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• W1994521227 abstract "The major molecular risk factor for Alzheimer disease so far identified is the amyloidogenic peptide Aβ42. In addition, growing evidence suggests a role of cholesterol in Alzheimer disease pathology and Aβ generation. However, the cellular mechanism of lipid-dependent Aβ production remains unclear. Here we describe that the two enzymatic activities responsible for Aβ production, β-secretase and γ-secretase, are inhibited in parallel by cholesterol reduction. Importantly, our data indicate that cholesterol depletion within the cellular context inhibits both secretases additively and independently from each other. This is unexpected because the β-secretase β-site amyloid precursor protein cleaving enzyme and the presenilin-containing γ-secretase complex are structurally different from each other, and these enzymes are apparently located in different subcellular compartments. The parallel and additive inhibition has obvious consequences for therapeutic research and may indicate an intrinsic cross-talk between Alzheimer disease-related amyloid precursor protein processing, amyloid precursor protein function, and lipid biology. The major molecular risk factor for Alzheimer disease so far identified is the amyloidogenic peptide Aβ42. In addition, growing evidence suggests a role of cholesterol in Alzheimer disease pathology and Aβ generation. However, the cellular mechanism of lipid-dependent Aβ production remains unclear. Here we describe that the two enzymatic activities responsible for Aβ production, β-secretase and γ-secretase, are inhibited in parallel by cholesterol reduction. Importantly, our data indicate that cholesterol depletion within the cellular context inhibits both secretases additively and independently from each other. This is unexpected because the β-secretase β-site amyloid precursor protein cleaving enzyme and the presenilin-containing γ-secretase complex are structurally different from each other, and these enzymes are apparently located in different subcellular compartments. The parallel and additive inhibition has obvious consequences for therapeutic research and may indicate an intrinsic cross-talk between Alzheimer disease-related amyloid precursor protein processing, amyloid precursor protein function, and lipid biology. Aβ peptides are the main proteinaceous component of Alzheimer disease amyloid plaques. Aβ is derived from post-translational cleavage of the amyloid precursor protein (APP). 3The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer disease; FAD, familial Alzheimer disease; Aβ, β-amyloid peptide; CTF, C-terminal fragment; ER, endoplasmic reticulum; TGN, trans-Golgi network; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; SFV, Semliki-Forest virus; BACE, β-site APP cleaving enzyme; SP, signal peptide; apoE, apolipoprotein E; PS, presenilin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Cleavage of APP by BACE I (1Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3327) Google Scholar) at the N terminus of the Aβ sequence generates a C-terminal fragment (C99) that includes the entire Aβ sequence. In mouse cortical neurons BACE I is essential for APP β-cleavage (2Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (958) Google Scholar). A second proteolytic activity termed γ-secretase cleaves APP at the C-terminal end of the Aβ sequence, releasing Aβ40 and Aβ42 during normal cellular metabolism of APP (3Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar, 4Shoji M. Golde T.E. Ghiso J. Cheung T.T. Estus S. Shaffer L.M. Cai X.D. McKay D.M. Tintner R. Frangione B. Younkin S.G. Science. 1992; 258: 126-129Crossref PubMed Scopus (1327) Google Scholar). A fraction of APP is processed by the α-secretase pathway in which APP is cleaved within the Aβ region thus precluding Aβ formation. However, neurons predominately use the β-secretory pathway at the expense of the α-secretory pathway to process APP (5Simons M. de Strooper B. Multhaup G. Tienari P.J. Dotti C.G. Beyreuther K. J. Neurosci. 