Nguyen T. Tien , Ilker Karaca , Irfan Y. Tamboli , and …...Nguyen T. Tien#, Ilker Karaca&, Irfan...
Transcript of Nguyen T. Tien , Ilker Karaca , Irfan Y. Tamboli , and …...Nguyen T. Tien#, Ilker Karaca&, Irfan...
Trehalose alters trafficking of APP
1
Trehalose alters subcellular trafficking and the metabolism of the Alzheimer-associated amyloid
precursor protein*
Nguyen T. Tien#, Ilker Karaca
&, Irfan Y. Tamboli
§, and Jochen Walter
1
From the Department of Neurology, University of Bonn, Germany #present address: Department of Radiology and Human Genetics, Leiden University Medical Center, The
Netherlands &present address: Department of Psychiatry and Psychotherapy, University of Bonn, Germany
§present address: Department of Neuroscience, Georgetown University, Washington, DC, USA.
*Running title: Trehalose inhibits lysosomal degradation of APP
To whom correspondence should be addressed: Jochen Walter, Department of Neurology, University of
Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany, Tell: +49 228 2871 9782; Fax: +49 228 2871
4387 ; Email: [email protected]
Keywords: trehalose, autophagy, amyloid precursor protein, Alzheimer’s disease, subcellular trafficking,
endosome, lysosome.
_____________________________________________________________________________________
ABSTRACT
The disaccharide trehalose is commonly
considered to stimulate autophagy. Cell
treatment with trehalose could decrease
cytosolic aggregates of potentially pathogenic
proteins, including mutant huntingtin, alpha-
synuclein and phosphorylated tau that are
associated with neurodegenerative diseases.
Here, we demonstrate that trehalose also alters
the metabolism of the Alzheimer related
amyloid precursor protein (APP). Cell
treatment with trehalose decreased the
degradation of full-length APP and its C-
terminal fragments (CTFs). Trehalose also
reduced the secretion of the amyloid β-peptide.
Biochemical and cell biological experiments
revealed that trehalose alters the subcellular
distribution and decreases the degradation of
APP-CTFs in endolysosomal compartments.
Trehalose also led to strong accumulation of the
autophagic marker proteins LC3-II and p62,
and decreased the proteolytic activation of the
lysosomal hydrolase cathepsin D. The combined
data indicate that trehalose decreases the
lysosomal metabolism of APP by alterating its
endocytic vesicular transport.
Alzheimer disease (AD) is characterized
by the accumulation of extracellular amyloid
plaques that contain aggregates of the amyloid β-
peptide (Aβ) Aβ derives from the amyloid
precursor protein by sequential proteolytic
processing by β- and γ-secretases (1,2). The initial
cleavage of APP by β-secretase results in the
secretion of the soluble ectodomain and the
generation of a membrane-tethered C-terminal
fragment (APP-CTFβ). Subsequently, APP-CTFβ
can be cleaved by -secretase to liberate Aβ from
cellular membranes (2). In an alternative pathway,
APP can also be cleaved by α-secretase within the
Aβ domain thereby generating CTFα. The
subsequent cleavage of APP-CTFα by -secretase
results in secretion of a small peptide called p3 (2).
It is important to note that APP is also metabolized
by additional pathways including proteasomal and
lysosomal degradation (3-5).
The metabolism of APP is also regulated
by macroautophagy (herein referred to as
autophagy), a lysosome-dependent degradative
pathway for long-lived proteins, organelles and
nutrient recycling (6,7). Autophagy starts with the
formation of double-membrane vesicles, the so-
called autophagosomes, which further fuse with
lysosomes to become autolysosomes for
degradation of autophagosome contents by
http://www.jbc.org/cgi/doi/10.1074/jbc.M116.719286The latest version is at JBC Papers in Press. Published on March 8, 2016 as Manuscript M116.719286
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Trehalose alters trafficking of APP
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lysosomal hydrolases (8). Autophagy is an
essential pro-survival pathway induced by a
variety of stress factors, including nutrient
deprivation, growth factor withdrawal, oxidative
stress, infection, and hypoxia (9). These factors
contribute to the etiology of multiple diseases such
as cancer, stroke, heart disease, and infection (10).
Eukaryotic cells have a basal autophagic activity
under normal physiological conditions. Cells
deficient in autophagy show diffuse abnormal
protein accumulation and mitochondria
disorganization (11,12), suggesting that cells use
autophagy to maintain cellular homeostasis by
eliminating protein aggregates and damaged
organelles. Basal autophagy is particularly
important and active in the liver and non-dividing
cells such as neurons and myocytes (11,13,14).
Dysfunction in autophagy is implicated in the
pathogenesis of many human diseases, including
different types of neurodegeneration (15,16).
Incompletely degraded autophagic vacuoles
contain both amyloid precursor protein (APP) and
-secretase complex, and could thus, contribute to
enhanced processing of APP into toxic amyloid-
beta (Aβ) peptides (17-19).
Trehalose (α,α-trehalose) is a natural non-
reducing disaccharide containing two D-glucose
residues connected via an α,α-1,1 linkage. It
functions not only as an energy source, but is also
used to protect cells against heat, cold, oxidation
or dehydration (20). Trehalose is considered to
enhance autophagic activity (21). It has been
shown to promote the cellular clearance of
pathogenic proteins like mutant huntingtin, α-
synuclein (21,22), and hyphosphorylated-tau (22-
24) that are associated with Huntington (HD),
Parkinson (PD) and Alzheimer disease (AD),
respectively. However, the role of trehalose in the
cellular metabolism of the AD-associated APP
remains to be investigated.
In this study, we analyzed the role of
trehalose on the cellular metabolism of APP.
Interestingly, trehalose strongly decreased the
degradation of APP and its CTFs. By biochemical
and cell biological approaches we demonstrate that
trehalose interferes with endocytic vesicular
trafficking, indicated by decreased processing of
cathepsin D (Cat D), redistribution of LAM2, and
accumulation of the autophagy related proteins
microtubule-associated protein 1 light chain 3
(LC3) and p62. The combined data demonstrate
that trehalose exerts complex effects on vesicular
trafficking thereby decreasing the metabolism of
APP and the secretion of Aβ.
