Alkalizing drugs induce accumulation of amyloid precursor protein byproducts in lumenal vesicles

22 Pages·2007·2.58 MB·English

by  Nicolas Sergeant

JBC Papers in Press. Published on April 27, 2007 as Manuscript M609475200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M609475200
ALKALIZINGDRUGSINDUCEACCUMULATIONOFAMYLOIDPRECURSOR
PROTEINBYPRODUCTSINLUMENALVESICLESOFMULTIVESICULARBODIES
ValérieVingtdeux1,2,MalikaHamdane1,2,AnneLoyens1,2,PatrickGelé1,2,HervéDrobeck3,
SéverineBégard1,2,Marie-ChristineGalas1,2,AndréDelacourte1,2,Jean-ClaudeBeauvillain1,2,
LucBuée1,2andNicolasSergeant1,2*
RunningTitle:APP-CTFsaresecretedinexosomevesicles
1- Inserm, U837, Neurodegenerative Disorders and Neuronal Death, place de Verdun, F-59045 Lille,
France. 2- Université Lille 2, Faculté de Médicine, Institut de Médecine prédictive et de Recherche
thérapeutique, Centre de Recherches Jean-Pierre Aubert, place de Verdun, 59045 Lille, France 3-
CNRS, UMR 8161 "Lille Institute of Biology", University of Lille 1, Pasteur Institute of Lille, 1, rue
duProfesseurCalmette,F-59021Lillecedex.
*Address correspondence and reprint requests to: Sergeant Nicolas, PhD, INSERM U837,
Neurodegenerative Disorders and Neuronal Death, Jean-Pierre Aubert Research Centre, 1, Place de
Verdun,F-59045Lille,France;[email protected]
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The amyloid precursor protein (APP) gamma-secretase further processes APP-CTFs nlo
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metabolism is central to the pathogenesis of (2,3), releasing Abeta from beta-CTF and the d
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Alzheimer’s disease. We recently showed amyloid intracellular domain (AICD or fro
that amyloid intracellular domain (AICD) epsilon-CTF) from all APP-CTFs (2,4-8). m
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which is released by a gamma-secretase Several lines of evidence suggest that AICDis ttp
cleavage of APP carboxy-terminal a trans-regulating factor of gene expression, ://w
fragments(APP-CTFs)isstronglyincreased including Neprelysin, KAI1, APP, GSK3-(cid:2)… ww
in cells treated with alkalizing drugs. (9-12). However, AICD is rapidly degraded .jb
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Herein, we aimed to determine the cell and thus seldom detected (13). We recently .org
compartment in which AICD accumulates. showed that AICD is strongly increased while b/
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We showed that APP-CTFs and AICD are using alkalizing drugs, suggesting that the g
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pMrueslteinvtesicuinlar bmoudliteisvecsoicnutalainr intsrtarluucmtuerneasl. ednegdorasdoamtieo/nly(s1o4s)o.me pathway regulates AICD est on
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vesicles known as exosomes when released The endosomal/lysosomal pathway is essential an
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in the extracellular space. We demonstrated for Abeta production and APP catabolism. For ary
that APP, APP-CTFs and AICD are instance, beta-site APP cleaving enzyme 1 23
integrated and secreted within exosomes in (BACE-1) resides within endosomes and , 20
1
differentiated neuroblastoma and primary endocytosis of BACE-1 and APP is a 9
neuronal culture cells. Together with recent prerequisite for generating Abeta (15-17). An
data showing that Abeta is also found in acidic pH is necessary for BACE-1 optimal
exosomes,ourdatashowthat multivesicular proteaseactivity(18)andBACE-1isdegraded
bodies is an essential organelle for APP in lysosomes (19). The gamma-secretase
metabolism and that all APP metabolites activity has been localized at the endosomal
canbesecretedintheextracellularspace. and lysosomal membrane (20-23). Treatment
with drugs that prevent endosomal/lysosomal
Metabolism of Amyloid Precursor Protein acidification (24-26) or deletion of APP
(APP) is central to Alzheimer’s disease (AD)
internalization motif (27,28) dramatically
etiopathogenesis. Extracellular amyloid
reduceAbetasecretion.
