Alkalizing Drugs Induce Accumulation of Amyloid Precursor Protein By-products in Luminal Vesicles of Multivesicular Bodies

• DOI: 10.1074/jbc.m609475200
• Publication Date: 2007-04-28T00:12:51Z
• Authors: Valérie Vingtdeux; Malika Hamdane; Anne Loyens; Patrick Gelé; Hervé Drobeck; Séverine Bégard; Marie-Christine G...; André Delacourte; Jean-Claude Beauv...; Luc Buée; Nicolas Sergeant
• Keywords: Amyloid (mycology)

Amyloid precursor protein (APP) metabolism is central to the pathogenesis of Alzheimer disease. We showed recently that amyloid intracellular domain (AICD), which released by γ-secretase cleavage APP C-terminal fragments (CTFs), strongly increased in cells treated with alkalizing drugs (Vingtdeux, V., Hamdane, M., Bégard, S., Loyens, A., Delacourte, Beauvillain, J.-C., Buée, L., Marambaud, P., and Sergeant, N. (2007) Neurobiol. Dis. 25, 686–696). Herein, we aimed determine cell compartment AICD accumulates. show APP-CTFs are present multivesicular structures. Multivesicular bodies contain intraluminal vesicles (known as exosomes) when extracellular space. demonstrate APP, APP-CTFs, integrated secreted within exosomes differentiated neuroblastoma primary neuronal culture cells. Together recent data showing amyloid-β also found exosomes, our essential organelles for all metabolites can be 3The abbreviations used are: protein; Aβ, amyloid-β; CTFs, fragments; AICD, domain; MVBs, bodies; BafA1, bafilomycin A1; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; APPWT, wild-type protein. disease etiopathogenesis. Extracellular deposits, a neuropathological hallmark disease, composed (Aβ) peptides derive from catabolism. type I transmembrane glycoprotein processed an α-ora β-secretase produce (CTFs) (for review, see Ref. 1Suh Y.-H. Checler F. Pharmacol. Rev. 2002; 54: 469-525Crossref PubMed Scopus (417) Google Scholar). γ-Secretase further processes (2Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref (424) Scholar, 3Kim S.H. Ikeuchi T. Yu Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text PDF (96) Scholar), releasing Aβ β-CTF (AICD or ϵ-CTF) 4Andrau D. Dumanchin-Njock Ayral E. Vizzavona Farzan Boisbrun Fulcrand P. Hernandez J.F. Martinez Lefranc-Jullien S. 25859-25866Abstract (66) 5Baulac LaVoie M.J. Kimberly W.T. Strahle Wolfe M.S. Selkoe D.J. Xia W. 14: 194-204Crossref (94) 6Gu Y. Misonou Sato Dohmae Takio Ihara 276: 35235-35238Abstract (267) 7Vingtdeux V. Hamdane Gompel Bégard Drobecq Ghestem Grosjean M.E. Kostanjevecki Grognet Vanmechelen Buée L. Delacourte Sergeant 2005; 20: 625-637Crossref (68) 8Weidemann Eggert Reinhard F.B. Vogel Paliga Baier Masters C.L. Beyreuther Evin Biochemistry. 41: 2825-2835Crossref (315) Several lines evidence suggest trans-regulating factor gene expression (neprilysin, KAI1, glycogen synthase kinase-3β) (9Baek Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 110: 55-67Abstract (493) 10Kim H.-S. Kim E.-M. Lee J.-P. Park C.H. Seo J.-H. Chang K.-A. Jeong S.-J. Chong Y.H. Suh FASEB 17: 1951-1953Crossref 11von Rotz R.C. Kohli B.M. Bosset Meier Suzuki Nitsch R.M. Konietzko U. Cell Sci. 2004; 117: 4435-4448Crossref (295) 12Pardossi-Piquard R. Petit Kawarai Sunyach Alves da Costa Vincent B. Ring D'Adamio Shen Müller St. George-Hyslop Neuron. 46: 541-554Abstract (299) However, rapidly degraded thus seldom detected (13Cupers Orlans I. Craessaerts Annaert De Strooper Neurochem. 78: 1168-1178Crossref (213) upon treatment drugs, suggesting endosomal/lysosomal pathway regulates degradation (14Vingtdeux Loyens Beauvillain J.