EXPERIMENTAL PROCEDURES
Antibodies, constructs and reagents -
APP-CT (C1/6.1) antibody has been described in
(25). Human cathepsin D-CT antibody was a
generous gift from Dr. S. Höning (University of
Cologne, Germany). Anti-LAMP2 antibody was
purchased from the Iowa Hybidoma Bank. Other
antibodies were purchased from the indicated
companies: anti-EEA1 and anti-LC3 (MBL
International Corporation; Nanotools), anti-beclin-
1, anti-mTOR, and anti-phospho-mTOR (S2448)
antibodies (Cell Signaling); 6E10 antibody
(Biolegend) anti-rab9 and anti-calnexin (Santa
Cruz Biotech), anti-rab7 (Abcam), anti-tau (BD
BioSciences), anti-α-synuclein (Rockland), anti-
β-actin, anti-TGN46, anti-mouse, anti-rabbit and
anti-goat IgG-peroxidase (Sigma-Aldrich), anti-
mouse, rabbit and goat Alexa Flour 488 and 546
antibodies (Invitrogen).
RFP-GFP-LC3 construct has been
described previously (26), and was generously
provided by Dr. Jörg Höhfeld (University of Bonn,
Germany). Trehalose, maltose, glucose and 4′,6-
Diamidino-2-phenyindole (DAPI) were obtained
from Sigma-Aldrich. Bafilomycin A1 and
chloroquine were purchased from Enzo Life
Sciences. Cycloheximide as obtained from Sigma-
Aldrich, DMEM, Earle’s balanced salt solution
(EBSS), fetal calf serum (FCS), penicillin,
streptomycin, ampicillin, puromycin were
purchased from Invitrogen.
Cell lines and cell culture - Human
neuroglioma H4, human neuroblastoma SH-
SY5Y, human embryonic kidney 293 (HEK-293)
cells and human hepatocellular carcinoma cells
(Hep-G2) were obtained from ATCC. Mouse
embryonic fibroblasts–wild type (Mef-WT) and
Presenilin 1 and 2 double knock-out (Mef-PSdKO)
were a generous gift from Dr. De Strooper (VIB,
Belgium) and described previously (27). Mouse
embryonic fibroblasts of control wild-type (Mef-
WT) and Atg5 knock-out (Mef-Atg5KO) were
obtained from the Riken Cell Bank.
Cells were cultured in DMEM
supplemented with 10% FCS and 1%
penicillin/streptomycin. H4-mCherry-GFP-LC3
stable clones were generated from transfection of
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H4 cells with mCherry-GFP-LC3 constructs. The
protocol was performed with Lipofectamine
(Invitrogen) according to the manufacturer’s
instructions. Clones were selected with 25 µg/ml
puromycin.
Immunocytochemistry - Cells were
processed for immunocytochemistry as described
previously (28). Briefly, cells were cultured in
poly-L-lysine–coated coverslips and fixed with
4% PFA for 20 min. Subsequently, cells were kept
in blocking solution (PBS 90%, FCS 10%, Triton
X-100 0.1%) at room temperature (RT) for 1 h,
incubated with primary antibodies at 4oC
overnight or at RT for 1-2 h, and then with
secondary antibodies for 1 h at RT. Cells were
imaged with Carl Zeiss Axio Imaging 2 ApoTome
Fluorescence Microscope for optical sectioning.
Signal intensity and co-localization was analyzed
with AxioVision software. For quantification,
randomly selected images (n=10) were used.
Signals were quantified in two randomly chosen
boxes (150 µm2) per cell. Values represent means
± S.D.
Cell lysis, isolation of cellular membranes
and Western immunoblotting - For lysis, cells were
incubated in RIPA buffer (150 mM NaCl, 10 mM
Tris pH 8.0, 1% NP-40, 0.5% Na-DOC, 0.1%
SDS, 5 mM EDTA) supplemented with 1%
phosphatase inhibitor and 1% proteinase inhibitor
(Roche) for 15 minutes. Particulate materials were
removed by centrifugation at 13,200 rpm for 15
minutes, and supernatants were used for further
analyses.
To isolate cellular membranes, cells were
incubated in hypotonic buffer D (Tris HCl 10 mM
pH 7.5, NaCl 10 mM, EGTA 0.1 mM, Glycerol 2-
Phosphate 25 mM and DTT 1 mM) supplemented
with 1% proteinase inhibitor on ice for 10 minutes.
Cells were then homogenized by using 21 gauche
needles and centrifuged at 1000 rpm for 10
minutes to pellet nuclei. The supernatant was then
centrifuged at 13,200 rpm for 30 minutes to obtain
the membrane fraction as a pellet.
Membrane fractions or cellular lysates
were separated by sodium dedecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to nitrocellulose membranes
(Schleicher & Schuell). Membranes were blocked
in 5% non-fat milk for 1 h, incubated with primary
antibodies overnight and appropriate secondary
antibodies for 1 h. Proteins were detected by
enhanced chemiluminescence (ECL) using ECL
reagent (GE Healthcare) and an ECL imaging
station (ChemiDoc XRS, Bio-Rad). Quantification
of signals was done by the Quantity One software
package (Bio-Rad).
Subcellular fractionation – For subcellular
fractionation, cells were subjected to a hypotonic
shock as described above. Post nuclear fractions
(3800 rpm, 5min) were loaded onto a
discontinuous gradient (30%, 20%, 17.5%, 15%,
12,5%, 10%, 7,5% 5%, 2,5% OptiPrepTM,
Sigma
Aldrich, 1.2 ml each) and separated at 100000 x g
at 4°C for 8 h without break. Single fractions were
collected at a volume of 1ml and proteins were
precipitated with trichloroacetic acid.
Measurement of Aβ variants – Cells were
grown on 6-well culture plates until 70%
confluency in DMEM as described above. For
collection of Aβ, 750 µl of fresh medium was
added over-night. Conditioned media were cleared
by centrifugation. Cells were briefly washed and
lysed in RIPA buffer (150 mM NaCl, 10 mM Tris
pH 8.0, 1% NP-40, 0.5% Na-DOC, 0.1% SDS, 5
mM EDTA). Both cell lysates and conditioned
medium was analyzed by
electrochemiluminescence technology (MesoScale
Discovery) for Aβ38, Aβ40 and Aβ42 according
manufacturers protocol.
