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• W2028781640 abstract "Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr668 and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648–695)) with Thr(P)668 did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease. Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr668 and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648–695)) with Thr(P)668 did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease. Alzheimer disease (AD) 3The abbreviations used are: ADAlzheimer diseaseAβamyloid β-proteinAICDAPP intracellular domain peptideAPPamyloid β-precursor proteinCTFC-terminal fragment of APP truncated at primary α- or β-cleavage siteDRMdetergent-resistant membranePSpresenilinmAPPmature APPimAPPimmature APPMβCDmethyl-β-cyclodextrinCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonateTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinepCTFphosphorylated C-terminal fragmentnCTFnonphosphorylated C-terminal fragmentpAPPAPP phosphorylated at Thr668. is the most common of the senile neurodegenerative disorders. Amyloid β-protein (Aβ) is a causative peptide of AD and is believed to show toxicity to neurons by forming oligomers (1Haass C. Selkoe D.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 101-112Crossref PubMed Scopus (3876) Google Scholar). Aβ is generated from the amyloid β-precursor protein (APP), which is a type I membrane protein composed of three major spliced isoforms, APP770, APP751, and APP695, with APP695 being expressed exclusively in neurons (2Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Müller-Hill B. The precursor of Alzheimer disease amyloid A4 protein resembles a cell-surface receptor.Nature. 1987; 325: 733-736Crossref PubMed Scopus (3943) Google Scholar, 3Rohan de Silva HA Jen A Wickenden C Jen LS Wilkinson SL Patel AJ Cell-specific expression of β-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes.Mol. Brain Res. 1997; 47: 147-156Crossref PubMed Scopus (96) Google Scholar). APP is primarily cleaved by either non-amyloidogenic α-secretase or amyloidogenic β-secretase at the juxtamembrane region, with a secondary cleavage by γ-secretase in the transmembrane domain. APP cleavage by the combination of β- and γ-secretases generates Aβ, whereas that of the combination of α- and γ-secretases generates an amyloidolytic (non-amyloidogenic) product (4Thinakaran G. Koo E. Amyloid precursor protein trafficking, processing, and function.J. Biol. Chem. 2008; 283: 29615-29619Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar). Thus, suppression of APP amyloidogenic cleavages and its associated Aβ generation or facilitation of the non-amyloidogenic cleavage of APP are thought to potentially be useful therapies for AD (5Aydin D. Weyer S.W. Müller U.C. Exp. Brain Res. 2012; 217: 423-434Crossref PubMed Scopus (69) Google Scholar). Alzheimer disease amyloid β-protein APP intracellular domain peptide amyloid β-precursor protein C-terminal fragment of APP truncated at primary α- or β-cleavage site detergent-resistant membrane presenilin mature APP immature APP methyl-β-cyclodextrin 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphorylated C-terminal fragment nonphosphorylated C-terminal fragment APP phosphorylated at Thr668. Cleavage of APP by β-secretase generates a large N-terminal sAPPβ fragment and a short, membrane-associated C-terminal fragment, CTFβ, whereas the cleavage by α-secretase occurs within the Aβ domain and generates sAPPα and CTFα. Both amyloidogenic CTFβ and non-amyloidogenic CTFα are subsequently cleaved by the γ-secretase complex composed of nicastrin (NCT), presenilin enhancer 2 (pen-2), anterior pharynx defective 1 (APH-1), and presenilin 1 or 2 (PS1 or PS2) (6Steiner H. Fluhrer R. Haass C. Intramembrane proteolysis by γ-secretase.J. Biol. Chem. 2008; 283: 29627-29631Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Following endoproteolysis to generate its N- and C-terminal fragments, presenilin forms the catalytic site of the γ-secretase complex. Both PS1 and PS2 are linked, along with APP, to causative genes for familial AD (FAD) (7De Strooper B. Annaert W. Novel research horizons for presenilins