshly prepared 1180 Previous studies have shown that APP, BACE and c-secretase partially localize to lipid rafts, and it has been suggested that the clustering of these proteins in lipid rafts increase Ab production [15,21] These studies were performed in cell lines, which in many cases overexpressed APP or c-secretase proteins Recently, c-secretase was also found to be associated with DRMs in adult mouse brain [23] However, the association of c-secretase with lipid rafts in human brain has not been investigated, and biochemical evidence for c-secretase activity in DRMs is limited Due to their lipid composition, lipid rafts are resistant to certain detergents Therefore, isolation of DRMs by treatment with detergents such as Triton X-100 followed by flotation in a discontinuous sucrose gradient is frequently used for studying lipid raft components It should be noted that these preparations are dependent on the nature and concentration of the detergent used [20] Previously, 2% CHAPSO has been used to isolate DRMs containing an active c-secretase from SH-SY5Y neuroblastoma cells [22], and 0.5% Lubrol WX has been used to isolate c-secretase-rich DRMs from N2a neuroblastoma cells and mouse brain FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al Human brain c-secretase in DRMs Fig Confocal microscopy shows partial co-localization of lipid rafts and c-secretase in human brain tissue Immunofluorescence labeling was performed on human brain sections The nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) The cholera toxin subunit B (CT-B) that labels the lipid rafts is shown by the green fluorescence of the Alexa Fluor 488-coupled goat anti-rabbit serum Expression of PS1-CTF, nicastrin and APP is shown by the red fluorescence using secondary anti-mouse Alexa Fluor 594 conjugates (A) PS1-CTF (B) Nicastrin (C) APP Scale bar = lm [23,33] We and others have previously studied the effect of various detergents on the activity of c-secretase prepared from rat brain, and found that 0.4% CHAPSO resulted in the highest activity [31] but Triton X-100 abolished the activity [34] Hence, we used CHAPSO to prepare DRMs from human and rat brain To preserve c-secretase as an active complex, we started with CHAPSO concentrations in the range 0.25–1.0% However, it was necessary to increase the CHAPSO concentration to 2.0% to obtain a good separation between raft and non-raft markers We also noted that the separation was better in SH-SY5Y cells than in brain samples Difficulties in obtaining pure DRM fractions from brain tissue are probably due to the heterogeneity of the sample and high levels of myelin In the case of human brain, the DRM fraction resulting from treatment with 2.0% CHAPSO was FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1181 Human brain c-secretase in DRMs J.-Y Hur et al enriched in lipid raft marker proteins and all four known c-secretase complex components, while APP, APP-CTFs and BACE were mainly found in other fractions The association of c-secretase with DRMs is in line with previous results from studies in cells or mouse brain [22,23,33], and suggests that the majority of c-secretase in human brain is localized to lipid rafts Importantly, the localization of PS1 and nicastrin to lipid rafts was confirmed by immunofluoresence confocal microscopy on human brain sections In accordance with our data, previous studies show that < 25% of BACE is associated with lipid rafts [21,23,24,26] Interestingly, it has been suggested that raft association is necessary for BACE activity [21], and thus decreasing the amount of raft-associated BACE could result in lower levels of Ab Our observation that most of the APP occurs outside the DRM fraction is in line with previous studies, where the reported association of APP with DRMs varied from zero up to 20% [21,23,24,26] The low levels of BACE, its substrate and the product (see below) in the DRM fraction could indicate that the initial step in the amyloidogenic pathway occurs outside lipid rafts Alternatively, the processing may be initiated by a transient localization of APP and BACE to lipid rafts With regard to APP-CTFs, the published information is limited due to the difficulties in detecting endogenous APP-CTFs in cell lines Using a c-secretase inhibitor, APP-CTFs accumulated