1996; 16: 899-908Crossref PubMed Google Scholar). Moreover, neurons produce significant amounts of intracellular Aβ in vivo and in vitro (6Wertkin A.M. Turner R.S. Pleasure S.J. Golde T.E. Younkin S.G. Trojanowski J.Q. Lee V.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9513-9517Crossref PubMed Scopus (195) Google Scholar, 7Tienari P.J. Ida N. Ikonen E. Simons M. Weidemann A. Multhaup G. Masters C.L. Dotti C.G. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4125-4130Crossref PubMed Scopus (145) Google Scholar, 8Gouras G.K. Tsai J. Naslund J. Vincent B. Edgar M. Checler F. Greenfield J.P. Haroutunian V. Buxbaum J.D. Xu H. Greengard P. Relkin N.R. Am. J. Pathol. 2000; 156: 15-20Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar). A specific feature of γ-secretase is that it is capable of cleaving APP only after a major part of the APP luminal domain is removed. Under normal circumstances it is therefore not possible to assay γ-secretase activity directly. Analyses of APP-FAD mutations (9Hardy J. Acta Neurol. Scand. Suppl. 1996; 165: 13-17Crossref PubMed Scopus (31) Google Scholar) as well as of PS-FAD mutations (10Borchelt D.R. Thinakaran G. Eckman C.B. Lee M.K. Davenport F. Ratovitsky T. Prada C.M. Kim G. Seekins S. Yager D. Slunt H.H. Wang R. Seeger M. Levey A.I. Gandy S.E. Copeland N.G. Jenkins N.A. Price D.L. Younkin S.G. Sisodia S.S. Neuron. 1996; 17: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar) have corroborated the assumption that a small increase in Aβ42 levels causes AD (11Younkin S.G. Ann. Neurol. 1995; 37: 287-288Crossref PubMed Scopus (303) Google Scholar). The subcellular activities of both β- and γ-secretase have been extensively studied. Processing of APP to Aβ differs for different intracellular compartments (12Grimm H.S. Beher D. Lichtenthaler S.F. Shearman M.S. Beyreuther K. Hartmann T. J. Biol. Chem. 2003; 278: 13077-13085Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and depends among others on the interaction of membrane composition and the APP transmembrane domain (13Grziwa B. Grimm M.O. Masters C.L. Beyreuther K. Hartmann T. Lichtenthaler S.F. J. Biol. Chem. 2003; 278: 6803-6808Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Variable amounts of β-secretase activity were found along the secretory pathway starting in the ER/intermediate compartment, post-Golgi vesicles, TGN, and endosomes (14Chyung A.S. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (137) Google Scholar, 15Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (454) Google Scholar). In contrast, γ-secretase activity was found to be prominent in the ER, TGN, and plasma membrane and to produce different Aβ isoforms in different compartments (reviewed by Hartmann (16Hartmann T. Eur. Arch. Psychiatry Clin. Neurosci. 1999; 249: 291-298Crossref PubMed Scopus (64) Google Scholar) and in Ref. 74Bunnell W.L. Pham H.V. Glabe C.G. J. Biol. Chem. 1998; 273: 31947-31955Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). APP processing by the ER/intermediate compartment pathway in neurons generated only Aβ42 (17Hartmann T. Bieger S.C. Bruhl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 18Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar), whereas Aβ40 was produced in TGN (17Hartmann T. Bieger S.C. Bruhl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 19Greenfield J.P. Tsai J. Gouras G.K. Hai B. Thinakaran G. Checler F. Sisodia S.S. Greengard P. Xu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 742-747Crossref PubMed Scopus (331) Google Scholar). There is a strong connection between AD and lipid metabolism. There are a number of findings that indicate that cholesterol lowering might be of use in AD therapy. Several epidemiological studies showed a strongly decreased prevalence of AD and other dementias in patients that underwent treatment with statins (20Wolozin B. Kellman W. Ruosseau P. Celesia G.G. Siegel G. Arch. Neurol. 