and γ-secretases in cell biology and disease.Annu. Rev. Cell Dev. Biol. 2010; 26: 235-260Crossref PubMed Scopus (199) Google Scholar). Over 150 FAD mutations in PS1 and PS2 that increase the generation of pathogenic types of Aβ species are known (8De Strooper B. Loss-of-function presenilin mutations in Alzheimer disease. Talking point on the role of presenilin mutations in Alzheimer disease.EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (280) Google Scholar). Sporadic AD also shows a similar pathogenic process but is associated with later onset and has no mutation(s) in the causative APP and PS genes. This points to other sources in the sporadic AD brain, which alter the APP processing. It is known that some of the APP-interacting proteins regulate the APP metabolism, including Aβ generation. Dysfunction of and/or aberrant regulation by these APP-interacting proteins may alter the APP metabolism without mutations in causative genes (9Saito Y. Sano Y. Vassar R. Gandy S. Nakaya T. Yamamoto T. Suzuki T. X11 proteins regulate the translocation of amyloid β-protein precursor (APP) into detergent-resistant membrane and suppress the amyloidogenic cleavage of APP by β-site-cleaving enzyme in brain.J. Biol. Chem. 2008; 283: 35763-35771Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Kondo M. Shiono M. Itoh G. Takei N. Matsushima T. Maeda M. Taru H. Hata S. Yamamoto T. Saito Y. Suzuki T. Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain.Mol. Neurodegener. 2010; 5: 35Crossref PubMed Scopus (25) Google Scholar) and suggest(s) a possible cause for sporadic AD (11Suzuki T. Nakaya T. Regulation of amyloid β-protein precursor by phosphorylation and protein interactions.J. Biol. Chem. 2008; 283: 29633-29637Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12Taru H. Suzuki T. Regulation of the physiological function and metabolism of AβPP by AβPP binding proteins.J. Alzheimers Dis. 2009; 18: 253-265Crossref PubMed Scopus (18) Google Scholar). Post-translational modifications of APP are also involved in the regulation of APP function and metabolism. APP is subject to N-glycosylation (immature APP or imAPP) in the endoplasmic reticulum and the imAPP is further modified with O-glycosylation in the Golgi complex. The mature APP (mAPP), with both N- and O-glycosylation, localizes to the late protein secretory pathway in which mAPP is cleaved by secretases (4Thinakaran G. Koo E. Amyloid precursor protein trafficking, processing, and function.J. Biol. Chem. 2008; 283: 29615-29619Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 11Suzuki T. Nakaya T. Regulation of amyloid β-protein precursor by phosphorylation and protein interactions.J. Biol. Chem. 2008; 283: 29633-29637Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The mAPP is further modified by phosphorylation, also occurring in the late secretory pathway. Phosphorylation of mAPP at Thr668 (numbering for APP695 isoform) in the cytoplasmic region is predominantly observed in neurons in the brain (13Iijima K. Ando K. Takeda S. Satoh Y. Seki T. Itohara S. Greengard P. Kirino Y. Nairn A.C. Suzuki T. Neuron-specific phosphorylation of Alzheimer β-amyloid precursor protein by cyclin-dependent kinase 5.J. Neurochem. 2000; 75: 1085-1091Crossref PubMed Scopus (205) Google Scholar). Both neurite outgrowth of differentiating PC12 cells and interaction of APP with a neural adaptor FE65 protein are affected by the phosphorylation of APP at Thr668 (14Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. Role of phosphorylation of Alzheimer amyloid precursor protein during neuronal differentiation.J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 15Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of β-amyloid.J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 16Nakaya T. Suzuki T. Role of APP phosphorylation in Fe65-dependent gene transactivation mediated by AICD.Genes Cells. 