deposits, a neuropathological hallmark of AD,
The endosomal/lysosomal system is likely to
are composed of amyloid-beta peptides
be altered in AD(for reviewsee (29)). Several
(Abeta) that derive from the catabolism of
APP derivatives accumulate in multivesicular
APP. APP is a type I transmembrane
bodies (MVB), in transgenic animal models of
glycoprotein processed by an alpha- or beta-
amyloidosis (30,31), in AD (30) as well as in
secretase to produce carboxy-terminal
cell models (32). MVB belongs to the
fragments (CTFs) (for review see (1)). The
endocytic pathway (33) and is at the crossroad
1
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
of several cellular mechanisms such as APP cDNA was transfected into SY5Y cells
membrane receptor recycling or protein using the ethyleneimine polymer ExGen 500
degradation and MVB can release their (Euromedex) according to the manufacturer’s
intralumenal vesicles, known as exosomes (for instructions. Cells expressing APP were
review see (34-36). More recently, exosomes selected by the addition of 200 μg/ml G418 in
were demonstrated to contain Abeta peptides the cell medium. For differentiation, SY5Y
(37). Taken together, a growing body of cells were maintained for 15 days in
evidencessuggeststhatAPPprocessingmainly DMEM/F12 medium supplemented with 2mM
take place between the plasma membrane and L-glutamine, 50 units/mL
late endosome compartments such as penicillin/streptomycin, 7 mg/mL
multivesicular endosomes. Herein, we studied progesterone, 1% Insulin/Transferrin/selenium
the localization of APP and its derivatives in (Invitrogen) and 1 mM retinoic acid (Sigma).
SY5Y neuroblastoma cells stably Mediumwasreplenishedevery3days.
overexpressing human APP and demonstrated
that APP, APP-CTFs and AICD accumulates PrimaryEmbryonicNeuronalCulture
in luminal vesicles of multivesicular Rat primary cortical neurons were prepared
endosomesandarealsofoundinexosomes. from 17- to 18-day-old Wistar rat embryos as
followed. Briefly, brain and meninges were
Experimentalprocedures removed. Cortex was carefully dissected out D
o
and mechanically dissociated in culture w
n
Antibodies medium by triturating with a polished Pasteur loa
d
The APPCter-C17 is a well-characterized pipette. Once dissociated and after blue trypan ed
rabbitantibodyraisedagainstthelast17amino counting, cells were plated in 25-cm2 flasks at fro
m
acids of the human APP sequence (7,38-40). a density of 900 cells/mm2 in poly-D-lysine h
Anti-Heat Shock Protein 70 (HSP70) antibody and laminin coated plates. For dissociation, ttp
was purchased at Euromedex (Euromedex, plating and maintenance, we used Neurobasal ://w
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Mundolsheim, France), anti-Flotillin was from medium supplemented with 2% B27 w
Translab (Erembodegem, Belgium), anti- containing 200 mM glutamine and 1% .jbc
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LAMP-2 and anti-Tsg101 were from Santa antibiotic-antimycotic agent (GIBCO). rg
Cruz (Santa Cruz Biotechnology) and Anti- Medium was replenished every 3 days and 24 by/
g
EEA1 (Early Endosome Antigen 1) was from hbeforecelltreatment. u
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Upstate (Lake Placid, USA). The mouse t o
monoclonal antibody 8E5 was a generous gift Drugtreatments n J
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fromDr.PeterSeubert(ElanPharmaceuticals). Thefollowingdrugswereusedattheindicated nu
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8E5 is raised against the 444-592 residues of final concentration: N-[N-(3,5- ry
2
human APP770. The beta-Tubulin antibody Difluorophenacetyl)]-L-alanyl)]-3-(S)-amino- 3, 2
was purchased at SIGMA Immunochemical 1-methyl-5-phenyl-1,3-dihydro-benzo[e] (1,4) 01
9
(Saint Quentin Fallavier, France). Secondary diazepin-2-one (Compound E, Gamma-
antibodiescoupledwithhorseradishperoxidase secretase inhibitor XVIII) (30 nM),
werefromSIGMA. bafilomycin A1 (BafA1) (100 nM), were
purchased at Calbiochem (VWR, International
Cell culture of neuroblastoma SKNSH-SY5Y S.A.S. Fontenay-sous-Bois, France).
anddifferentiation Chloroquine (10 μM) was purchased at
The human neuroblastoma cell line SKNSH- SIGMA (Saint Quentin Fallavier, France).