2000; 57: 1439-1443Crossref PubMed Scopus (1345) Google Scholar, 21Jick H. Zornberg G.L. Jick S.S. Seshadri S. Drachman D.A. Lancet. 2000; 356: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (1591) Google Scholar). Two high dosage pilot intention-to-treat clinical trials using atorvastatin or simvastatin in patients with mild to moderate Alzheimer disease showed a slower cognitive decline in the treatment group (23Sparks D.L. Sabbagh M. Connor D.J. Lopez J. Launer L.J. Browne P. Wasser D. Johnson-Traver S. Lochhead J. Ziolwolski C. Arch. Neurol. 2005; 62: 753-757Crossref PubMed Scopus (385) Google Scholar, 24Simons M. Schwarzler F. Luthjohann D. von Bergmann K. Beyreuther K. Dichgans J. Wormstall H. Hartmann T. Schulz J.B. Ann. Neurol. 2002; 52: 346-350Crossref PubMed Scopus (381) Google Scholar) and statin treatment may favor the non-amyloidogenic pathway of APP processing in AD patients treated with simvastatin (25Hoglund K. Thelen K.M. Syversen S. Sjogren M. von Bergmann K. Wallin A. Vanmechelen E. Vanderstichele H. Luthjohann D. Blennow K. Dement. Geriatr. Cogn. Disord. 2005; 19: 256-265Crossref PubMed Scopus (90) Google Scholar). Although these findings indicate a molecular link between cholesterol and AD too little is still known about the molecular mechanisms involved and it currently remains unclear whether cholesterol lowering is sufficiently effective for AD therapy or prevention. Statins are inhibitors of the key enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). They are widely used and well tolerated drugs, active in various cell types including brain cells (26Vaughan C.J. Delanty N. Stroke. 1999; 30: 1969-1973Crossref PubMed Scopus (360) Google Scholar), but the first potential link between lipids and AD was established with apolipoprotein E (apoE), a protein involved in cholesterol transport between tissues (27Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). In AD the apoE ϵ-4 allele reduces the age of disease onset (28Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Science. 1999; 261: 921-923Crossref Scopus (7342) Google Scholar) and cholesterol and apoE genotype interact to influence progression of Alzheimer disease (29Evans R.M. Hui S. Perkins A. Lahiri D.K. Poirier J. Farlow M.R. Neurology. 2004; 62: 1869-1871Crossref PubMed Scopus (87) Google Scholar). The link between lipids and AD has recently been significantly extended with findings connecting elevated cholesterol levels with increased amyloid plaque formation in animal models (30Sparks D.L. Scheff S.W. Hunsaker 3rd, J.C. Liu H. Landers T. Gross D.R. Exp. Neurol. 1994; 126: 88-94Crossref PubMed Scopus (517) Google Scholar, 31Kuo Y.M. Emmerling M.R. Bisgaier C.L. Essenburg A.D. Lampert H.C. Drumm D. Roher A.E. Biochem. Biophys. Res. Commun. 1998; 252: 711-715Crossref PubMed Scopus (298) Google Scholar, 32Refolo L.M. Pappolla M.A. Malester B. LaFrancois J. Bryant-Thomas T. Wang R. Tint G.S. Sambamurti K. Duff K. Neurobiol. Dis. 2000; 7: 321-331Crossref PubMed Scopus (889) Google Scholar) and Aβ aggregation in vitro (33Yamazaki T. Chang T.V. Haass C. Ihara Y. J. Biol. Chem. 2001; 276: 4454-4460Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). On a cellular level cholesterol has been identified to be a relevant factor regulating Aβ production in vitro (34Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6460-6464Crossref PubMed Scopus (1085) Google Scholar, 35Frears E.R. Stephens D.J. Walters C.E. Davies H. Austen B.M. Neuroreport. 