Metabolic radiolabeling and pulse-chase
experiments - Cells were grown in petri dishes
until 80% confluency. Cells were washed with
PBS and incubated in serum and methionine-free
medium for 30 min. Cells were then incubated
with 20 µCi of [35
S]-radiolabeled
methionine/cystein for 30 min to pulse-label newly
synthesized proteins. After pulse-labeling, cells
were washed with label free-medium containing
10% serum and a five-fold excess of unlabeled
methionine, and chased for the indicated time
periods. Cells were then lysed, and proteins
separated by SDS-PAGE. After transfer to PVDF
membrane, radiolabeled proteins were quantified
by phospho-imaging.
In vitro -secretase assay - The in vitro -
secretase assay was carried out as described
previously (18,29). Briefly, cells were lysed in
hypotonic buffer D and membranes were isolated
as described previously. The membrane pellet was
then re-suspended in citrate buffer (sodium citrate
150 mM, dH2O, pH 6.4 adjusted with citric acid)
supplemented with 1% protease/phosphatase
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inhibitors and incubated in the absence or presence
of -secretase modulators at 37°C for 2 h. Samples
were centrifuged at 13,200 rpm for 1 h. The
resulting pellets and supernatants were separated
by SDS-PAGE and proteins were detected by
Western-immunoblotting.
Data analysis and statistics - Statistical
analyses were carried out by calculation of
standard deviation (SD) and Student’s t test.
Significance is indicated by asterisks as follows: *
for p<0.05, ** for p<0.01, *** for p<0.001 and
n.s.: not significant.
RESULTS
Trehalose impairs the metabolism of APP
and decreases the secretion of Aβ - The
degradation of APP and its CTFs involves
autophagic and lysosomal pathways (17-19,25,26).
To assess the effect of trehalose on APP
metabolism, human neuroglioma H4 cells that
endogenously express APP were incubated with
trehalose for different time periods, and APP
levels were analyzed by Western immunoblotting.
Cell treatment with trehalose strongly increased
levels of APP-full length (FL) (Fig. 1A, B), and
even more pronounced that of APP-CTFs (Fig.
1A, C, D). Trehalose also significantly increased
the secretion of soluble APP derived from α-
secretory processing (sAPPsα) into conditioned
media. (Fig. 1E, F), suggesting that the increased
levels of cellular APP-FL were not caused by
inhibition of APP secretion.
To test whether trehalose exerts similar
effects in other cell types, we also used human
neuroblastoma SH-SY5Y, liver Hep-G2, and
embryonic kidney 293 cells. Trehalose induced
accumulation of APP-FL and particularly of APP-
CTFs in all tested cell lines (Fig. 1G), very similar
to the effects observed in human H4 cells.
We next wanted to test whether the
trehalose-dependent increase in APP-FL and APP-
CTFs is due to altered degradation. To inhibit de
novo protein biosynthesis, H4 cells were incubated
with cycloheximide (CHX) and chased in the
absence or presence of trehalose. In the absence of
trehalose, levels of APP-FL and its CTFs declined
within 2 h after block of de novo protein synthesis
(Fig. 1F). In the presence of trehalose, levels of
APP-FL also declined. Notably, levels of APP-
CTFs were stabilized and even increased after 1
and 2 h of CHX treatment in the presence of
trehalose (Fig. 1H). The increase in APP-CTFs
after block of protein biosynthesis could be
explained by the ongoing processing of APP-FL
by α- and β-secretase. These data indicate that
trehalose inhibits the degradation of APP-CTFs
without inhibition of secretory processing of APP-
FL.
Levels of Aβ derived from endogenous
APP in the different cell lines were below the
detection limit (not shown). Thus, we used
previously described fibroblasts that express either
wild-type APP (APP-WT) or the familial AD
associated Swedish mutant variant of APP (APP-
SW) (30). As expected, levels of Aβ in
conditioned media from cells expressing the APP-
SW variant were much higher than that in media
from APP-WT expressing cells (Fig. 2A, B).
Incubation of cells with trehalose reduced the
secretion of the different length variants Aβ38,
Aβ40, and Aβ42 from both APP-WT and APP-
SW expressing cells (Fig. 2A, B). Levels of cell-
associated or intracellular Aβ were very low. Only
Aβ40 could be detected in lysates of APP-SW
expressing cells. Here, levels were slightly
increased upon cell treatment with trehalose (Fig.
2B). These data demonstrate that trehalose
decreases the secretion of Aβ, and also impairs the
degradation of APP-CTFs.
Trehalose does not inhibit γ-secretase
activity or stimulate mTOR dependent autophagy -
‐Secretase is critically involved in the cleavage of
APP-CTFs and Aβ generation. Thus, we next
tested the potential effect of trehalose on γ-
secretase activity. Purified membranes of H4 cells
were incubated in the presence or absence of
trehalose, and γ-secretase activity was monitored
by the detection of the APP intracellular domain
(AICD) released into the supernatant. In control
samples, AICD was readily detected indicating
efficient cleavage of APP-CFTs by γ-secretase
(Fig. 3A, B). As expected, pharmacological
inhibition of γ‐secretase by the specific inhibitor
DAPT efficiently reduced the generation of AICD
(Fig. 3A, B). However, trehalose did not decrease
the formation of AICD (Fig. 3A, B),
demonstrating that trehalose does not inhibit
γ‐secretase activity. To further test whether
trehalose induced accumulation of APP-CTFs
independent on γ-secretase inhibition, we next
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used fibroblasts from wild-type (WT) and
presenilin-1/2 double knock-out (PSdKO) mice.
PSdKO cells lack both catalytically active variants
of presenilins and thus have no γ-secretase
activity. Accordingly, basal levels of APP-CTFs
were higher in PSdKO than in WT cells (Fig. 3C,
D). Interestingly, trehalose further increased
APP‐CTF levels in PSdKO cells (Fig. 3C, D),
indicating that the observed accumulation of APP-
CTFs upon cell treatment with trehalose is not
caused by inhibition of γ‐secretase activity. The
combined data demonstrate that trehalose
decreases the degradation of APP-CTFs without
inhibiting γ-secretase.
We next analyzed the effects of trehalose
on LC3, which is widely used as a marker for
autophagosomes. Upon induction of autophagy,
LC3 is converted from a cytosolic form (LC3-I) to
a phosphatidylethanolamine-conjugated form
(LC3-II), and thereby recruited to membranes of
autophagosomes (31). Consistent with previous
findings (21,22), trehalose induced a strong
increase of LC3-II levels over time (Fig. 4A),
resulting in elevated LC3-II/LC3-I ratios (Fig.