SY5Y (SY5Y) was maintained in Dulbecco’s Cellswereplatedinto6-wellplates24hbefore
modified Eagle medium (DMEM, GIBCO drug exposure. Cells were washed with
BRL, Cergy Pontoise, France) supplemented warmed PBS and scraped with a policeman
with10%fetal calf serum, 2 mM L-glutamine, rubber in 70 μl of Laemmli sample buffer
1 mM non-essential amino-acids, 50 units/mL containing protease inhibitors (Complete Mini,
penicillin/streptomycin (Invitrogen SARL, Roche Molecular Biochemicals, Meylan,
Cergy Pontoise, France) in a 5% CO France). The cell homogenate was sonicated
2
humidified incubator at 37°C. APP695 cDNA and heat-treated for 5 min at 100°C. Protein
was sub cloned into eukaryotic expression concentration was determined using the
vector pcDNA3 (Invitrogen), allowing for a PlusOne™ 2-D Quant Kit (Amersham
G418 (Invitrogen) selection of clones. This
2
Biosciences, Orsay, France) and samples were pellet was resuspended in 0.25M of sucrose in
keptat–80°Cuntilused. Hepes/NaOHpH7.4 solution. Step gradient of
sucrose of 2.25, 2.00, 1.75, 1.5, 1.25, 1.00,
WesternblottingandMassspectrometry 0.75, 0.5 and 0.25M was layered with 0.25M
Equal quantity of total proteins (20 μg/lane) exosome-containingsolution.Thegradientwas
was loaded on a 16.5 % Tris-tricine or 8-16 % spun at 200,000g for 18h using SW41 Ti rotor
Tris-glycine polyacrylamide gel. Tris-tricine (Beckman-Coulter, USA). Fractions of 1ml
SDS-polyacrylamide gel electrophoresis were diluted with 3ml of Hepes/NaOH pH 7.4
(PAGE) was performed following the buffer and spun at 200,000g for 1h using a
procedure of Schägger and von Jagow (1987) SW60 Ti rotor. Pellets were used for Western-
(41) with a protean II Xi Cell (Bio-Rad blottingorelectronmicroscopy.
Bioresearch division, Marnes la coquette,
France). Following the final sucrose-density Immunoflorescence(IF)
gradient, see below, proteins were loaded onto SY5YAPPWT cells weredifferentiatedonpoly
Criterion XT-Mes gels (Biorad) as previously L-Lys-coated glass coverslips. After drug
described (14). Proteins were transferred to treatment, cells were fixed in 0.1 M phosphate
nitrocellulose membrane (Hybond, Amersham buffer (PBS) containing 4% paraformaldehyde
Biosciences) at 2.5 mA/cm2 per gel using the for 30 min at room temperature, and further
Semidry Novablot Transfert system permeabilized with 0.25% (v/v) Triton X-100 D
o
(Amersham Biosciences), according to the in PBS. After blocking in 1% (w/v) bovine wn
manufacturer’s instructions. Proteins were serum albumin (BSA), fixed materials were loa
d
reversibly stained with Ponceau Red to check incubated for 2 h at room temperature with ed
the quality of protein transfer. Membranes primary antibody in PBS added with 1%(w/v) fro
m
were blocked in 25 mM Tris-HCl pH 8.0, 150 BSA and 0.25% Triton X-100. After washing, h
m(wM/v)NoafCskl,im0m.1ed%mTilkwefoenr-3200m(ivn/.vM) eamndbr5ane%s sraebcobnitdIagrGy oanrtAibloexdiaesF,luAolre5x6a8Falnutoi-rm4o8u8seaIngtGi- ttp://w
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were incubated overnight at 4°C with (MolecularProbes)wereused.Coverslipswere w
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appropriatedilutionsoftheprimaryantibodies, mounted to slides with Vectashield (Vector c
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and incubated for 1h at room temperature with laboratories). Images were acquired with a rg
secondary antibody. The immunoreactive Leica TCS NT laser scanning confocal by/
g
complexes were revealed using the ECL™ microscope. u
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Western Blotting kit and Hyperfilms For APP internalization analysis, cells were t o
n
(Amersham Biosciences). For Mass incubated with 8E5 antibody for 1 h at 4°C in J
a
n
spectrometry analyses, protein were resolved ice-cold DMEM added with 1% (w/v) BSA, u
a
with Criterion-XT gels (Bio-Rad) and stained andthen washed andincubated at37°Cforthe ry 2
with Coomassie Brillant Blue G250 (SIGMA). times indicated. Cells were then fixed as 3, 2
In-gel trypsin digestion and mass spectrometry describe above and permeabilized by 01
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analyses were performed as previously incubation in 0.3% (v/v) Triton X-100 in PBS
described(42). and incubated with Alexa Fluor 568 anti-
mouseIgG.