2006; 11: 633-645Crossref PubMed Scopus (69) Google Scholar). These observations suggest that phosphorylation of APP at Thr668 plays an important role in the expression and/or regulation of APP function (11Suzuki T. Nakaya T. Regulation of amyloid β-protein precursor by phosphorylation and protein interactions.J. Biol. Chem. 2008; 283: 29633-29637Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In contrast to this, the role of the phosphorylation in APP metabolism is controversial. Mutant mice possessing Ala substitution for Thr668 of APP did not show significant differences in APP metabolism, including Aβ generation, when compared with the wild-type mouse brain (17Sano Y. Nakaya T. Pedrini S. Takeda S. Iijima-Ando K. Iijima K. Mathews P.M. Itohara S. Gandy S. Suzuki T. Physiological mouse brain Aβ levels are not related to the phosphorylation state of threonine 668 of Alzheimer APP.PLoS ONE. 2006; 1: e51Crossref PubMed Scopus (52) Google Scholar). This analysis would suggest that the phosphorylation state of APP at Thr668 does not play an obvious role in the direct governing of the APP metabolism in the brain in vivo. However, contrary to this observation, several reports have indicated that phosphorylation of Thr668, or amino acid substitutions mimicking phosphorylated Thr, could regulate the cleavage by γ-secretase in cultured cells (18Liu F. Su Y. Li B. Zhou Y. Ryder J. Gonzalez-DeWhitt P. May P.C. Ni B. Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35Cdk5 and p25Cdk5.FEBS Lett. 2003; 547: 193-196Crossref PubMed Scopus (90) Google Scholar, 19Lee M.S. Kao S.C. Lemere C.A. Xia W. Tseng H.C. Zhou Y. Neve R. Ahlijanian M.K. Tsai L.H. APP processing is regulated by cytoplasmic phosphorylation.J. Cell Biol. 2003; 163: 83-95Crossref PubMed Scopus (361) Google Scholar, 20Pastorino L. Sun A. Lu P.J. Zhou X.Z. Balastik M. Finn G. Wulf G. Lim J. Li S.H. Li X. Xia W. Nicholson L.K. Lu K.P. The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production.Nature. 2006; 440: 528-534Crossref PubMed Scopus (408) Google Scholar, 21Pierrot N. Santos S.F. Feyt C. Morel M. Brion J.P. Octave J.N. Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal amyloid-β accumulation.J. Biol. Chem. 2006; 281: 39907-39914Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 22Rockenstein E. Torrance M. Adame A. Mante M. Bar-on P. Rose J.B. Crews L. Masliah E. Neuroprotective effects of regulators of the glycogen synthase kinase-3β signaling pathway in a transgenic model of Alzheimer disease are associated with reduced amyloid precursor protein phosphorylation.J. Neurosci. 2007; 27: 1981-1991Crossref PubMed Scopus (255) Google Scholar, 23Feyt C. Pierrot N. Tasiaux B. Van Hees J. Kienlen-Campard P. Courtoy P.J. Octave J.N. Phosphorylation of APP695 at Thr668 decreases γ-cleavage and extracellular Aβ.Biochem. Biophys. Res. Commun. 2007; 357: 1004-1010Crossref PubMed Scopus (29) Google Scholar). Therefore, the ability of phosphorylation at Thr668 to induce alterations in the cleavage of APP at β- and/or γ-secretase sites is still controversial in AD brains. The phosphorylation of APP at Thr668 can likely induce a change in the overall structure of its cytoplasmic domain because Thr668 is located in the 667VTPEER672 motif, which forms a type I β-turn and amino-terminal helix-capping box structure to stabilize its carboxyl-terminal helix structure (24Ramelot T.A. Gentile L.N. Nicholson L.K. Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.Biochemistry. 2000; 39: 2714-2725Crossref PubMed Scopus (76) Google Scholar, 25Ramelot T.A. Nicholson L.K. Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR.J. Mol. Biol. 2001; 307: 871-884Crossref PubMed Scopus (129) Google Scholar). The usual procedure to explore the function of phosphorylation of a protein is to mimic the phosphorylation state by amino acid substitutions of Asp or Glu for the appropriate Thr and Ser residues (26Tomita S. Stein V. Stocker T.J. Nicoll R.A. Bredt D.S. Bidirectional synaptic plasticity regulated by phosphorylation of stargazin-like TARPs.Neuron. 