1999; 10: 1699-1705Crossref PubMed Scopus (329) Google Scholar, 36Puglielli L. Konopka G. Pack-Chung E. MacKenzie Ingano L.A. Berezovska O. Hyman B.T. Chang T.Y. Tanzi R.E. Kovacs D.M. Nat. Cell. Biol. 2001; 3: 905-912Crossref PubMed Scopus (396) Google Scholar, 37Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (365) Google Scholar) and in vivo (32Refolo L.M. Pappolla M.A. Malester B. LaFrancois J. Bryant-Thomas T. Wang R. Tint G.S. Sambamurti K. Duff K. Neurobiol. Dis. 2000; 7: 321-331Crossref PubMed Scopus (889) Google Scholar, 38Refolo L.M. Pappolla M.A. LaFrancois J. Malester B. Schmidt S.D. Thomas-Bryant T. Tint G.S. Wang R. Mercken M. Petanceska S.S. Duff K.E. Neurobiol. Dis. 2001; 8: 890-899Crossref PubMed Scopus (482) Google Scholar), whereas amyloid oligomers can change membrane function (75Sokolov Y. Kozak J.A. Kayed R. Chanturiya A. Glabe C. Hall J.E. J. Gen. Physiol. 2006; 128: 637-664Crossref PubMed Scopus (182) Google Scholar). β-Secretase activity can be stimulated by cholesterol (39Kalvodova L. Kahya N. Schwille P. Ehehalt R. Verkade P. Drechsel D. Simons K. J. Biol. Chem. 2005; 280: 36815-36823Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), the γ-secretase complex and BACE I have been found associated with lipid microdomains (40Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 22: 44945-44954Abstract Full Text Full Text PDF Scopus (365) Google Scholar, 41Cordy J.M. Hussain I. Dingwall C. Hooper N.M. Turner A.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11735-11740Crossref PubMed Scopus (312) Google Scholar). Moreover, γ-secretase activity is increased in cellular compartments in which cholesterol accumulates (42Runz H. Rietdorf J. Tomic I. de Bernard M. Beyreuther K. Pepperkok R. Hartmann T. J. Neurosci. 2002; 22: 1679-1689Crossref PubMed Google Scholar). The enzyme cascade of γ-secretase, APP processing, and Aβ is mechanistically linked to lipid homeostasis because Aβ regulates cholesterol and sphingomyelin homeostasis via HMG-CoA reductase inhibition and sphingomyelinase activation (43Grimm M.O. Grimm H.S. Patzold A.J. Zinser E.G. Halonen R. Duering M. Tschape J.A. De Strooper B. Muller U. Shen J. Hartmann T. Nat. Cell Biol. 2005; 7: 1118-1123Crossref PubMed Scopus (357) Google Scholar). Here we analyzed the cellular mechanism by which cellular cholesterol levels influence intracellular Aβ production. For this purpose we utilized a set of APP deletion mutants that allowed us also to analyze the influence of cholesterol on each of the Aβ generating enzymes independently of each other. Our results demonstrate that γ-secretase activity is regulated by cellular cholesterol levels and corroborate that cellular cholesterol levels also regulate β-secretase activity. Importantly, our results show that regulation of β- and γ-secretase activities by cellular cholesterol levels are, at least in part, independent of each other. This indicates that cellular cholesterol levels regulate the enzymatic activity of both secretases by different mechanisms and may indicate ways to specifically target β-secretase activity. Constructs—To clarify the modulation of cellular cholesterol levels on Aβ generation we took advantage of comparisons between the processing of APP (34Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6460-6464Crossref PubMed Scopus (1085) Google Scholar) and of two truncated APP constructs termed C99 and C111 (Fig. 1). Whereas β-secretase is able to cleave full-length APP, γ-secretase requires previous APP cleavage by another secretase. Therefore, we used a construct termed SP-C99, which mimics β-cleaved APP, a substrate for γ-secretase, which directly yields Aβ. Thus, in comparison to full-length APP, this construct allowed us to examine Aβ generation independent of β-secretase. It was previously shown that directly expressed SP-C99 protein is processed to Aβ40 and Aβ42 in the same way as full-length APP (48Lichtenthaler S.F. Ida N. Multhaup G. Masters C.L. Beyreuther K. Biochemistry. 