and were detectable in both DRMs and the soluble fraction from SHSY5Y neuroblastoma cells or Chinese hamster ovary (CHO) cells [22,23,25] In brain tissue, the situation is different, and endogenous APP-CTFs are readily detected In a previous study using adult mouse brain, the majority of APP-CTFs were found in DRMs, while full-length APP was found in soluble fractions [23] In contrast, we detected APP-CTFs mainly in the soluble fraction, and it is possible that the choice of detergent (2% CHAPSO versus 0.5% Lubrol WX) could explain this discrepancy Despite the low substrate levels, AICD production was easily detected in DRMs from human and rat brain, while only minor amounts of AICD were generated in the other fractions To our knowledge, this is the first study to show c-secretase processing of an endogenous substrate in DRMs, and the first to show c-secretase activity in mammalian brain DRMs Interestingly, we could not detect APP and APP-CTFs in DRMs after incubating the sample at 37 °C for 16 h As shown in Fig 2C,D, APP and APP-CTFs are present in the DRM fraction before the start of the activity assay However, the levels of those fragments were clearly lower in DRMs than in fraction and We 1182 speculate that the low levels of APP-CTFs might be degraded by non-specific protease activity during incubation at 37 °C, which also explains why blocking c-secretase activity using an inhibitor did not lead to the accumulation of APP-CTFs in DRMs APP and APP-CTFs are generally more difficult to detect in human brain material than in rat brain, probably due to proteolysis during the long postmortem time We were not able to detect production of endogenous Ab, but, after addition of an exogenous substrate, Ab could be detected in the DRM fraction from rat brain using sandwich ELISA Both AICD and Ab production were inhibited by the c-secretase inhibitor L-685,458 In cell studies, only mature nicastrin, which is the form that associates with the active c-secretase complex [35], is localized to DRMs, while immature nicastrin is detected in other fractions [22,33] However, in accordance with our previous results [36], nicastrin was highly glycosylated in all fractions in the brain study Thus, there is a clear difference in the maturation process of nicastrin between cell lines and mammalian brain The predicted molecular weight of the c-secretase complex at a stoichiometry of : : : (PS : nicastrin : Aph-1 : Pen-2) is approximately 220 kDa [37,38] Previous studies on soluble c-secretase have estimated the size of the complex to vary between 200 and 2000 kDa, and the stoichiometry of the c-secretase complex is not clear [10,36,39–41] The diverse results might be due to differences in starting material, preparation procedures and the techniques used (e.g SEC, blue native PAGE or gradient centrifugation) By SEC, the molecular weight of the DRM fraction was estimated to be > 2000 kDa This high-molecular-weight fraction contained the raft marker flotillin-1, the c-secretase complex and low amounts of APP and APPCTFs We suggest that the estimated molecular weight reflects the size of the DRMs (including other proteins, lipids and CHAPSO) rather than the size of the c-secretase complex Elution of the soluble c-secretase complex has been shown to shift from the void volume to a lower-molecular-weight fraction when the CHAPSO concentration in the mobile phase is increased [42] We detected the majority of the c-secretase components in the high-molecular-weight fraction from DRMs even when 2% CHAPSO was used as the mobile phase These data show that the c-secretase complex is stably associated with DRMs In line with these SEC results, it was also possible to co-immunoprecipitate PS1, Aph-1aL and Pen-2 using an anti-nicastrin serum in 2% CHAPSO Another indication of the stability of the c-secretase complex is that activity can be observed FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al in preparations from human brain with long postmortem times (22 h) Freshly prepared membranes from rat brain showed significantly higher c-secretase activity than membranes prepared at various postmortem times As access to human brain material is often limited, and rat and human brain showed similar results in our experiments, we suggest that rat brain is a useful