4B). However, this result would indicate that
trehalose either increases autophagosome
formation or/and decreases autophagic flux. To
further verify the effects of trehalose on
autophagy, we assessed p62 levels by
immunoblotting. P62 binds directly to LC3 and
also degraded during autophagy (32). Consistent
with increased LC3-II, we detected a significant
increase of p62 upon treatment with trehalose (Fig.
4A, C). Thus, the accumulation of p62 supports a
decreased clearance capacity of trehalose-treated
cells. Interestingly, immunocytochemistry
revealed that accumulated LC3 - partially co-
localized with APP positive vesicles upon
trehalose treatment, while both proteins were
segregated in control cells (Fig. 4D-F).
Although trehalose is commonly used to
modulate autophagic activity, very little is known
how it regulates the autophagic machinery. We
next analyzed proteins involved in the induction of
autophagy, including the mammalian target of
rapamycin (mTOR) found in mTOR complexes
and BECN1, a major regulator protein of the class
III phosphatidyl-inositol-3-kinase (PI3K) (8,33).
Western blot analyses revealed no significant
changes in the levels of BECN1 (Fig. 5A, B), total
mTOR and phosphorylated mTOR (S2448) (Fig.
5A, C, D) upon cell treatment with trehalose,
suggesting that trehalose did not affect these
proteins involved in the induction of autophagy.
The results are consistent with previous findings
showing that effects of trehalose are independent
on mTOR activity (21).
To further analyze the role of trehalose in
the autophagic process and the metabolism of
APP, we used Atg5KO cells. Atg5 plays a role at
early steps of autophagosome formation and is
required for the lipidation of LC3 (31).
Accordingly, cells deficient for Atg5 are defective
in the formation of autophagosomes (34).
Fibroblasts of WT and Atg5-KO mice were treated
with trehalose, and LC3 and APP were analyzed
by Western immunoblotting. In WT cells,
trehalose induced an increase in LC3-II resulting
in an increased LC3-II/LC3-I ratio (Fig. 5E, F). In
contrast, LC3-II was not detected in Atg5 KO
cells. Due to defective conversion of LC3-I to
LC3-II, LC3-I levels were strongly elevated in
Atg5-KO cells (Fig. 5E, F). Interestingly, the
treatment of Atg5-KO cells with trehalose still led
to accumulation of APP-FL and particularly of its
CTFs (Fig. 5E, G). The response of Atg5 KO cells
was very similar to that of WT cells, indicating
that the effects of trehalose on APP metabolism
are independent on Atg5-dependent induction of
autophagy. Together with the unchanged levels of
BECN1 and mTOR phosphorylation (S2448) in
trehalose-treated cells, an increase in LC3-II, APP-
CTFs as well as elevation of p62 strongly suggest
alterations of vesicular transport and/or autophagic
flux upon cell treatment with trehalose. Trehalose
also decreased the degradation of long-lived
proteins (Fig. 5H), further indicating an
impairment of autophagic flux.
Inhibition of APP-CTF degradation is
selective for the disaccharide trehalose - The
intracellular pathways by which trehalose is
metabolized and regulates cell signaling are still
unknown. Since trehalose is a disaccharide
composed of two D‐glucose residues, it was tested
whether supplementation of glucose induces
similar effects on the metabolism of APP and
LC3. H4 cells were incubated with trehalose or
additional glucose in the culture media. As
observed before, trehalose treatment led to strong
accumulation of LC3‐II, APP-FL, and APP-CTFs.
In contrast, glucose treatment did not change the
ratio of LC3‐II/LC3‐I (Fig. 6B, C). Similarly,
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glucose also did not alter the ratio of APP-
CTFs/APP‐FL (Fig. 6B, D). Together, the data
indicate that the effects of trehalose on autophagy-
lysosomal function did not involve increased
generation of glucose.
Maltose is a disaccharide similar to
trehalose that is also composed of two glucose
molecules. The two disaccharides only differ in
the type of the glycosidic linkage between the two
glucose residues. While the glucose residues in
maltose are coupled by an α‐1,4 linkage, they are
coupled by an α‐1,1 glycosidic linkage in trehalose
(35) (Fig. 6A). Therefore, we tested whether
trehalose‐induced effects could be mimicked by
the disaccharide maltose. However, in contrast to
trehalose, maltose did not affect the levels of LC3
and APP (Fig. 6E‐G). These findings indicate that
the inhibition of LC3 and APP degradation is
specific for the disaccharide trehalose.
Trehalose alters the subcellular
localization and degradation of APP-CTFs - The
present data strongly suggest an inhibitory effect
of trehalose on APP metabolism in endo-
lysosomal compartments. To test whether direct
inhibition of lysosomal degradation could mimic
the effects of trehalose on LC3 and APP, H4 cells
were treated with trehalose or different lysosomal
inhibitors including bafilomycin A1 and
chloroquine. Bafilomycin A1 is a specific inhibitor
of the vacuolar-type H(+)-ATPase, which inhibits
acidification and therefore protein degradation in
lysosomes (36). Chloroquine is a lysosomotrophic
compound that also elevates/neutralizes the
intralumial pH of endolysosomal compartments
(37). Both bafilomycin A1 and chloroquine led to
accumulation of LC3-II and APP-CTFs (Fig. 7A-
C). However, the effects of bafilomycin A1 and
chloroquine differed quantitatively from that of
trehalose. While LC3-II showed strongest
accumulation upon direct inhibition of lysosomal
activity with bafilomycin A1 or chloroquine, APP-
CTF showed strongest accumulation upon cell
treatment with trehalose (Fig. 7A-C).
Cathepsin D (Cat D) is an abundant
aspartic endopeptidase in lysosomes (38,39). The
precursor form of Cat D is proteolytically
processed to an intermediate during its transport
from the Golgi to acidic endolysosomal
compartments. In the lysosomes, the intermediate
Cat D is further processed to generate the active
fragment as the C-terminal heavy chain (40,41).