Exosome Isolation and sucrose-density
gradient Electron microscopy and Gold-
The protocol used was derived from that of immunolabeling
Thery and collaborators (43). The cell culture For conventional observations the
media were centrifuged for 10 min at 1200g, differentiated SY5Y cells pretreated or not
thesupernatantwasfilteredona0.22μmfilter with 10% FCS for 2 min, were fixed in 2%
(Millex® GP, hydrophilic membrane of glutaraldehyde in PBS (pH 7.2, 0.1M) for 10
polyethersulfone, Millipore, Molsheim, min. After washing they were post-fixed with
France) and centrifuged for 30 min at 12,000g 1% osmic acid in the same buffer. The cells
beforeultracentrifugationat100,000gfor1hat were then embedded in Araldite after
4°C. The pellet was added once with a large dehydrationinethanol.Sectionswerecollected
volume of PBS and further centrifuged at on perlodion coated Nickel grids and
100,000g for 1h. For purification of exosomes counterstained with uranyl acetate and lead
by sucrose-density gradient, the 100,000g citrate before observation on a Zeiss 9025
electronmicroscope(EM).
3
For immunoelectron microscopy (IEM) both control and BafA1 treated cells, we
observations, the same cells (treated or not) observed that the membrane is delineated by
were fixed in 0.2% glutaraldehyde, 4% immuno-complexes, demonstrating that
paraformaldehyde in PBS for 15 min and were endocytosis is repressed at low temperature.
directly embedded in LR white after After 5 min at 37°C, APP is rapidly
dehydration. Polymerisation occurs under UV internalized in small vesicles in both
for 48h. Sections were collected on coated conditions. Noteworthy, in control condition
nickel grids and were treated for gold the plasma membrane was no more visualized
immunolabeling. Briefly the sections were whereas in BafA1 treated cells, the plasma
successively incubated for 30 min in solution membrane was detected. Although indirect
A (sol A: Tris 0.1M, NaCl 0.15M, BSA 1%, immunofluorescence is not quantitative, we
goat serum 4%), antibody diluted in the sol A observed that APP staining strongly
(for 24h), colloidal gold (18 nm) labeled goat disappeared in control condition after 60 min
anti-rabbit immunoglobulins in sol A (1.5h). at 37°C. In contrast, APP staining remained
Afterwashingthesectionswerecounterstained intenseinvesicularstructuresinBafA1treated
in uranyl acetate 4% in H O. For observation cells and the signal at the plasma membrane
2
ofisolatedexosomes,carboned-400meshgrids was diminished. BafA1 drug induced the
wereputfor5minonadropofexosomepellet accumulation of 8E5 antibody in vesicles
diluted 1/50 in PBS. After blotting, the grids resembling those observed in Figure 1 D
o
were successively put in fixative solution suggesting that those vesicles belongs to the w
n
(0.2%glutaraldehyde,2%paraformaldehydein endosome/lysosome pathway. To further loa
d
PBS), glycine solution (0.2M in PBS), characterize those vesicles, we used antibodies ed
APPCter-C17antiserumfor12h,colloidalgold against markers of the early and late fro
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labeled-goat-anti-rabbit(1.5h).Counterstaining endosomes compartments (Fig. 2B). In control h
wasmadeinuranylacetate. andBafA1treatedcells,APPCter-C17labeling ttp
partially overlap that of EEA1 early ://w
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RESULTS endosomes marker in both control and BafA1 w.jb
treated cells. However, EEA1 staining did not c
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Accumulation of APP in late endosomes after corresponded to large engulfed vesicles. rg
alkalizingdrugtreatments Tsg101 is a marker of late endosomes by/
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Alkalizing drugs induced an accumulation of compartments and more especially that of u
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APP-CTFs and AICD as determined by multivesicular endosomes (46). In control t o
biochemical analyses (14). Herein, we SY5Y APPWT, few vesicles were stained with n J
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investigated the cellular compartments in both Tsg101 and APPCter-C17. In sharp nu
a
which this accumulation may arise. We contrast, large vesicles were all stained by ry 2
investigated the cellular localization of APP in Tsg101 antibody and APPCter-C17 antibody 3, 2
chloroquine and BafA1-treated SY5Y APPWT in BafA1 treated cells. We showed previously 01
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by immunofluorescence (IF) using 8E5 and that those large vesicles were also partially
APPCter-C17 antisera (Fig. 1). After drug positive for Lamp2 lysosome marker (14).