substitute for human brain in studies on c-secretase The distribution of c-secretase and its substrates between lipid rafts and disordered domains of the membrane seems to regulate processing We speculate that lowering the levels of c-secretase or APP ⁄ APPCTFs in lipid rafts may be one way to decrease Ab production Possibly, c-secretase inhibitors that preferentially distribute to lipid rafts might show increased selectivity for inhibition of Ab production, and thus be useful for pharmacological treatment of AD In conclusion, c-secretase is present in DRMs prepared from human and rat brain, and confocal microscopy on sections from human brain confirms that c-secretase is indeed localized to lipid rafts The DRM fraction shows high c-secretase activity although the substrate levels are low, and DRMs prepared from brain tissue are suitable for studies on active c-secretase Experimental procedures Human brain material The cortex from a postmortem human brain (postmortem time 22 h) of a non-Alzheimer case was obtained from Huddinge Brain Bank (Huddinge, Sweden) and stored at )70 °C before use Animals Male Sprague–Dawley rats (200–250 g) were obtained from B&K Universal (Sollentuna, Sweden) The ethical permit was granted by the Animal Trial Committee of Southern Stockholm (no S60-05) The rats were killed by carbon dioxide treatment The brains were dissected to remove blood vessels and white matter Cell culture The human neuroblastoma cell line, SH-SY5Y, was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution (GIBCO ⁄ Invitrogen, Carlsbad, CA, USA) Cells were grown in 5% CO2 ⁄ 95% air at 37 °C Nearly confluent SH-SY5Y cells in three 150 mm dishes were Human brain c-secretase in DRMs washed with cold phosphate buffered saline and centrifuged two times at 1500 g for at room temperature The cell pellet was stored at )20 °C before use Preparation of membranes Membranes were prepared as described previously [36] with some modifications Briefly, the brain material was homogenized by 25 strokes at 1500 r.p.m using a mechanical pestle homogenizer (RW20; IKALaborteknik, Hattersheim, Germany) in lysis buffer (1 mL buffer ⁄ 0.2 g tissue) containing 20 mm Hepes (pH 7.5), 50 mm KCl, mm EGTA and CompleteÔ protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA) All procedures were carried out on ice The samples were centrifuged at 1000 g for 10 to remove nuclei and poorly homogenized material The pellet was homogenized and then centrifuged at 1000 g for 10 min, and the post-nuclear supernatants were pooled and centrifuged once more at 10 000 g for 30 in order to remove mitochondria The supernatant was centrifuged once more, and the final supernatant was then centrifuged at 100 000 g for h to yield the final pellet (P3) Preparation of detergent-resistant membranes DRMs were prepared as described previously [22] with some modifications To isolate DRMs from brain material or cells, P3 or the cell pellet, respectively, were resuspended in 600 lL of buffer containing 20 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl, mm EDTA, 2.0% CHAPSO or 1% Triton X-100, and CompleteÔ protease inhibitor mixture (Roche Applied Science) The samples were incubated with end-over-end rotation for 20 at °C The sample was adjusted to 45% sucrose and placed at the bottom of a 14 mL Beckman Ultra-ClearÔ centrifuge tube Then, 6.9 mL of 35% sucrose followed by 2.3 mL of 5% sucrose was overlaid The sample was centrifuged at 100 000 g for 16 h at °C in a SW40Ti rotor (Beckman Coulter, Fullerton, CA, USA) Six fractions were collected from the top of the tube using a mL syringe (CODAN, Hørsholm, Denmark) In order to remove sucrose from the six fractions, PD-10 desalting columns (GE Healthcare, Piscataway, NJ, USA) were used according to the manufacturer’s instructions A buffer containing 20 mm Hepes (pH 7.4), 150 mm NaCl, mm EDTA and CompleteÔ protease inhibitor mixture (Roche Applied Science) was diluted sevenfold and used to equilibrate the columns The samples were applied, eluted and concentrated to 1· buffer (seven times) using a vacuum centrifuge (Maxi Dry Lyo, Heto-Holten AIS, Allerød, Denmark) The protein concentration was determined by BCA protein assay according to the manufacturer’s instructions (Pierce, Rockford, IL, USA) FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1183 Human brain c-secretase in DRMs J.