Thus, we assessed the effect of trehalose on the
proteolytic processing of Cat D. Interestingly, cell
treatment with trehalose strongly decreased the
processing of Cat D as indicated by elevated level
of the Cat D intermediate form, with concurrent
decrease of the catalytically active fragment of Cat
D (Fig. 7A). Accordingly, ratios of fully processed
active Cat D to precursor and intermediate forms
were strongly reduced upon cell incubation with
trehalose (Fig. 7D). Consistent with previous
reports showing that Cat D processing is affected
by lysosomal pH (40), bafilomycin A1 and
chloroquine also decreased the levels of the active
forms of Cat D (Fig. 7A, D).
GFP-LC3 is being widely used as a
fluorescent marker of autophagosomes. However,
GFP is acid-sensitive, and thus its fluorescence
decreases in acidic compartments, i.e. when GFP-
LC3 containing autophagosomes fuse with
endosomes and lysosomes (26). In contrast, the
monomeric red fluorescent protein mCherry is
acid-stable. Therefore, a tandem fusion of the
acid-insensitive red fluorescent protein (mCherry)
and the acid-sensitive green fluorescent protein
(GFP) at the N-terminus of LC3 is a useful tool to
assess fusion of LC3-positive autophagic vesicles
with lysosomes by microscopy (37). H4 cells
stably expressing mCherry-GFP-LC3 were
cultured in the absence or presence of trehalose.
As additional controls, cells were also incubated
with chloroquine or starved in EBSS medium.
Signals for mCherry and GFP were analyzed by
fluorescence microscopy. Control cells showed
predominantly mCherry positive punctae, but very
few GFP positive punctae (Fig. 8A).
Quantification revealed that the number of
mCherry positive punctae was approximately ten-
times higher than that of GFP positive punctae
(Fig. 8B). This is consistent with efficient
quenching of the GFP fluorescence in acidic
autolysosomes, while the mCherry flourescence is
not affected in these compartments. Interestingly,
cell treatment with trehalose not only increased the
number of GFP positive punctae as compared to
control cells, but also led to a large overlap with
mCherry positive structures (Fig. 8A, B),
indicating inefficient quenching of the GFP signal.
Cell incubation with chloroquine had very similar
effects as trehalose (Fig. 8A, B). Cell incubation
with EBSS increased both mCherry and GFP
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positive punctae compared to control cells (Fig.
8A, B). However, the number of mCherry positve
punctae was approximately three-time higher than
the number of GFP-LC3 positive punctae (Fig.
8B). As the incubation with EBSS enhances the
induction of autophagy, LC3-I is efficiently
converted to LC3-II and associates with
autophagosomes. However, the efficient
acidification of lysosomes and autolysosomes
could lead to quenching of the GFP signal, thereby
explaining the higher number of mCherry positive
vesicles as compared to that of GFP positive
punctae. The combined data strongly indicate that
trehalose impairs the delivery of LC3 to and its
degradation in lysosomal compartments
To further test alterations in the
subcellular distribution of APP-CTFs upon cell
treatment with trehalose, we performed
fractionation of vesicular compartments. In control
cells, mature full-length APP and CTFs were
detected in LC3, Rab7, Rab9 and LAMP2 positive
fractions (Fig. 9). The treatment with trehalose
resulted in redistribution of APP-CTFs, Rab7,
Rab9, and LAMP2. Cell incubation with Baf A1
also led to accumulation of APP-CTFs in fractions
that clearly segregated from APP-CTF positive
fractions from trehalose treated cells (Fig. 9). The
combined treatment of cells with trehalose and Baf
A1 resulted in APP-CTF distribution similar to
that from cells treated with Baf A1 alone.
Fractionation of marker proteins for the trans-
Golgi network (TGN46), and endoplasmic
reticulum (calnexin) was not affected by the
treatment with trehalose or Baf A1. These data
suggest that cell treatment with trehalose affects
the subcellular distribution and/or trafficking of
APP in late endosomal and lysosomal
compartments.
DISCUSSION
The present data demonstrate that
trehalose decreases clearance of APP-CTFs and
the secretion of Aβ. Cell biological experiments
showed redistribution of APP-CTFs and other
endosomal and lysosomal proteins upon trehalose
treatment. Trehalose also led to accumulation of
LC3-II and decreased the processing of Cat D.
These data are consistent with impaired vesicular
transport and fusion of endosomal and lysosomal
compartments. The cleavage of APP by α- and β-
secretases can occur at distinct cellular locations.
While α-secretase could predominantly cleave
APP in secretory vesicles and at the cell surface,
β-secretase has an acidic pH optimum and shows
preferential cleavage in acidic compartments,
including late endosomes and lysosomes. The C-
terminal fragments of APP generated by α- and β-
secretase cleavage represent substrates for γ-
secretase that resides predominantly in
endolysosomal compartments or at the cell surface
(42-46). Although trehalose led to strong
accumulation of APP-CTFs, we observed rather
decreased secretion of Aβ. Since trehalose did not
inhibit γ-secretase, the findings suggest that
trehalose rather decreases the delivery of the APP-
CTFs to vesicular compartments containing γ-
secretase activity. We also observed decreased
proteolytic activation of cathepsin D that also
occurs during endosomal transport and delivery to
lysosomes upon cell treatment with trehalose.
Subcellular fractionation indeed showed
redistribution of APP-CTFs together with marker
proteins of late endosomal (Rab7, Rab9) and
lysosomal (LAMP2) compartments. The
molecular mechanisms underlying these effects
remain to be determined in more detail. However,
the effects of trehalose on subcellular distribution
differ from that induced by bafilomycin A1,
suggesting that trehalsoe does not or at least not
only alter lysosomal pH regulation.
Additional experiments using a tandem
fusion mCherry-GFP tagged LC3 also
demonstrated significant changes in the transport
of LC3 to lysosomes upon trehalose treatment.
While control cells showed predominantly
mCherry punctae due to quenching of GFP in
efficiently acidified autolysosomes, trehalose
treatment increased the colocalization of mCherry
and GFP. These data indicate inefficient delivery
of LC3 to lysosomes.
Trehalose is a disaccharide composed two
D-gluclose molecules linked by an α-1,1
glycosidic linkage (35). Therefore, we tested
whether trehalose-regulated effects might be
caused by potential metabolites of trehalose or
could be mimicked by other disaccharides.
However, neither addition of glucose itself nor of
the disaccharide maltose had overt effects on APP
and LC3 levels, indicating that the observed
effects are specific for trehalose, and not due to
increased supply with glucose. Our data also
indicate that the effects of trehalose are
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Trehalose alters trafficking of APP
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independent on mTOR, BECN1 and Atg5. The
data thus confirm and extend that trehalose
induced effects do not involve changes in mTOR
complex 1 activity (21).