treatment the APP staining accumulates in Together, we suggest that BafA1 treatment
numerous engulfed vesicles (Fig. 1). Those results in the accumulation of APP and its
large engulfed vesicles are not seen in control derivatesinlateendosomecompartments.
conditions and could result from a defective
endocytosisorroutingofAPP. APP and its derivatives are found in
BafA1 wasdemonstratedtoreduceslightlythe multivesicularbodiesinBafA1treatedcells
rate of internalization, recycling and We next observed cellular organelles at the
intracellular sorting of transmembrane protein ultrastructural level in control and BafA1
andtocompletelyblockthetransportfromlate treated cells. Numerous large vesicles
endosome to lysosomes (44,45). We assessed containing smaller intralumenal vesicles were
the internalization of APP in control and observed in SY5Y APPWT BafA1 treated cells
BafA1treated cells. SY5Y APPWT cells were but not in control cells (Fig. 3A and
incubatedwith8E5antibodyat4°Candkinetic supplementary Fig. 1A and 1D). Immunogold
of endocytosis of 8E5-APP complexes was labeling with our APPCter-C17 antibody
followedbyindirectimmunofluorescence(Fig. showed that few gold particles were localized
2A). Following incubation of 8E5 at 4°C in in vesicular structures and multivesicular
4
bodies in control cells (Supplementary Fig. final pellet was homogenized in SDS-lysis
1C). SY5Y APPWT cells were not stained with buffer. Proteins were resolved by SDS-PAGE
the secondary antibody alone (supplementary and stained with Coomassie blue (Fig. 5A).
Fig. 1A and 1D). In BafA1 treated cells, The identity of proteins was determined by
intralumenal vesicles were stained with our peptide mapping after in-gel trypsin digestion
APPCter-C17 antibody. We hypothesized that and mass spectrometry analyses (42). A low
those large vesicles corresponded to engulfed ppm error (inferior to 30 ppm) was applied to
multivesicularbodies. match a protein with a minimum of four
Multivesicularbodiescanfusewiththeplasma matching peptides and a highprobabilityscore
membrane to release their luminal content, (Fig. 5A Table). The proteome of exosomes
including the small vesicles named exosomes isolated from multiple biological sources has
(33,36). The latter originate from the budding been described (43,48,49). The protein that
of endosome membrane to form intralumenal were herein identified, such as the Clathrin
vesicles. If multivesicular bodies are the cell heavy chain, Translation initiation factor 3,
compartments in which APP metabolites HSP90, Pyruvate kinase, Tubulin, Actin and
accumulate, those products should be Elongation factor-1delta, Ras-related nuclear
associatedwithexosomes. protein were previously characterized in
We first investigated the presence or not of exosomes. Together, our data indicate that the
exosome vesicles in the cell medium of SY5Y isolated-vesicles are SY5Y-derived exosomes. D
o
APPWT cell line (Fig. 4). SY5Y APPWT cells Those exosomes are most likely not w
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were differentiated with retinoic acid for 15 contaminated by proteins of other unrelated loa
d
days in a medium that did not contained fetal organelles, as the vast majority of the proteins ed
calf serum, which is a source of exogenous we have characterized were previously shown fro
m
exosomes (43). To ascertain the presence of inexosomesfromothersources.Moreover, we h
exosome-like vesicles in differentiated SY5Y performed a staining with EEA1 antibody. ttp
APPWT, the medium was collected after 24, 48 EEA1 was detected in SY5Y APPWTlysate but ://w
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or 72 hrs of culture and processed as for the not in exosomes obtained from 10 ml of w
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isolationofsecretedvesicles(43). Flotillinand culturemedium(Fig.5B). c
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HSP70, which are often found in exosome rg
vesicles, were used as exosomes markers APP,APP-CTFsandAICDaresecretedwithin by/
g
(36,43,47). We also used electron microscopy exosomevesicles u
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(EM) to determine the morphology of the Wenextaddressedthequestionofthepresence t o
secreted vesicles. Following, two low speed of APP metabolic products in SY5Y APPWT n J
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centrifugations, filtration and exosomes (Fig. 6A). Immuno-gold labeling nu
a
ultracentrifugation; the pellet was analyzed for with our APPCter-C17 antibody was ry
2
the presence of Flotillin and HSP70. We performed to localize APP metabolic products. 3, 2
observedbothFlotillinandHSP70onwestern- Numerous vesicles of 50- to 90-nm were 01
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blot(Fig.4B). Electronmicroscopyanalysisof stained with gold particles of 18-nm diameter
the same pellet revealed the presence of (Fig. 6A). The gold particles localized inside
numerous membrane vesicles witha meansize thevesicles.Using,thesameantibody,wenext
of 50-90 nm and a typical ‘cup shape’ (Fig. characterized the APP metabolic products by
4B),hencecorrespondingtothemorphological western-blot (Fig. 6A). This analysis was
characteristics of exosomes (35). We exclude performed on exosome vesicles obtained from