2005; 45: 269-277Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). However, this strategy might not be suitable in the case of APP phosphorylation as the substitution of Asp for Thr668 did not alter the carboxyl-terminal helix state as remarkably as did phosphorylation of Thr668 (supplemental Fig. S1). In contrast, substitution with Ala668 for Thr668 of APP is found to effectively mimic the nonphosphorylated state in the helix structure of the APP cytoplasmic domain (supplemental Fig. S1) and in localization and metabolism of APP in the brain (17Sano Y. Nakaya T. Pedrini S. Takeda S. Iijima-Ando K. Iijima K. Mathews P.M. Itohara S. Gandy S. Suzuki T. Physiological mouse brain Aβ levels are not related to the phosphorylation state of threonine 668 of Alzheimer APP.PLoS ONE. 2006; 1: e51Crossref PubMed Scopus (52) Google Scholar). Therefore, to reveal the role of APP phosphorylation at Thr668, we carefully examined the phosphorylation state of both APP and APP metabolic fragments in the brain, in vivo. Furthermore, because the phosphorylation state of the CTFs is higher than that of mAPP, we focused on the features of the phosphorylated forms of the CTFs as regards to their susceptibility to γ-secretase cleavage and lipid binding, in vitro. Finally, we analyzed the age-dependent changes of the phosphorylation state of CTFβ in cynomolgus monkey brain in which amyloid plaque formation has progressed in an age-dependent manner (27Kimura N. Tanemura K. Nakamura S. Takashima A. Ono F. Sakakibara I. Ishii Y. Kyuwa S. Yoshikawa Y. Age-related changes of Alzheimer disease-associated proteins in cynomolgus monkey brains.Biochem. Biophys. Res. Commun. 2003; 310: 303-311Crossref PubMed Scopus (74) Google Scholar). The cDNA encoding the cytoplasmic domain of APP (APPcyt) was inserted into pGEX4T-1 (GE Healthcare) at the EcoRI/BamHI sites to produce a GST fusion protein (28Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. Interaction of a neuron-specific protein containing PDZ domains with Alzheimer amyloid precursor protein.J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 29Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. Interaction of Alzheimer β-amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade.J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The cDNA encoding APPcyt ΔC8 (APP(649–687), a cytoplasmic domain of APP695 lacking C-terminal 8 amino acids) was produced by PCR and cloned into pGEX4T-1. pcDNA3-FLAG-APP695 was described (14Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. Role of phosphorylation of Alzheimer amyloid precursor protein during neuronal differentiation.J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar). To prepare pcDNA3-FLAG-APPΔC8, the cDNA in pcDNA3-FLAG-APP695 was amplified by PCR using specific primers. The PCR products were digested with HindIII/XbaI and cloned into the pcDNA3 (Invitrogen). All of the animal studies were conducted in compliance with the guidelines of the Animal Studies Committee of Hokkaido University, Shiga University of Medical Science, and the National Institute of Biomedical Innovation. The C57BL/6 line of mice housed in an SPF environment were used throughout these studies. Brain samples of cynomolgus monkey (Macaca fascicularis) were obtained from the Shiga University of Medical Science and the National Institute of Biomedical Innovation. Monkeys were housed in individual cages and maintained according to guidelines for experimental animal welfare. Membrane fractions were prepared from brains of wild-type mice (C57BL/6, 4 months old) as described (16Nakaya T. Suzuki T. Role of APP phosphorylation in Fe65-dependent gene transactivation mediated by AICD.Genes Cells. 2006; 11: 633-645Crossref PubMed Scopus (69) Google Scholar). To dephosphorylate proteins, membrane lysates (30 μg of protein) were treated with λ-protein phosphatase (400 unit; Sigma) for 4 h at 30 °C. The immunoblotting procedure was described previously (17Sano Y. Nakaya T. Pedrini S. Takeda S. Iijima-Ando K. Iijima K. Mathews P.M. Itohara S. Gandy S. Suzuki T. Physiological mouse brain Aβ levels are not related to the phosphorylation state of threonine 668 of Alzheimer APP.PLoS ONE. 2006; 1: e51Crossref PubMed Scopus (52) Google Scholar). In brief, for detection of APP CTFs, membrane lysate proteins were separated by electrophoresis on a 17.5% (w/v) polyacrylamide Tris-Tricine gel, transferred onto a nitrocellulose membrane, and incubated with anti-APP C-terminal A8717 (Sigma) or G369 (30Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. The cytoplasmic domain of Alzheimer amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar) (gift from Dr. S. Gandy), anti-Thr(P)668 of APP (anti-pAPP, Cell Signaling), or anti-actin (Chemicon) antibodies. Immunoreactants were further reacted with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibody (GE Healthcare) and detected with ECL Plus (GE Healthcare). Proteins were separated by a standard SDS electrophoresis with 8 (w/v) or 15% (w/v) polyacrylamide Tris glycine gel and detected by immunoblotting with the indicated antibodies; PS1 N-terminal Ab14 (31Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar) (gift from Dr. S. Gandy), PS1 C-terminal (Chemicon), pen-2 (Zymed Laboratories Inc.), and GST (Upstate Biotechnology). The protein band intensities detected by ECL were quantified with an imaging analyzer, VersaDoc (Bio-Rad). Cynomolgus monkey brain lysates were prepared from frozen stocks of temporal cortices. The tissue blocks were homogenized in a 10-fold volume of a lysis buffer (50 mm Tris-HCl, pH 8.0, containing 0.5% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 150 mm NaCl) containing a protease inhibitor mixture (Sigma) and 1 μm microcystin-LR (Wako Pure Chemical) and then lysed by sonication in ice. The resulting supernatant was used for immunoblot analysis with mouse monoclonal anti-Tau HT7 (Thermo Scientific) and anti-phosphorylated Tau AT8 (Ser(P)202 and Thr(P)205, Innogenetics) antibodies, along with immunoblotting with anti-APP C-terminal A8717 (Sigma), anti-phosphorylated APP (Thr(P)668, Cell Signaling), and anti-actin (Chemicon) antibodies. APP CTFs detected by immunoblotting with anti-APP C-terminal and anti-pAPP antibodies were quantified with VersaDoc (Bio-Rad). The values of phosphorylated C99 (pC99) CTFβ were set to 1.0 and the level of the other CTF species are defined as a ratio to that of pC99. In practice, we estimated the ratio of CTFs using the following calculations: pC99:nC99:pC89:(pC83 + nC89):nC83 = 1:a:b:c:d, these ratios being determined by immunoblotting with anti-C-terminal antibody, and pC99:pC89:pC83 = 1:b:e, these ratios being determined by immunoblotting with anti-pAPP antibody. Therefore, the relative ratios of all six CTFs were expressed as: pC99:nC99:pC89:nC89:pC83:nC83 = 1:a:b:(c-e):e:d. Data were obtained from three independent experiments. The levels of phosphorylated (pCTFs) and nonphosphorylated CTFs (nCTFs) were combined and indicated as total levels of CTFs. Wild-type C57BL/6 mouse (4 months old) brains were suspended in 5 volumes of buffer H (20 mm Hepes-NaOH, pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% (v/v) glycerol) including a protease inhibitor mixture (Sigma) and 1 μm microcystin LR. The suspension was then homogenized with a Dounce homogenizer on ice. After centrifugation at 1,000 × g for 10 min, the postnuclear supernatant was further centrifuged at 100,000 × g for 60 min. The precipitated membrane fraction (P100) was suspended in the same buffer, including a protease-inhibitor mixture and 1 μm microcystin LR, and used as the mouse brain membrane preparation. Aliquots (25 μg of protein) of the mouse brain membrane preparation were incubated at 37 °C for the indicated times, as described (32Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. Distinct intramembrane cleavage of the β-amyloid precursor protein family resembling γ-secretase-like cleavage of Notch.J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), in buffer H containing 5 mm EGTA, 5 mm phenanthroline, protease inhibitor mixture (Sigma), and 2× PhosSTOP (Roche Diagnostics) to prevent protein degradation and dephosphorylation. Samples were then separated by electrophoresis in Tris-Tricine gels (17.5% (w/v) polyacrylamide). The separated proteins were transferred onto a nitrocellulose membrane, boiled in PBS for 5 min, and probed with the indicated antibodies. Reactive proteins were detected using an ECL plus detection system (GE Healthcare). Detergent-resistant membrane (DRM) fractions were prepared according to the established DRM isolation procedure (9Saito Y. Sano Y. Vassar R. Gandy S. Nakaya T. Yamamoto T. Suzuki T. X11 proteins regulate the translocation of amyloid β-protein precursor (APP) into