-C. Marambaud 2007; 25: 686-696Crossref (58) Scholar).The production For instance, BACE-1 (beta-site APP-cleaving enzyme 1) resides endosomes, endocytosis prerequisite generating (15Ehehalt Keller Thiele Simons 160: 113-123Crossref (911) 16Vassar Bennett B.D. Babu-Khan Kahn Mendiaz E.A. Denis Ross Amarante Loeloff Luo Fisher Fuller Edenson Lile Jarosinski M.A. Biere A.L. Curran Burgess Louis J.C. Collins Treanor Rogers Citron Science. 1999; 286: 735-741Crossref (3256) 17Walter Fluhrer Hartung Willem Kaether Lammich 14634-14641Abstract (247) An acidic pH necessary optimal protease activity (18Yan Bienkowski Shuck Miao Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney Nature. 402: 533-537Crossref (1328) lysosomes (19Koh von Arnim C.A. Hyman B.T. Tanzi R.E. Tesco 280: 32499-32504Abstract (142) The has been localized at membrane (20Lah J.J. Levey A.I. Mol. Neurosci. 2000; 16: 111-126Crossref (80) 21Pasternak Bagshaw R.D. Guiral Zhang Ackerley Pak B.J. Callahan J.W. Mahuran 26687-26694Abstract (250) 22Vetrivel K.S. Cheng Lin Sakurai Li Nukina Wong P.C. Xu Thinakaran 279: 44945-44954Abstract (360) 23Kaether Schmitt Traffic. 2006; 7: 408-415Crossref (124) Treatment prevent acidification (24Golde T.E. Estus Younkin L.H. S.G. 1992; 255: 728-730Crossref (618) 25Haass 1995; 270: 6186-6192Abstract (89) 26Schrader-Fischer Paganetti P.A. Brain Res. 1996; 716: 91-100Crossref (65) Scholar) deletion internalization motif (27Koo Squazzo S.L. 1994; 269: 17386-17389Abstract 28Perez R.G. Soriano Hayes J.D. Ostaszewski Chen X. Stokin G.B. 274: 18851-18856Abstract (341) dramatically reduces secretion.The system likely altered 29Nixon R.A. Aging. 26: 373-382Crossref (304) derivatives accumulate (MVBs), transgenic animal models amyloidosis (30Takahashi R.H. Milner T.A. Nam E.E. Edgar Yamaguchi Beal M.F. Greengard Gouras G.K. Am. Pathol. 161: 1869-1879Abstract (580) 31Langui Girardot El Hachimi K.H. Allinquant Blanchard Pradier Duyckaerts 165: 1465-1477Abstract (141) (32Verbeek Otte-Holler Fransen J.A. de Waal Histochem. Cytochem. 50: 681-690Crossref (23) MVBs belong endocytic (33Gruenberg Stenmark Nat. 5: 317-323Crossref (578) Scholar); crossroad several cellular mechanisms such receptor recycling degradation; release their vesicles, known Refs. 34de Gassart Geminard Hoekstra Vidal 896-903Crossref (108) 35Fevrier Raposo Curr. Opin. 415-421Crossref (810) 36Johnstone Blood Cells 34: 214-219Crossref (116) More recently, were demonstrated (37Rajendran Honsho Zahn T.R. Geiger K.D. Verkade Proc. Natl. Acad. 103: 11172-11177Crossref (916) Taken together, growing body suggests processing takes place mainly between plasma late endosomal compartments endosomes. studied localization its SY5Y stably overexpressing human luminal endosomes exosomes.EXPERIMENTAL PROCEDURESAntibodies—APPCter-C17 well characterized rabbit antibody raised against last 17 amino acids sequence (7Vingtdeux 38Casas Itier J.M. Wirths O. van der Kolk Vingtdeux Steeg Ret Canton Clark Bonici Benavides Schmitz Tremp Bayer Benoit 1289-1300Abstract (350) 39Santiard-Baron Langui Delehedde Delatour Schombert Touchet Paul Mercken 93: 330-338Crossref (46) 40Sergeant David J.P. Champain Wattez 81: 663-672Crossref (51) Anti-HSP70 (heat shock 70 kDa) was purchased Euromedex (Mundolsheim, France), Anti-flotillin Translab (Erembodegem, Belgium); anti-LAMP-2 anti-Tsg101 antibodies Santa Cruz Biotechnology, Inc.); anti-EEA1 (early