the possibility that the release of such vesicles differentiatedSY5YAPPWTcellsmaintainedin
was a characteristic of our cell model as the culture medium for 24, 48 or 72 hours. An
secreted exosome vesicles were isolated in increased of HSP70 staining was observed
mock SY5Y cells as well as in primary suggesting that exosomes accumulated in the
neuronalculturecells(Figure4Cand4D). cell medium with time (Fig. 6A). APPCter-
To further demonstrate that those vesicles C17 stained full-length APP, APP-CTFs and
corresponded to exosomes, we analyzed the AICD (Fig. 6A). Whereas full-length APP
protein in 100,000g pellet. SY5Y APPWTwere staining only slightly increased in the three
differentiated with retinoic acid for 15 days conditions,APP-CTFsstronglyincreasedat48
andthe medium was collectedfrom thelast72 and 72 h. AICD was mainly observed after 72
hours. Exosome vesicles were isolated h.TofurtherdemonstratethatAPP,APP-CTFs
following the established procedure and the and AICD were associated with exosomes, the
5
pelletfrom100,000gcentrifugationwasloaded morphological and biochemical properties of
on the top of a sucrose-density gradient exosomes. We also showed that proteins often
comprise between 0.25 and 2.5M sucrose. found in exosomes, as for instance HSP70 and
After centrifugation at 200,000g for 18 h, one Flotillin (36), were detected in those small
ml fractions were collected and analyzed by vesicles secreted by SY5Y cells. In contrast, a
western blot for the presence of EEA1, APP, marker of early endosome, EEA1, was not
APP-CTFs, AICD, HSP70 and Flotillin (Fig. found associated with exosomes. Further
6B). Exosomes are found principally between similarities were deduced from the
1.0 and 1.25M sucrose, corresponding to characterization of the proteome SY5Y-
densityof1.13-1.16g/ml,asdescribed(50,51). derived vesicles. Fifty percent of the proteins
APP, APP-CTFs and AICD were strongly characterized were previously identified in
detected in the 1.0-1.25M fraction where exosomes of other biological sources (43,48).
HSP70 and Flotillin were strongly detected as Similar vesicles were also isolated from
well (Fig. 6B). Moreover, we verified the primary neuronal culture cells demonstrating
presence of exosomes in this 1.0M sucrose that the production of exosomes is not a
fraction by EM (Fig. 6B) and showed also that specificity of SY5Yneuroblastoma cells but is
EEA1 wasnotdetectedinthisfractionbutwas most possibly a physiological process of
slightly detected in fractions below 2.0M of neuronalcells(51).Moreimportantly,ourdata
sucrose, when the blot was overexposed (Fig. demonstrate that APP derivatives including D
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6B). Altogether, weshowed thatSY5Y APPWT full-length APP, APP-CTFs and more w
n
cells produce exosomes in which APP and surprisingly, AICD are found in exosomes loa
d
APP metabolites were co-sedimented with secreted by differentiated SY5Y. These APP ed
exosome markers. Finally, to demonstrate that fragmentsarealsoco-sedimented withmarkers fro
m
the AICD we observed in exosomes was of exosomes at a sucrose density h
released by the gamma-secretase and not from corresponding to that described for exosomes ttp
an unrelated proteolytic cleavage, we used a (50,51). The presence of soluble full-length ://w
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specific gamma-secretase inhibitor. SY5Y APP in the medium was previously evidenced w
APPWT cells were treated for 24h either with in cells transfected with APP but remained .jbc
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compound E or chloroquine and we analyzed unexplained (52,53). Secretion of APP within rg
the exosome content after treatment (Fig. 7A). exosomes is consistent with these observations by/
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In presence of a gamma-secretase inhibitor andcouldbeexplanation.Wedemonstratealso u
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compoundE,AICDwasneitherdetectedinthe that AICD found in exosomes was produced t o
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cell lysate nor in the exosomes. Conversely, by the cleavage APP-CTFs by the gamma- J
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the amount of APP-CTFs was strongly secretase since the use of a gamma-secretase nu
a
increased in SY5Y APPWT and exosomes (Fig. inhibitor abrogates the release AICD. ry
2
7B), demonstrating that AICD in exosomes is Moreover, AICD is not produced during the 3, 2
produced by gamma-secretase cleavage of purification of exosomes. First, we failed to 01
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APP-CTFs. Moreover, we showed that in detect presenilin in exosomes and secondly,
chloroquinetreatedcellsAICDwasincreasein when exosomes were incubated at 37°C for
SY5Y APPWT lysate and in exosomes, 24h APP-CTFs were not processed further to
suggesting further that chloroquine might produce AICD or other catabolites (data not
repress AICD degradation in MVB. shown). However, the current negative
Altogether, our data demonstrate that APP, findings do not exclude the possibility that
APP-CTFs and AICD are found in gamma-secretase cleavage occurs prior to
intralumenal vesicles of multivesicular bodies exosomesecretion.