1997; 36: 15396-15403Crossref PubMed Scopus (80) Google Scholar). To facilitate efficient removal of the signal peptide by SP peptidase, the dipeptide LE was introduced directly between the original APP signal peptide and C99 (49Dyrks T. Dyrks E. Masters C. Beyreuther K. FEBS Lett. 1992; 309: 20-24Crossref PubMed Scopus (30) Google Scholar). To further address the mechanisms involved in cholesterol-dependent regulation of Aβ production we used another construct termed SP-C111, which has the structure of δ-secretase-cleaved APP. This δ cleavage is a naturally occurring secretase cleavage event 12 amino acids N-terminal of the β-cleavage (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). Therefore C111 is identical to C99 except that it contains the 12 amino acids preceding the Aβ domain. This construct resembles APP as it requires β- and γ-secretase cleavage for Aβ production. However, due to the small luminal domain the β-cleavage is not an essential prerequisite for γ-secretase cleavage. Without β-cleavage, γ-cleavage of Sp-C111 produces a 5.5-kDa Aβ-like peptide consisting of the Aβ-domain and the first 12 amino acids preceding the Aβ-domain. In this regard C111 resembles more the C99 construct that does not require β-secretase cleavage for γ-secretase activation (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). Thus, the relevant Aβ generating secretases may act here independent of each other. This allowed us to analyze whether the observed inhibition of β-secretase and γ-secretase activities are due to the same or distinct mechanisms triggered by reducing the cellular cholesterol content. Moreover, C111 allowed us to analyze further the possible additive effects of β- and γ-secretase inhibition that were indicated by comparison of the APP and C99 inhibition profiles. The SpeI site in the 3′-untranslated region of the APP cDNA was removed by Klenow polymerase and the resultant blunt ends were re-ligated prior to cloning into the expression vector pSFV1 (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). This was necessary as the pSFV1/APP have to be linearized from the SpeI site of pSFV1. The APP cDNA was cloned into the SmaI site of the pSFV1 expression vector (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). Preparation of Recombinant SFV—The pSFV1/APP, pSFV1/C99, and pSFV1/C111 and pSFV helper DNAs were linearized with SpeI and in vitro transcribed as described (76Liljestrom P. Garoff H. Biotechnology. 1991; 9: 1356-1361Crossref PubMed Scopus (747) Google Scholar). The transcription mixture of both pSFV1/APP and pSFV1/C99 or pSFV1/C111 and pSFVhelper was co-transfected into baby hamster kidney cells using electroporation (77Olkkonen V.M. Liljestrom P. Garoff H. Simons K. Dotti C.G. J. Neurosci. Res. 1993; 35: 445-451Crossref PubMed Scopus (70) Google Scholar). The baby hamster cells were cultured in Glasgow's modified Eagle's medium supplemented with 5% fetal bovine serum, 2 mm glutamate, and 10% tryptose phosphate broth. The culture supernatant containing infective recombinant SFV particles was collected 36 h after electroporation (77Olkkonen V.M. Liljestrom P. Garoff H. Simons K. Dotti C.G. J. Neurosci. Res. 1993; 35: 445-451Crossref PubMed Scopus (70) Google Scholar). Cell Culture and Infections—Hippocampal neurons were prepared as described elsewhere (44De Hoop, M. J., Meyn, L., and Dotti, C. G. (1998) in Cell Biology, A Laboratory Handbook (Cellis, J., ed) Vol. 1, 2nd Ed., pp. 154–163, Academic Press, San DiegoGoogle Scholar). The glial feeding layer was replaced by addition of the B27 supplement (45Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. J. Neurosci. Res. 1993; 35: 567-576Crossref PubMed Scopus (1912) Google Scholar). Primary mixed cortical neurons were prepared from 14-day-old fetal rats as described earlier with slight modifications (46De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 87-90Crossref Scopus (1560) Google Scholar). Briefly pregnant Sprague-Dawley rats were sacrificed by etherization at gestation day E14, brains were dissected, cells were plated on 10 μg/ml poly-l-lysine (Sigma)-coated dishes and kept in minimal essential medium supplemented with 0.2% ovalbumin, 1% N2 supplement, and 1% B27 supplement at 5% CO2 and 36.5 °C. In the absence of a glial feeding layer, but with B27 supplement, neurons survived for 30 days or more. Experiments were performed at the age of 6–10 days. At this time neurons were mature and fully polarized (47Dotti C.G. Sullivan C.A. Banker G.A. J. Neurosci. 