The present findings are surprising,
because trehalose has been shown to decrease
cellular levels of the neurodegenerative disease-
associated proteins huntingtin, α-synuclein and
phosphorylated-tau in cellular and animal models
(22-24), suggesting that trehalose promoted
autophagy and lysosomal clearance of these
proteins. A possible explanation for this
contradiction might emerge from a recent study
demonstrating the involvement of unconventional
secretion of aggregated α-synuclein via exophagy
in cases of impaired autophagosome-lysosome
fusion or lysosomal function. Importantly,
trehalose was shown to enhance α-synuclein
secretion in PC12 catecholaminergic nerve cells
(47). In addition, increased secretion of tau was
also observed upon starvation and lysosomal
inhibition in primary neurons, likely caused by
release of autophagosomal contents into
extracellular fluids (48). In our experiments
cellular level of endogenously expressed tau and
α–synuclein were not affected by trehalose (data
not shown). Both proteins were not detected in
conditioned media. However, we observed
elevated levels of an overexpressed reporter
protein for polyQ-containing aggregates upon cell
treatment with trehalose (data not shown),
suggesting that trehalose might increase the
release of protein aggregates. Secretion of
pathogenic polyQ protein from cultured cells has
been reported previously (49).
Decreased levels of neurodegeneration-
associated protein aggregates upon trehalose
treatment might also result from upregulation of
the ubiquitin proteasome system. In contrast to
APP and its CTFs; tau, huntingtin and α-synuclein
are cytosolic proteins. It has been shown that
inhibition of the proteasome by epoxomicin
increased the levels of α-synuclein and
phosphorylated-tau, and correlated with enhanced
cell death (22). Thus, although autophagy is
considered as the major pathway to degrade
protein aggregates, a contribution of proteasomal
degradation to the reduction of aggregate-prone
proteins could not be excluded. APP as an integral
membrane protein is mainly targeted to
endosomal-lysosomal compartments via
endocytosis (42), and eventually degraded by
lysosomal hydrolases (17-19,44,50,51).
Decreased formation of cytosolic
aggregates and lower toxicity was also observed
with a mouse model for Huntington’s disease upon
treatment with trehalose (21). However, this effect
was at least partially attributed to the function of
trehalose as a chemical chaperone that could bind
to expanded polyQ stretches and thereby stabilize
the partially unfolded protein (52,53).
Trehalose appears to exert complex
functions in cellular protein homeostasis. The
effects of trehalose on vesicular trafficking and
protein metabolism shown in this study should be
considered not only for the interpretation of
experimental results obtained with this
disaccharide, but also for its application in future
therapeutical approaches.
Acknowledgments: We thank Drs. S. Höning and J. Höhfeld for providing Cathepsin D antibodies and
mCherry-GFP-LC3 cDNA, respectively. We are also grateful to Dr. B. de Strooper and the RIKEN cell
Bank for providing PS1/2 and Atg5 deficient cells, respectively.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contribution: NT and IK contributed equally to this study, performed and designed the
experiments, and analyzed data. IYT also designed and analyzed the experiments. JW conceived the
study, designed experiments, interpreted data and wrote the manuscript. All authors read and edited the
manuscript.
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The abbreviations used are: APP, amyloid precursor protein; Aβ, amyloid β-peptide; AICD, APP
intracellular domain; ATG, autophagy related gene; BECN1, beclin-1; CTF, C-terminal fragment; EBSS,
Earle’s balanced salt solution; FL, full-length; cat D, cathepsin D; LC3, microtubule-associated protein 1
light chain 3; NPC, Niemann-Pick type C; mTOR, mammalian target of rapamycin, PI3K, phosphatidyl
inositol 3 kinase; WT, wild-type; PS, presenilin.
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FIGURE LEGENDS
FIGURE 1. Trehalose induces accumulation of APP and APP-CTFs. A, Western immunoblot of APP
and APP-CTFs. H4 cells were incubated in the presence or absence of 100 mM trehalose (Tre) for the
indicated time periods. Cell lysates were subjected to SDS-PAGE and Western immunoblotting. β-Actin
was used as loading control. B-D, Quantification of APP-full length (FL) (B), APP-CTFs (C) and the
ratios of APP-CTFs/FL (D) by ECL imaging. Values represent means ± S.D. of six biological replicates.
(E) H4 cells were incubated in the presence or absence of 100 mM trehalose (Tre) for 24 hrs. Soluble
APPα (sAPPα) in conditioned media, and APP-FL and APP-CTFs in cell lysates were analyzed by
western immunoblotting. (F) Quantification of sAPPα in media by ECL imaging. Values represent means
± S.D. of three biological replicates. G, The indicated cell types (SH-SY5Y, HepG2, HEK293) were
incubated in the presence or absence of 100 mM trehalose for 24 h. Cells were lysed and proteins detected
by Western immunoblotting. (H) H4 cells were incubated in the presence or absence of 100 mM trehalose
(Tre) for 2 hrs. After addition of cycloheximide (CHX, 20 µg/ml), cells were further incubated for the
indicated time periods. Cellular membranes were isolated and APP-FL and APP-CTFs detected by
Western immunoblotting.
FIGURE 2. Trehalose decreases the secretion of Aβ. Mouse embryonic fibroblast stably expressing APP-
wild type (WT) (A) or APP-swedish (SW) (B) were incubated for 24 hrs in the absence or presence of
trehalose. Levels of extracellular (medium) and intracellular (lysate) Aβ variants were determined by
electrochemiluminescence. n.d., not detectable. Values represent means ± S.D. of three biological
replicates.
FIGURE 3. Trehalose impairs degradation of APP CTFs without inhibition of -secretase activity. A, -
Secretase in vitro assay. Isolated membranes of H4 cells were incubated in citrate buffer supplemented
with either 10 µM DAPT or 100 mM trehalose at 37oC for 2 h and then centrifuged to separate pellets and
supernatants. Proteins of the pellet and supernatant fractions were separated by SDS-PAGE.