where they are likely degraded, as other type I Exosomes derive from the inward budding of
transmembraneproteins. the membrane of endosome en route for
multivesicular endosomes (54). Several
proteins are marker of this cellular
DISCUSSION compartment such as Flotillin, HSP70 and
Tsg101 (36,43). Interestingly, Flotillin was
In the present study, we show that recently shown to associate with AICD (55).
differentiated SY5Y stably expressing or not We showhere that APP and its derivatives are
APP produced and secreted small vesicles of found in exosomes, in multivesicular
50 to 90 nm of diameter, consistent with endosomes and accumulate in engulfed
6
vesicles following treatments with alkalizing related situation, although we showed that
drugs. Together with our recent data, we alkalizing drugs induce an intracellular
suggest that APP, APP-CTFs that are not accumulation of APP catabolites. However, an
processed by the gamma-secretase, are altered routing of APP to late endosome
addressed to this late endosome compartment compartment could be associated to an
for being degraded by fusion of MVB with increase riskofdevelopingAD. Arecent work
lysosomes. Abeta and AICD, which result by Rogaeva and collaborators demonstrates
from the gamma-secretase cleavage of APP- that genetic polymorphisms of SORL1 are at
CTFs, are also addressed to multivesicular risk for late onset AD (58). SORL1 acts as a
endosomes and are integrated in the luminal sorting receptor for full-length APP and favors
vesicles, namely exosomes. The presence of its recycling. A loss of function of SORL1
AICD in exosomes is consistent with gamma- results in an increased production of Abeta in
secretase cleavage of APP-CTFs that would late endosome compartment. Further work
occur during the budding of late endosome will be needed to study markers of exosomes
membrane. However, the contribution of in AD and the relationship, if any, with a
Flotillin to this mechanism remains to be potentialdefectof lateendosomecompartment
established. The process of APP following suchMVB.
endocytosisisillustratedinFigure6. The release of exosomes is most likely
Recent data suggest that multivesicular regulated as demonstrated by Faure and D
o
endosomes are important cellular collaborators. Thus, depolarization stimulates w
n
compartmentsinneurodegenerativesdisorders. exosomes released (51). The release of full- loa
d
Exosomes that are produced in those length APP was also demonstrated to be ed
compartments contain prion protein and are stimulated by cholinergic agonists (52). fro
m
suggest to mediate the diffusion of the scrapie Together with the present study, APP and its h
fhoarsmboefentheshporwotneinto(5b6e). Ma ocroemrpeocennetnlty,oAfbtehtae cexatoasboomlietessancdanthibseprroecleesasseids liaksesloycimatoeddulwatietdh ttp://w
w
exosome membrane (37). Protease implicated by several parameters. However, further work w
.jb
in APP metabolism and moreover, other willbeneededtoinvestigatethosehypotheses. c
.o
protein showing similarities with APP In conclusion, we demonstrated a novel rg
metabolism, such as L1 and CD44, are found secretion pathway of APP metabolites, by/
g
in exosomes (57). Exosomes are known in the including APP, APP-CTFs and AICD that is u
e
s
immune system to contribute to antigen mediated by exosome vesicles. Our work open t o
n
presentation. Neurons also produce exosomes new research perspectives toward the J
a
but their potential roles remain unclear (51). understanding of APP biological function. A nu
a
Herein, we show that several metabolites of more complete characterization of the ry
2
APP,includingAICD,arefoundinexosomes. proteome of neuronal-derived exosomes could 3, 2
Multivesicular bodies and exosomes are of also be helpful to discover new biomarkers 01
9
growinginterestinneurodegenerativediseases. useful for diagnostic purposes and the
We don’t know whether “alkalizing” development of therapeutic drugs in
conditions used herein reflect an Alzheimer- Alzheimer’sdiseases.