endosome antigen Upstate (Lake Placid, NY). mouse monoclonal 8E5 (raised residues 444–592 APP770) generous gift Dr. Peter Seubert (Elan Pharmaceuticals). Anti-β-tubulin horseradish peroxidase-coupled secondary Sigma (Saint-Quentin-Fallavier, France).Cell Culture SKNSH-SY5Y Neuroblastoma Differentiation—The line (referred SY5Y) maintained Dulbecco's modified Eagle's medium (Invitrogen, Cergy-Pontoise, France) supplemented 10% fetal calf serum, 2 mml-glutamine, 1 mm nonessential acids, 50 units/ml penicillin/streptomycin (Invitrogen) 5% CO2 humidified incubator 37 °C. APP695 cDNA subcloned into eukaryotic vector pcDNA3 (Invitrogen), allowing G418 selection clones. This transfected using ethyleneimine polymer ExGen 500 (Euromedex) according manufacturer's instructions. expressing selected addition 200 μg/ml medium. differentiation, 15 days medium/nutrient mixture F-12 penicillin/streptomycin, 7 mg/ml progesterone, 1% insulin/transferrin/selenium retinoic acid (Sigma). replenished every 3 days.Primary Embryonic Neuronal Culture—Rat cortical neurons prepared 17–18-day Wistar rat embryos follows. Briefly, brain meninges removed, cortex carefully dissected out mechanically dissociated triturating polished Pasteur pipette. Once after blue trypan counting, plated 25-cm2 flasks density 900 cells/mm2 poly-d-lysine- laminin-coated plates. dissociation, plating, maintenance, Neurobasal 2% B27 containing glutamine antibiotic/antimycotic agent (Invitrogen). 24 h before treatment.Drug Treatments—N-[N-(3,5-Difluorophenacetyl)-l-alanyl)]-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydrobenzo[e]-(1,4)-diazepin-2-one (compound E, inhibitor XVIII; final concentration 30 nm) A1 (BafA1; 100 Calbiochem (Fontenay-sous-Bois, France). Chloroquine (10 μm) Sigma. 6-well plates drug exposure. washed warmed saline (PBS) scraped policeman rubber μl Laemmli sample buffer inhibitors (Complete mini, Roche Applied Science, Meylan, homogenate sonicated heat-treated 5 min determined PlusOne™ 2D Quant kit (Amersham Biosciences, Orsay, samples kept –80 °C until used.Western Blotting Mass Spectrometry—An equal quantity total proteins (20 μg/lane) loaded onto 16.5% Tris/Tricine- 8–16% Tris/glycine-polyacrylamide gel. Tris/Tricine/SDS-PAGE performed following procedure Schägger Jagow (41Schägger Anal. Biochem. 1987; 166: 368-379Crossref (10442) PROTEAN II xi (Bio-Rad, Marnes-la-Coquette, Following sucrose gradient (see below), Criterion XT/MES gels (Bio-Rad) described previously Proteins transferred nitrocellulose (Hybond, Amersham Biosciences) 2.5 mA/cm2/gel semidry NovaBlot transfer reversibly stained Ponceau red check quality transfer. Membranes blocked 25 Tris-HCl (pH 8.0), 150 NaCl, 0.1% (v/v) Tween 20, (w/v) skim milk min. then incubated overnight 4 appropriate dilutions room temperature antibody. immunoreactive complexes revealed ECL™ Western blotting Hyperfilm Biosciences). mass spectrometry analyses, resolved XT Coomassie Brilliant Blue G-250 In-gel trypsin digestion analyses (42Sergeant Bombois Missiaen Sergheraert 85: 1581-1591Crossref (176) Scholar).Exosome Isolation Sucrose Density Gradients—The protocol derived Thery et al. (43Thery Boussac Veron Ricciardi-Castagnoli Garin Amigorena Immunol. 