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Curcumin Decreases Amyloid-

Peptide Levels by
Attenuating the Maturation of Amyloid-

Precursor Protein
*
□
S
Received for publication, April 13, 2010, and in revised form, June 30, 2010 Published, JBC Papers in Press, July 9, 2010, DOI 10.1074/jbc.M110.133520
Can Zhang, Andrew Browne, Daniel Child, and Rudolph E. Tanzi
1
From the Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Department of Neurology,
Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129-2060
Alzheimer disease (AD) is a devastating neurodegenerative
disease with no cure. The pathogenesis of AD is believed to be
driven primarily by amyloid-

(A

), the principal component of
senile plaques. A

is an ⬃4-kDa peptide generated via cleavage
of the amyloid-

precursor protein (APP). Curcumin is a com-
pound in the widely used culinary spice, turmeric, which possesses
potent and broad biological activities, including anti-inflammatory
and antioxidant activities, chemopreventative effects, and effects
on protein trafficking. Recent in vivo studies indicate that curcu-
min is able to reduce A

-related pathology in transgenic AD mouse
models via unknown molecular mechanisms. Here, we investigated
the effects of curcumin on A

levels and APP processing in various
cell lines and mouse primary cortical neurons. We show for the first
time that curcumin potently lowers A

levels by attenuating the
maturation of APP in the secretory pathway. These data provide a
mechanism of action for the ability of curcumin to attenuate amy-
loid-

pathology.
Alzheimer disease (AD)
2
is a devastating neurodegenerative
disorder and the primary cause of dementia in the elderly (1).
Genetic studies of the disease have revealed a complex and
strong genetic etiology. Four genes have been established to
either cause early-onset familial AD with complete penetrance
(amyloid-

(A4) protein precursor (APP), presenilin 1 (PSEN1),
and presenilin 2 (PSEN2) or to increase susceptibility for late-
onset AD with partial penetrance (APOE) (2–4). Despite its
heterogeneous inheritance, the functional neuropathology of
AD is represented commonly by disruption in neural circuits,
such as loss of neurons and synapses primarily in the neocortex
and hippocampus (5, 6). The pathophysiology of AD is charac-
terized by two distinctive features: amyloid plaques comprised
primarily of a small peptide named A

and neurofibrillary tan-
gles composed of hyperphosphorylated Tau (2, 6, 7). Whereas
A

42 and A

40 are the two primary A

species, A

42 is more
prevalent than A

40 in amyloid plaques. Mounting genetic,
biochemical, and molecular biological evidence suggests that
the excessive accumulation of A

is the primary pathological
event leading to AD (3, 6, 7). A

is produced by a sequential
proteolytic cleavage of the type I transmembrane protein, amy-
loid-

(A4) precursor protein (APP) (8). The initial cleavage of
APP can be mediated by
␣
-or

-secretase (or BACE1).
␣
-Secre-
tase cleavage produces sAPP
␣
and
␣
C-terminal fragment (or
C83);

-secretase cleavage produces sAPP

and

C-terminal
fragment (C99). C83 and C99 can be further cleaved by
␥
-secre-
tase to produce P3 or A

(2, 7).
For more than a decade after the discovery of A

and the
establishment of the A

hypothesis, one essential strategy for
AD therapeutics has focused on modulating APP processing
and decreasing A

levels (2, 9). Recently, considerable effort
has been concentrated on identifying natural dietary supple-
ments that can prevent, inhibit, or reverse A

accumulation or
aggregation (10). Emerging evidence supports a use for curcu-
min in AD therapeutics. Curcumin (diferulomethane) is a yel-
low pigment in turmeric (or curcuma longa), the widely used
spice, and a food additive used primarily in Indian culinary
preparations (11). Curcumin is a low molecular weight mole-
cule with broad and beneficial biological activities including
potent antioxidant, anti-inflammatory, and chemo-preventa-
tive effects (12–15) with a favorable toxicity profile (13, 14).
Epidemiological studies have suggested that curcumin con-
tributes to the reported 4.4-fold reduced (age-adjusted)
prevalence of AD in India compared with the United States
(16). Both in vitro and in vivo studies have shown that cur-
cumin can bind to amyloid and inhibit A

aggregation (14,
17), as well as fibril and oligomer formation (14). In vivo studies
have shown that dietary curcumin can cross the blood-brain
barrier and significantly decrease A

deposition and plaque
burden in AD transgenic mice (14, 15, 18, 19), markedly inhibit
Tau phosphorylation (20), and attenuate inflammation and
reduce oxidative damage (14, 18), and reduce genomic instabil-
ity events (21).
These findings support a beneficial role for the use of cur-
cumin in treating AD. However, to date, the effects of cur-
cumin on APP metabolism have not been elucidated. Here,
we investigated the effects of curcumin on A

levels and APP
processing in mouse primary cortical neurons and various
cell lines. Curcumin potently lowered A

levels and hindered
APP maturation. Curcumin also markedly attenuated the
effects of brefeldin A (BFA), a specific Golgi-disrupting agent,
on APP maturation and trafficking. Taken together, we have
*This work was supported by the Cure Alzheimer’s Fund, the Funds for Med-
ical Discovery from Massachusetts General Hospital, and a Neurodegen-
erative Disease Pilot Study Grant from the Harvard NeuroDiscovery Center
and Massachusetts Alzheimer’s Disease Research Center.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1 and 2.
1
To whom correspondence should be addressed: Harvard Medical School,
Genetics and Aging Research Unit, MassGeneral Institute for Neurode-
generative Disease, Massachusetts General Hospital, 114 16th St.,
Charlestown, MA 02129. Tel.: 617-726-6845; Fax: 617-724-1949; E-mail:
tanzi@helix.mgh.harvard.edu.
2
The abbreviations used are: AD, Alzheimer disease; APP, amyloid-