-Y Hur et al SDS–PAGE and western blotting Equal amounts of protein (20 lg) were mixed with 4· LDS sample buffer (Invitrogen) and kept at room temperature for 20 The samples were then loaded onto a NuPAGEÔ 4–12% Bis–Tris gel or a 16% Tricine gel (Invitrogen) The samples were electrophoresed, transferred to nitrocellulose membranes (Whatman Ltd, Maidstone, UK), and the proteins of interest were detected by specific antibodies Antibodies The following antibodies were used: PS1-NTF (529591; Calbiochem, Darmstadt, Germany), raised against amino acid residues 1-65 of human PS1; PS1-loop (MAB5232; Chemicon, Billerica, MA, USA), raised against the loop (amino acid residues 263–378) of human PS1; nicastrin (N1660; Sigma, St Louis, MO, USA), raised against C-terminal residues 693–709 of human nicastrin; Aph-1aL (PRB-550P; COVANCE, Berkeley, CA, USA), raised against the C-terminal region of human Aph-1aL; UD1 (a gift from J Naslund, Karolinska Institutet, Sweden), raised against ă the N-terminal residues ERVSNEEKLNL of Pen-2; BACE-1 (B0681; Sigma), raised against the N-terminal regions of human BACE-1 (amino acids 46–62, with C-terminally added lysine); C1 ⁄ 6.1 (a gift from P M Mathews, Nathan Kline Institute, NY, USA), raised against the C-terminus of b-APP; calnexin (SPA-860; Stressgen, San Diego, CA, USA), raised against canine calnexin (residues 575-593); adaptin-c (610385; BD Biosciences); flotillin-1 (610820; BD Biosciences); caveolin-1 (sc-894; Santa Cruz Biotechnology, Santa Cruz, CA, USA); cholera toxin, subunit B (C3741; Sigma) Size-exclusion chromatography The DRM fraction was injected onto a Superose 6HR column (Amersham Biosciences, Piscataway, NJ, USA), using a buffer containing 20 mm Hepes, pH 7.0, 150 mm KCl, mm EGTA, CompleteÔ protease inhibitor mixture (Roche Applied Science) and 0.25% or 2% CHAPSO as the mobile phase at a flow rate of 0.5 mLỈmin)1 Fractions (0.5 mL) were collected from 10–50 min, and analyzed by SDS–PAGE as described above Co-immunoprecipitation Rat membranes (P3) were resuspended in 600 lL of immunoprecipitation buffer containing 20 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl, mm EDTA, 2.0% CHAPSO and CompleteÔ protease inhibitor mixture (Roche Applied Science) The samples were incubated with end-over-end rotation for 20 at °C P3, fraction and fraction were pre-cleared with a : ratio of protein A ⁄ protein G Sepharose (GE Healthcare) for 30 at °C, and incu- 1184 bated with anti-nicastrin or control rabbit IgG overnight at °C Protein A ⁄ G Sepharose was added for h at 37 °C After washing three times with immunoprecipitation buffer, the beads were eluted in SDS–PAGE sample buffer and subjected to SDS–PAGE as described above c-Secretase activity assay The production of AICD was assayed by incubating the samples for 16 h at 37 °C in the absence or presence of the c-secretase inhibitor L-685,458 (Bachem, Torrance, CA, USA) After incubation, samples were centrifuged at 100 000 g for h to remove the membranes, and the supernatant was collected, concentrated using a vacuum centrifuge (Maxi Dry Lyo) and analyzed by SDS–PAGE Sandwich enzyme-linked immunosorbent assay Ab40 levels were analyzed by commercial Sandwich enzyme-linked immunosorbent assay (sandwich ELISA; Wako Chemicals, Osaka, Japan) according to the manufacturer’s instructions The DRM fraction was incubated for 16 h at 37 °C in the absence or presence of the c-secretase inhibitor L-685,458 Twenty nanograms of recombinant and purified C99-FLAG dissolved in 2,2,2-trifluoroethanol were added to the samples The reaction was stopped by adding RIPA buffer (150 mm NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris ⁄ HCl, pH 8.0) and boiling for The samples were centrifuged at 1000 g for at room temperature and the supernatants were dispensed into wells (12 lg protein ⁄ well) coated with BNT77 antibody (directed to amino acids 11–28 of Ab) and incubated overnight at °C Bound Ab40 was detected by the 3,3¢,5,5¢-tetramethylbenzidine (TMB) reaction using horseradish peroxidase-conjugated BA27 antibody (directed to the C-terminus of Ab40) All measurements were performed in duplicate, and Ab40 levels were calculated from the synthetic Ab(1–40) (Bachem Bioscience, King of