1988; 8: 1454-1468Crossref PubMed Google Scholar). Cell culture agents were purchased from Invitrogen (Karlsruhe). Infections were performed by diluting the recombinant SFV particles with serum-free N2 medium. To allow viral entry infection was continued for 1 h, the virus solution was then removed and incubation continued for 4–6 h to allow SFV-driven protein synthesis (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). Antibodies—Monoclonal antibodies G2-10 and G2-11 specific for Aβ40 and Aβ42, respectively, were used for immunoprecipitation. Antibody W0-2 directed against amino acids 4–10 of human Aβ was used for detection (50Jensen M. Hartmann T. Engvall B. Wang R. Uljon S.N. Sennvik K. Naslund J. Muehlhauser F. Nordstedt C. Beyreuther K. Lannfelt L. Mol. Med. 2000; 6: 291-302Crossref PubMed Google Scholar). W0-2 antibody (1 μg/ml) was used for detection of G2-10 and G2-11 precipitates to facilitate a greater linear range, better detection levels, and to avoid detection of endogenous rat Aβ40,Aβ42, and APP. W0-2 is specific for the human Aβ sequence. P3 was detected with G2-10. Polyclonal antibody 22/13 raised against the C-terminal 13 residues of C99 (13Grziwa B. Grimm M.O. Masters C.L. Beyreuther K. Hartmann T. Lichtenthaler S.F. J. Biol. Chem. 2003; 278: 6803-6808Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was used for immunoprecipitation and detection of C-terminal fragments. Cholesterol Depletion—For cholesterol depletion of primary neurons we used two different strategies. De novo cholesterol synthesis was inhibited by lovastatin, an inhibitor of HMG-CoA reductase. A basic level of mevalonate was maintained by adding mevalonate to the cell culture media to avoid toxicity due to inhibition of non-steroidal pathways (51Langan T.J. Volpe J.J. J. Neurochem. 1987; 49: 513-521Crossref PubMed Scopus (46) Google Scholar, 52Rao S. Porter D.C. Chen X. Herliczek T. Lowe M. Keyomarsi K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7797-7802Crossref PubMed Scopus (336) Google Scholar). Plasma membrane cholesterol was extracted by methyl-β-cyclodextrin. Methyl-β-cyclodextrin is a well established tool that selectively and quickly extracts cholesterol from plasma membranes in preference to other lipids (53Kilsdonk E.P. Yancey P.G. Stoudt G.W. Bangerter F.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). These treatments reduce intracellular cholesterol without affecting cell viability and polarity within the time window selected for the experiments (54Fassbender K. Simons M. Bergmann C. Stroick M. Lutjohann D. Keller P. Runz H. Kuhl S. Bertsch T. von Bergmann K. Hennerici M. Beyreuther K. Hartmann T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5856-5861Crossref PubMed Scopus (1036) Google Scholar, 55Keller P. Simons K. J. Cell Biol. 1998; 140: 1357-1367Crossref PubMed Scopus (472) Google Scholar). After 4–8 days in culture, 4 μm lovastatin (Calbiochem) and 0.25 mm mevalonate (Sigma) were added for 48 h. Control cells were left untreated. Recombinant SFV encoding SP-APP695, SP-C99, or SP-C111 were prepared as described elsewhere (56Tienari P.J. De Strooper B. Ikonen E. Simons M. Weidemann A. Czech C. Hartmann T. Ida N. Multhaup G. Masters C.L. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (124) Google Scholar). Neurons between 6 and 10 days of age were infected for 90 min at 36.5 °C and 5% CO2 with recombinant SFV. This solution was replaced by maintenance medium and cells incubated for 2 h. Cells were then treated with 5 mm methyl-β-cyclodextrin (Sigma) for 10 min and further incubated for 4.5 h. These extraction times did not completely abolish Aβ production, therefore allowing quantification of the effects of cholesterol depletion on the production of Aβ40/Aβ42 and their ratio. For some experiments different amounts of extraction times were used as indicated. Immunoprecipitation and Quantification—Cell extracts were prepared in 1% Nonidet P-40, 1% Triton X-100, 10 mm Tris, 2 mm EDTA, supplemented with 2× Complete protease inhibitor (Roche Diagnostics). Because Aβ42 constitutes only a minor amount of total Aβ, different amounts of cell lysate were used for immunoprecipitation to obtain similar signal intensity. Cell lysates were divided for Aβ42 (80%) and Aβ40 immunoprecipitation (20%). Immunoprecipitates were recovered on protein G-Sepharose (Sigma) and analyzed on 10–20% Tris-Tricine polyacrylamide gels (Novex, Frankfurtg/Main). Quantitative Western blotting was performed as described (57Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), except that 3% milk powder instead of bovine serum albumin was used for blocking. For detection of CTFs cell lysates were immunoprecipitated with polyclonal antibody 22/13 raised against the C-terminal 13 residues of C99 (13Grziwa B. Grimm M.O. Masters C.L. Beyreuther K. Hartmann T. Lichtenthaler S.F. J. Biol. Chem. 2003; 278: 6803-6808Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and recovered on protein A-Sepharose (Amersham Biosciences). For detection, antibody 22/13 was used in a dilution of 1:5000. For detection of p3, conditioned medium was collected and centrifuged at 10,000 × g for 10 min. The supernatant was then immunoprecipitated with G2-10 and Western blot analysis was performed as described above with the exception that biotinylated G2-10 (50Jensen M. Hartmann T. Engvall B. Wang R. Uljon S.N. Sennvik K. Naslund J. Muehlhauser F. Nordstedt C. Beyreuther K. Lannfelt L. Mol. Med. 2000; 6: 291-302Crossref PubMed Google Scholar) and streptavidin-coated horseradish peroxidase (Pierce) were used for detection. Quantification was performed using Macbas-2 software with densitometric scans from Western blots standardized against control Aβ protein. Staining of Hippocampal Neurons with Filipin—Neurons grown on coverslips for 8 days were treated for 48 h with lovastatin and mevalonate and for 10 min with 5 mm methyl-β-cyclodextrin. After fixation on ice with 4% paraformaldehyde and permeabilization with 0.1% saponin (Sigma) neurons were stained with 125 μg/ml filipin (Sigma) in phosphate-buffered saline. Filipin is a fluorescent polyene antibiotic that forms complexes with cholesterol that can be visualized with ultraviolet light (58Yeagle P.L. Biochim. Biophys. Acta. 1985; 822: 267-287Crossref PubMed Scopus (1261) Google Scholar). For Golgi58 co-staining, filipin staining was followed by blocking with 5% goat serum (Sigma) and 3% bovine serum albumin (Biomol, Hamburg). Afterward cells were incubated with monoclonal antibody Golgi58 (Sigma) and rhodamine isothiocyanate-conjugated second antibody (Sigma). Cells were mounted in Mowiol (Hoechst, Frankfurt/Main). Digital images were taken with a Zeiss axioskop microscope equipped with a three-chip color camera (Micromax, Princeton Instruments, Trenton, NJ). β-Secretase Activity Assay—Confluent grown SH-SY5Y cells were incubated with 5 mm methyl-β-cyclodextrin or solvent as a control for 10 min and afterward cultivated at 37 °C in growth medium without fetal calf serum for 2 h. The cells were washed 3 times with phosphate-buffered saline and scraped off and after homogenization the protein amoun" @default.
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• W1994521227 date "2008-04-01" @default.
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• W1994521227 title "Independent Inhibition of Alzheimer Disease β- and γ-Secretase Cleavage by Lowered Cholesterol Levels" @default.
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• W1994521227 doi "https://doi.org/10.1074/jbc.m801520200" @default.
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