Subsequently, APP-CTFs and APP intracellular domain (AICD) were detected by immunoblotting in
pellet and supernatant fractions, respectively. B, Quantification of the AICD/APP-CTFs ratio from the
blot (A) (n=2). C, Immunoblotting of APP in lysates of wild-type (WT) and presenilin 1/2 double knock-
out (PSdKO) MEFs after incubation in the absence or presence of 100 mM trehalose for 24 h. D,
Quantification of the APP-CTFs/APP-FL ratio from (C). Values represent means ± S.D. of duplicate
experiments.
FIGURE 4. Treahlose increases levels of autophagy markers. A, Western immunoblot analyses of the
autophagosomal marker proteins LC3 and p62 in H4 cells treated with 100 mM Trehalose for indicated
time periods. B-C, Quantification of the LC3-II/LC3-I ratio (B) and p62 (C) were performed by ECL
imaging. Values represent means ± S.D. of triplicates. D, Immunocytochemical analyses of LC3 and APP
in control and trehalose-treated cells. Cells were co-stained with anti-LC3 and anti-APP C-terminal
specific primary antibodies, and Alexa Fluor 546 and Alexa Fluor 488-coupled secondary antibodies,
respectively. Scale bar represents 10 µm. E, The colocalization of APP and LCe in control and trehalose-
treated cells was estimated through the Pearson's correlation coefficient by using GraphPad Prism
software. F, Quantification of LC3/APP punctae in control and trehalose-treated cells (n=20).
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FIGURE 5. Effects of trehalose are independent on mTOR and Atg5. A, Western immunoblot of Beclin-
1 (BECN1) and mTOR from lysates of H4 cells after cell incubation with or without trehalose for 24 h.
B-D, Quantification of BECN1 (B), mTOR (C) and phosphorylated mTOR (S2448) (D) by ECL imaging.
E, Western immunoblot of LC3 and APP from lysates of MEF wild-type (WT) and MEF Atg5 knock-out
(Atg5KO). F-G, Quantification of the ratios LC3-II/LC3-I (F) and APP-CTFs/FL (G). Values represent
means ± S.D of triplicate experiments. H, Trehalose reduces the degradation of long-lived proteins. H4
cells were pulse-labeled with [35
S]methionine/cysteine, and then chased in the presence or absence of 100
mM trehalose for the indicated time periods as described under experimental procedures. Values represent
means ± S.D. of triplicate experiments; p<0.001.
FIGURE 6. Selective effect of trehalose on the degradation of LC3 and APP-CTFs. A, Chemical
structures of trehalose, characterized by an α-1,1 glycosidic linkage between two glucose (Gluc) residues
(a) and maltose, characterized by an α-1,4 linkage between two glucose residues (b) (54). B, Western
immunoblot of LC3 and APP from H4 cellular lysates after treatment in the absence or presence of 100
mM trehalose or 150 mM glucose for 24 h. C-D, Quantification of the ratios of LC3-II/LC3-I (C) and
APP-CTFs/APP-FL (D). E, Western immunoblot of LC3 and APP from H4 cellular lysates after cell
incubation in the absence or presence of 100 mM trehalose or 100 mM maltose for 24 h. F-G,
Quantification of LC3-II/LC3-I (F) and APP-CTFs/APP-FL (G) ratios. Values represent means ± S.D of
triplicate experiments.
FIGURE 7. Impairment of Cathepsin D processing upon Trehalose treatment or inhibition of lysosomal
acidification-. A, H4 cells were incubated in the absence or presence of 100 mM trehalose for 24 h, 50
nM bafilomycin A1 (Baf) for 12 h, or 50 μM chloroquine (Chlo) for 12 h. Western immunoblot detection
of LC3, APP and Cathepsin D (Cat D). B-D, Quantification of LC3 (B), APP (C) and Cat D (D). Values
represent means ± S.D of duplicate experiments.
FIGURE 8. Altered trafficking of LC3 upon cell treatment with trehalose. A, H4 cells stably expressing
mCherry-GFP-LC3 were treated with 100 mM trehalose for 24 h, 50 μM chloroquine (Chlo) for 12 h or
starved in EBSS medium for 3 h. Images are representative maximum-intensity layers of Apotome optical
sections. White arrowheads indicate mCherry- and GFP-positive vesicles. B, mCherry- and GFP-positive
punctae were quantified as described in the experimental procedures. Scale bar 10 µm.
FIGURE 9. Trehalose alters subcellular localization of APP-CTFs. H4 cells were incubated in medium
only (Ctr), with trehalose (Tre) or bafilomycin A1 (Baf A1), separately, or with a combination of
trehalose and bafilomycin A1 (Tre+BafA1) for 24 hours, and subjected subcellular fractionation by
density gradient centrifugation (see experimental procedures). The indicated proteins were detected by
Western immunoblotting. Results are representative of two independent experiments.
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Trehalose alters trafficking of APP
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Figure 1B
0 .0
0 .5
1 .0
1 .5
AP
P-F
L
lev
els
[a
.u.]
*** *** ***
24 48 96Tre [h] 0
C
0 .0
0 .5
1 .0
1 .5
AP
P-C
TF
lev
els
[a.u
.]
****** ***
24 48 96Tre [h] 0
0 .0
0 .5
1 .0
1 .5
AP
P-C
TF
s/
FL
rati
o [
a.u
.]
*** ******
24 48 96Tre [h] 0
D
F
0 .0
0 .5
1 .0
1 .5
2 .0*
sA
PP
α (
a.u
)
TreCtr
A
G
APP-FL
APP-CTFs
SH
-SY
5Y
He
p-G
2H
EK
-293
β-Actin
Ctr Tre
APP-FL
APP-CTFs
β-Actin
APP-FL
APP-CTFs
β-Actin
100
100
100
15
15
15
37
37
37
HCtr Tre
0 1 2 0 1 2CHX [h]
APP-FL
APP-CTFs
100
15
sAPPα
APP-FL
APP-CTFs
β-Actin
Ctr Tre
H4 cells
medium
lysate
E
100
15
37
100
0 24 48 96
β-Actin
Tre [h]
APP-FL
APP-CTFs 15
37
100
kDa
kDakDa
kDa
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Trehalose alters trafficking of APP
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A
38
A
40
A
42
A
38
A
40
A
42
A
0
10
20
30
Ctr
Tre
A
(p
g/m
l)
***
n.d. n.d. n.d.
***
***
B
38
A
40
A
42
A
38
A
40
A
42
A
0
100
200
300
400
Ctr
Tre
A
(p
g/m
l)
n.d.n.d.