Prussia, PA, USA) Deglycosylation The glycosylation status of nicastrin was analyzed as described previously [36] Briefly, samples from the DRM preparation were denatured by heating for 10 at 100 °C in the presence of 0.5% v ⁄ v SDS and 1.0% v ⁄ v b-mercaptoethanol, cooled on ice and adjusted to 50 mm sodium citrate, pH 5.5 For Endo H treatment, 100 milliunits (as defined by the supplier) of Endo H (Roche Applied Science) was added For PNGase F treatment, NP-40 was added to a final concentration of 1.0%, followed by addition of 15.4 milliunits (as defined by the supplier) of PNGase F (Roche Applied Science) The samples were incubated overnight at 37 °C and analyzed by SDS–PAGE FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-Y Hur et al Postmortem time study The rats were killed and kept at room temperature for h In order to simulate a slow cooling curve (as in the case of the human brain), the head was removed, put in plastic bag, placed in a styrofoam box filled with water (37 °C), and the box was placed in a cold room (4 °C) [43] After 6, 12, 24 or 48 h, the brains were removed, and stored at )70 °C before use To obtain fresh tissue (postmortem time h), the rat was killed and the brain was immediately removed and homogenized Immunofluorescence labeling and confocal microscopy Cryopreserved human brain sections from the frontal cortex embedded in Tissue-TEK OCT compound (Miles, Elkhart, IN, USA), were cut in 12 lm thick sections, mounted on Hypertema Teflon-coated glass slides (Novakemi, Stockholm, Sweden) and air-dried For staining of lipid rafts, the brain tissues were labeled using fluorescent cholera toxin subunit B (CT-B) (Vybrant Alexa Fluor 488 lipid raft labeling kits, Invitrogen Molecular Probes, Carlsbad, CA, USA) and incubated with anti-CT-B The brain tissues were fixed in 4% formaldehyde and 4% sucrose, and permeabilized with 0.2% Triton X-100 After the blocking step with blocking buffer (DAKO protein block serum-free; Dako, Gidstrup, Denmark), primary and secondary antibodies were diluted in DAKO blocking buffer For labeling of PS1, the PS1-loop antibody followed by an Alexa Fluor 594-conjugated anti-mouse serum (Invitrogen Molecular Probes) was used The nicastrin antibody (AB5890; Chemicon International, Temecula, CA, USA) and an Alexa Fluor 594-conjugated anti-pig serum were used for labeling of nicastrin Anti-APP-CT20 C-terminal (171610; Calbiochem) and Alexa Fluor 488-conjugated anti-rabbit sera were used for APP To reduce the background of staining, we used autofluorescence eliminator reagent (Chemicon International) All samples were visualized using an inverted laser scanning microscope (LSM 510 META; Zeiss, Thornwood, NY, USA) Acknowledgements We thank Dr Jan Naslund (Karolinska Institutet) for ă the UD1 antibody, Dr Paul M Mathews (The Nathan S Kline Institute) for the C1 ⁄ 6.1 antibody, and Dr Takeshi Nishimura (Dainippon Sumitomo Pharma) for C99-FLAG We thank Dr Nenad Bogdanovic and Inga Volkmann (Karolinska Institutet) for skillful assistance with human brain tissue preparation This work was supported by Dainippon Sumitomo Pharma, by the Osher Foundation, Gamla Tjanarinnor (H.W.), ă Socialstyrelsen (H.W and L.O.T.), the Foundation for Human brain c-secretase in DRMs Alzheimer’s and Dementia Research (SADF) and the Foundation for Geriatric Diseases at the Karolinska Institutet (J.F.) 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stability of dopamine, glutamate decarboxylase and choline acetyltransferase in the mouse brain under conditions simulating the handling of human autopsy material J Neurochem 31, 381–383 FEBS Journal 275 (2008) 1174–1187 ª 2008 The Authors Journal compilation ª 2008 FEBS 1187 ... of c-secretase components and a lipid raft marker in thin sections of human brain In summary, our data indicates that the active c-secretase complex is localized to lipid rafts in human brain. .. access to human brain material is often limited, and rat and human brain showed similar results in our experiments, we suggest that rat brain is a useful substitute for human brain in studies on c-secretase. .. rafts and c-secretase in human brain tissue Immunofluorescence labeling was performed on human brain sections The nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) The cholera toxin subunit