7
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FOOTNOTES
Keywords:amyloidprecursorprotein,amyloidintracellulardomain,multivesicularbodies,exosomes,
proteomic,Alzheimer’sdisease
Abbreviations used: APP; amyloid precursor protein; AICD; amyloid precursor protein intracellular
domain;Abeta;Amyloid-betapeptide;CTF;carboxy-terminalfragment;MVB:multivesicularbody
Acknowledgments: This work was supported by INSERM and CNRS, IMPRT, University of Lille II D
o
and APOPIS (contract LSHM-CT-2003-503330). Valérie Vingtdeux is a recipient fellowship of w
n
Association France Alzheimer. 8E5 antibody was kindly provided by Peter Seubert (ELAN loa
d
Pharmaceuticals,USA). ed
fro
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FIGURELEGENDS h
ttp
Figure 1: APP localizes in large vesicular structures in SY5Y APPWT cells treated with Chloroquine ://w
w
and Bafilomycin A1. Analysis of APP localization in SY5Y APPWT cells treated or not with w
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Chloroquine or Bafilomycin A1. Cells were processed for immunofluorescence and labeled with the c
.o
APP-Cter-C17 and 8E5 antibodies. Note the intense staining of APP in large vesicular structures in rg
Chloroquine(Chloro)andBafilomycinA1(BafA1)treatedcells. by/
g
u
e
s
Figure 2: APP is internalized in presence of Alkalizing drugs and colocalizes with late endosome t o
n
marker. A. Kinetic of APP endocytosis indirectly visualized by internalization of 8E5 APP antibody. J
a
Cells were pretreated or not with BafA1 for 24 h. Cells were placed at 4°C in a cold room and nu
a
incubatewith8E5antibodyfor1h.Temperaturewasthenshiftedto37°Candcellswereprocessedfor ry
2
immunofluorescenceat0,5,15,30or60mintoallowAPPinternalization.B.UntreatedSY5YAPPWT 3, 2
(Ctrl panel) or cells treated with BafA1 were processed for immunofluorescence and labeled with the 01
9
APPCter-C17antibodyandEEA1orTsg101antibodies.
Figure 3: Multivesicular bodies localization of APP in SY5Y APPWT cells. A. Ultrastructural analysis
of SY5Y APPWTBafA1-treated cells. B. Immunogold-labeling of APP in SY5Y APPWT cells treated
withBafA1.18-nmgoldparticlesarelocalizedinsmallintralumenalvesiclesofalargerone.Notethat
the shape of those vesicles is very similar to multivesicular structures observed in the same cells and
sameconditionsbyultrastructuralEM(panelA).Bars=1μm.
Figure 4: Secretion of exosome-like vesicles by SY5Y APPWT cells. SY5Y APPWT cells were
differentiatedfor15dayswithretinoicacidandthemediumwascollectedafterthelast24,48or72h.
A. Summary of the protocol used to isolated exosomes from the cell media. B. The 100,000g pellet
(exosomes)obtainedafterdifferentialcentrifugationsfromcrudemedium(lane1)orfromsupernatant
of 24, 48 and 72 h of cell culture (lane 2, 3 and 4), and the cell lysate (lane 5) were analyzed by
Western blotting with anti-Flotillin and anti-HSP70 antibodies. Morphological analysis by electron
microscopy of the 100,000g pellet obtained from the 72 h cell culture supernatant. The pellet is
composed of 30-100 nm vesicles. Bar = 100 nm. The 100,000g pellet obtained after differential
centrifugationsfrom crude medium (Panel Cand D:lane1)orfromsupernatantafter 24,48and72h
ofSY5Ycellculture(PanelC:lane2,3and4)orafter72hofprimaryneuronalcellculture(PanelD:
10