***
***
*** ***
Figure 2
APP-WT APP-SWE
medium lysate medium lysate
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Trehalose alters trafficking of APP
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Figure 3
0.0
0.2
0.4
0.6
0.8
AIC
D/A
PP
-CT
Fs
rati
o [
a.u
.]
Ctr DAPT Tre
B
AP
P-C
TF
s/
FL
rati
o [
a.u
.]
WT PSdKO
0 .0
0 .2
0 .4
0 .6
0 .8
C t r T r eD
CA
Ctr
APP -FL
APP -CTFs
β-Actin
Tre
WT PSdKO
Ctr Tre
15
37
100
Ctr DAPT Tre
APP -CTFs
AICD
4oC
15
15
kDa kDa
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Trehalose alters trafficking of APP
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Figure 4
0 .0
0 .5
1 .0
1 .5
96
B
480 24
LC
3-I
I/ L
C3
-I
rati
o [
a.u
.] ***
*** ***
Tre [h]
A
D
Ctr
Tre
LC3 APP Merged
Ctr Tre
LC
3/A
PP
po
sit
ive
pu
ncta
e/c
ell
E
p6
2 lev
els
[a.u
.] *** ******
0 .0
0 .5
1 .0
1 .5
96480 24Tre [h]
C
0 5 0 1 0 0 1 5 0
0
1 0
2 0
3 0
0 2 0 4 0 6 0 8 0
0
2
4
6
8
1 0
LC
3/
AP
P c
o-
loc
ali
za
tio
n
LC
3/
AP
P c
o-
loc
ali
za
tio
n
APP punctaeAPP punctae
Pearson’s correlation
coefficient (R=0.171)
Pearson’s correlation
coefficient (R=0.341)
Ctr Tre
F
0
1 0
2 0
3 0
4 0
***
LC3-ILC3-II
0 24 48 96Tre [h]
β-Actin
p62
15
55
37
kDa
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Trehalose alters trafficking of APP
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Figure 5
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Ctr Tre
mT
OR
lev
els
[a.u
.]
n.s.
C
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Ctr Tre
BE
CN
1
lev
els
[a.u
.]
n.s.
B
0 .0
0 .1
0 .2
0 .3
0 .4
Ctr Tre
p-m
TO
R(S
244
8)
lev
els
[a.u
.]
n.s.
D
E F
0 .0
0 .5
1 .0
1 .5
C t r T r e
***
***
***
WT Atg5KO
AP
P-C
TF
s/F
L
rati
o [
a.u
.]
G
WT Atg5KO
**
LC
3-I
I/L
C3-I
rati
o [
a.u
.]
***
0 .0
0 .5
1 .0
1 .5
C t r T r e
Ra
dio
lab
ele
d p
rote
ins
[a.u
.]
Chase [h]
0 .0
0 .5
1 .0
1 .5 C t r T r e
0 4 8
H
***
BECN1
mTOR
Ctr Tre
p-mTOR
(S2448)
β-Actin
A
55
250
250
37
WT Atg5KO
LC3-I
LC3-II
APP-FL
APP-CTFs
β-Actin
Ctr Tre Ctr Tre
15
100
37
15
kDa
kDa
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Trehalose alters trafficking of APP
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Figure 6
A
C
D
LC
3-I
I/L
C3
-I
rati
o [
a.u
.]
Ctr Tre Gluc0
2
4
6
AP
P-C
TF
s/F
L
rati
o [
a.u
.]
Ctr Tre Gluc0 .0
0 .5
1 .0
1 .5
B
E
0
2
4
6
8
L3-I
I/L
C3-I
rati
o [
a.u
.]
Ctr Tre Mal
***
n.s.
F
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
AP
P-C
TF
s/F
L
rati
o [
a.u
.]
Ctr Tre Mal
***
n.s.
G
(a) Trehalose (b) Maltose
APP-FL
APP-CTFs
Ctr Tre Gluc
LC3-I
LC3-II 15
100
15
Ctr Tre Mal
LC3 I
LC3 II
APP-CTFs
APP-FL
β-Actin
15
100
15
37
kDa
kDa
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Trehalose alters trafficking of APP
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Figure 7A
0
5
1 0
1 5
LC
3-I
I/L
C3-I
rati
o [
a.u
.]
Ctr Tre Baf Chlo
B C
AP
P-C
TF
s
exp
ress
ion
[a.u
.]
Ctr Tre Baf Chlo0 .0
0 .5
1 .0
1 .5
0 .0
0 .5
1 .0
1 .5
Ca
t D
-acti
ve
/FL
rati
o [
a.u
.]
Ctr Tre Baf Chlo
D
Ctr Tre Baf Chlo
β-Actin
APP-CTFs
LC3-I
LC3-II
precursor
active
Cat D intermediate
15
37
25
15
37
kDa
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Trehalose alters trafficking of APP
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Figure 8A
Ctr
GFP-LC3mCherry-LC3 MergeE
BS
S
Ch
loT
re
B
0
2 0
4 0
6 0
8 0
1 0 0m C h e r r y G F P
Ctr Tre EBSSChlo
Nu
mb
ers
of
GF
P a
nd
mC
he
rry-p
os
itiv
e
ve
sic
les /c
ell
***
***
n.sn.s.
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Trehalose alters trafficking of APP
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35
Figure 9
APP FL
1 2 3 4 5 6 7 8 9 10 11
APP CTFs
Baf A1
Tre + BafA1
Ctr
Tre
Rab7LC3
LAMP2
Baf A1
Tre + BafA1
Ctr
Tre
Rab9
Calnexin
Baf A1
Tre + BafA1
Ctr
Tre
TGN46
1 2 3 4 5 6 7 8 9 10 11
25
25
25
25
35
35
35
100
100
100
100
Baf A1
Tre + BafA1
Ctr
Tre
28
28
28
28
15
15
15
15
130
130
130
130
55
55
55
55
15
15
15
15
kDa
kDa
kDa
kDa
kDa
kDa
kDa
kDa
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Nguyen T. Tien, Ilker Karaca, Irfan Y. Tamboli and Jochen WalterAlzheimer-associated amyloid precursor protein
Trehalose alters subcellular trafficking and the metabolism of the
published online March 8, 2016J. Biol. Chem.
10.1074/jbc.M116.719286Access the most updated version of this article at doi:
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