Nguyen T. Tien , Ilker Karaca , Irfan Y. Tamboli , and …...Nguyen T. Tien#, Ilker Karaca&, Irfan Y. Tamboli , and Jochen Walter1 From the Department of Neurology, University of Bonn,

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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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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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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18

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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19

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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20

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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21

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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22

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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23

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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24

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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25

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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26

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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Nguyen T. Tien , Ilker Karaca , Irfan Y. Tamboli , and …...Nguyen T. Tien#, Ilker Karaca&, Irfan Y. Tamboli , and Jochen Walter1 From the Department of Neurology, University of Bonn,