7309-7318Crossref (1172) media centrifuged 10 1200 × g, supernatant filtered on 0.22-μm filter (Millex® GP, hydrophilic polyethersulfone; Millipore, Molsheim, 12,000 g ultracentrifugation 100,000 pellet added once large volume PBS h. purification gradient, resuspended 0.25 m HEPES/NaOH 7.4). A step 2.25, 2.00, 1.75, 1.5, 1.25, 1.00, 0.75, 0.5, layered exosome-containing solution. spun 200,000 18 SW 41 Ti rotor (Beckman Coulter). Fractions ml diluted 7.4) 60 rotor. Pellets electron microscopy.Immunofluorescence—SY5Y (APPWT; referred SY5Y-APPWT cells) poly-l-lysine-coated glass coverslips. After treatment, fixed 0.1 4% paraformaldehyde permeabilized 0.25% Triton X-100 PBS. blocking bovine serum albumin, materials albumin X-100. washing, (Alexa Fluor 488-conjugated anti-rabbit IgG Alexa 568-conjugated anti-mouse IgG; Molecular Probes) used. Coverslips mounted slides VECTASHIELD (Vector Laboratories). Images acquired Leica TCS NT laser scanning confocal microscope.For analysis, ice-cold times indicated. above, incubation 0.3% antibody.Electron Microscopy Gold Immunolabeling—For conventional observations, pretreated not glutaraldehyde 7.2) they post-fixed osmic same buffer. embedded Araldite dehydration ethanol. Sections collected Parlodion-coated nickel grids counterstained uranyl acetate lead citrate observation Zeiss 9025 immunoelectron microscopy (treated not) 0.2% directly LR White dehydration. Polymerization occurred under UV light 48 coated gold immunolabeling. sections successively (0.1 Tris, 0.15 goat serum), h), colloidal (18 nm)-labeled Ig (1.5 h). H2O. isolated 400 mesh carbon put drop exosome 1:50 blotting, fixative solution (0.2% PBS), glycine (0.2 APPCter-C17 antiserum (12 gold-labeled Counter-staining acetate.RESULTSAccumulation Late Endosomes Alkalizing Drug Treatments—Alkalizing induce accumulation biochemical investigated this may arise. chloroquine- BafA1-treated immunofluorescence antisera (Fig. 1). accumulated numerous engulfed These seen control conditions could result defective routing APP.BafA1 reduce slightly rate internalization, recycling, sorting completely block transport (44van Weert A.W. Dunn K.W. Gueze H.J. Maxfield F.R. Stoorvogel 130: 821-834Crossref 45van Deurs Holm P.K. Sandvig Eur. 69: 343-350PubMed assessed °C, kinetics 8E5-APP followed indirect 2A). both cells, observed delineated immunocomplexes, demonstrating repressed low temperatures. internalized small conditions. Noteworthy, visualized, whereas detected. Although quantitative, staining disappeared In contrast, remained intense vesicular structures signal diminished. BafA1 induced resembling those Fig. 1, these pathway. To characterize markers early 2B). labeling partially overlapped EEA1 marker labeling. did correspond vesicles. Tsg101 particularly (46Babst Odorizzi Estepa E.J. Emr S.D. 1: 248-258Crossref (353) few antibodies. sharp positive LAMP-2 lysosome results compartments.FIGURE 2APP presence colocalizes markers. A, indirectly visualized h, placed cold room, shifted 0, 5, 15, 30, allow internalization. B, untreated (control (Ctrl)) labeled APPCter-C17, anti-EEA1, antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)APP Its Derivatives Are Found Cells—We next ultrastructural level Numerous smaller but 3A supplemental D). Immunogold particles (supplemental 1C). alone hypothesized MVBs.FIGURE 3MVB analysis cells; immunogold BafA1. 18-nm larger vesicle. Note shape very similar (A). Nu, nucleus. Scale bars = μm.View (PPT)MVBs fuse content, including termed latter originate budding form If accumulate, products should associated exosomes.We first absence 4). source exogenous ascertain exosome-like 24, 48, 72 isolation Flotillin HSP70, often (36Johnstone Di