detergent-resistant membrane and suppress the amyloidogenic cleavage of APP by β-site-cleaving enzyme in brain.J. Biol. Chem. 2008; 283: 35763-35771Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, the brain membrane preparation from each mouse (3 months old) was homogenized in TNE buffer (10 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 5 mm EDTA) containing 1% (v/v) CHAPSO, a protease inhibitor mixture, and 1 μm microcystin-LR. The sucrose concentration of the extract was adjusted to 42.5% (w/v) by the addition of 85% sucrose in TNE buffer; the extract was then placed at the bottom of an ultracentrifuge tube and overlaid with 5 (4 ml) and 35% (4 ml) discontinuous sucrose gradients in TNE buffer. The gradients were centrifuged at 39,000 × g for 20 h with an SW41 rotor (Beckman Coulter). Fractions (1 ml) were collected from the top (fraction 1) of the ultracentrifuge tubes and proteins in the respective fractions were analyzed by immunoblotting. The primary culture of cortical neurons was as described (9Saito Y. Sano Y. Vassar R. Gandy S. Nakaya T. Yamamoto T. Suzuki T. X11 proteins regulate the translocation of amyloid β-protein precursor (APP) into detergent-resistant membrane and suppress the amyloidogenic cleavage of APP by β-site-cleaving enzyme in brain.J. Biol. Chem. 2008; 283: 35763-35771Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, the cortex of mice at embryonic day 15.5 was dissected, and neurons were spread in a buffer containing papain and cultured at 1.5 × 105 cells in Neurobasal medium containing B27, Glutamax, and antibiotics (Invitrogen) on a poly-d-lysine-treated dish. Neurons at in vitro day 7 were incubated with Neurobasal medium containing MβCD for 2 h. After incubation, neuronal membrane fractions (P100) were isolated by ultracentrifugation. The membrane preparation was used for the in vitro γ-secretase assay and samples were analyzed by immunoblotting as described above. The labeling of membrane cholesterol in neurons (in vitro day 7) was investigated by microscopy analysis using the cholesterol cell-based detection assay kit including Filipin III (Cayman Chemical). Lipids were extracted with chloroform/ethanol mixtures from brain membrane preparations of C57BL/6 mouse and evaporated using a SpeedVac vacuum concentrator to prepare a lipid film. The lipid film was dissolved in phosphate-buffered saline (PBS) and agitated to prepare liposomes (33Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). The brain-lipid liposomes were incubated with the indicated peptide in PBS for 1 h at room temperature, and peptide bound to liposomes was recovered by ultracentrifugation at 100,000 × g for 10 min as described (34Sumioka A. Yan D. Tomita S. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers.Neuron. 2010; 66: 755-767Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Production and purification of recombinant GST fusion proteins were as described (28Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. Interaction of a neuron-specific protein containing PDZ domains with Alzheimer amyloid precursor protein.J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 29Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. Interaction of Alzheimer β-amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade.J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Briefly, GST fusion proteins were generated in Escherichia coli BL21 transformed with pGEX-4T-1 cDNA constructs and affinity purified with glutathione-Sepharose 4B (GE Healthcare). The cytoplasmic peptides of APP(648–695), with or without phosphate at the Thr668 residue, Aβ(1–40) and Aβ(1–42) peptides (for sELISA standard) were synthesized using solid phase N-tert-butyloxycarbonyl chemistry. The peptides were purified by reverse-phase high pressure liquid chromatography to greater than 90% purity, and their expected molecular weights were confirmed by mass spectroscopy. N2a cells (total" @default.
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• W2028781640 title "Membrane-Microdomain Localization of Amyloid β-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue" @default.
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