precur-
sor protein; A

, amyloid-

peptide; APPma, mature APP; APPim, immature
APP; sAPP
␣
,
␣
-secretase-soluble amyloid-

precursor protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 37, pp. 28472–28480, September 10, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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elucidated a novel curcumin-dependent mechanism for lower-
ing A

levels via attenuation of APP maturation.
EXPERIMENTAL PROCEDURES
Cell Culture and Mouse Primary Cortical Neuron Culture—
Human neuroglioma H4 cells that stably overexpress human
APP751 (H4-APP751) cells have been described previously (22,
23), as has the rat neuroblastoma cell line (B104-APP751) stably
overexpressing human APP751, and the Chinese hamster ovary
cell line (CHO-APP751) stably overexpressing human APP751
(24, 25). These cell lines were cultured on regular tissue culture
plates in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum, 2 mML-glutamine, 100
units/ml penicillin, 100
g/ml streptomycin, and 200
g/ml
G418. Mouse primary cortical neurons were from BrainBits
(E18) and were cultured on poly-D-lysine-coated plates in
B27/neurobasal medium supplemented with 1⫻GlutaMAX
(Invitrogen).
Chemicals and Antibodies—Curcumin was purchased from
Sigma (catalogue no. C7727). The APP C-terminal antibody
(targeting the last 19 amino acids of APP; 1:1000) was pur-
chased from Sigma (catalogue no. A8717) and used to detect
the full-length APP and APP C-terminal fragment. The anti-
APP antibody (antibody 6E10) was purchased from Covance
and utilized for detection of sAPP
␣
(1:1000). The APLP2 anti-
body was a gift from Dr. W. Wasco (26), and the ADAM10
antibody was purchased from Sigma (catalogue no. A2726;
1:1000). The

-actin antibody (1:10,000) was purchased from
Sigma. The HRP-conjugated secondary antibodies (anti-mouse
and anti-rabbit; 1:10,000) were purchased from Pierce.
A

Measurement—A

measurement was following the man-
ufacturer’s suggested protocols and was described previously
(27). In brief, A

40 and A

42 levels (pg/ml) were quantified
using a sandwich enzyme-linked immunosorbent assay (ELISA)
from Wako (catalogue no. 292-62301/294-62501 to detect A

40;
and catalogue no. 298-62401/290-62601 to detect A

42). A

lev-
els were further normalized to protein concentration from the
same cell lysates. Normalized A

40 and A

42 levels of the cur-
cumin treatment were compared and normalized to those lev-
els of control treatment.
Cell Lysis and Protein Amount Quantification—Cells were lysed
in M-PER (mammalian protein extraction reagent, Thermo
Fisher Scientific) with 1⫻Halt protease inhibitor mixture
(Thermo Fisher Scientific). The lysates were collected, centri-
fuged at 10,000 rpm using a microcentrifuge from Eppendorf
(model 5417) for 20 min, pellets were discarded, and superna-
tants were transferred to a new Eppendorf tube. Total protein
was quantified using the BCA protein assay kit (Pierce) (28, 29).
Western Blotting Analysis—Western blotting analysis was
carried out by the method described previously (27, 28). Briefly,
after centrifugation and protein concentration measurement,
an equal amount of protein was applied to electrophoresis, fol-
lowed by membrane transfer, antibody incubation, and signal
development.

-Actin was used as an internal control. We used
the VersaDoc imaging system (Bio-Rad) to develop the blots
and the software Quantity One (Bio-Rad) to quantify the pro-
teins of interest, following the protocols described previously
(27, 28).
Cell Surface Biotinylation—Cell surface biotinylation was
carried out using a protocol reported previously(27). Briefly,
stable H4-APP751 and CHO-APP751 cells were preincubated
in cold Mg
2⫹
/Ca
2⫹
containing PBS for 20 min and then incu-
bated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 30 min
with gentle rocking at room temperature. Excess biotin was
quenched with 0.1 Mglycine for 20 min. Cells were then lysed in
M-PER lysis buffer and immunoprecipitated with streptavidin
beads (Pierce) overnight at 4 °C. Samples were boiled at 95 °C
for 5 min then applied to Western blotting analysis.
alamarBlue Analysis—The alamarBlue assay was a nonin-
vasive way to assess cell viability and proliferation rate and
has been reported previously (30). The alamarBlue agent was
purchased from Invitrogen, and the assay was performed
according to the manufacturer’s recommended protocol.
Briefly, the alamarBlue agent was added to culture medium at a
final concentration of 10% (v/v), medium was collected after 6 h
of incubation, and the fluorescence intensity was read on the
Criterion Analyst AD high throughput fluorescence detection
system (Molecular Devices) using a 550-nm excitation wave-
length and a 590-nm emission wavelength. The fluorescence
readouts from the treatments with curcumin of different con-
centrations were compared with the readout from the treat-
ment of control (0
Mcurcumin; dimethyl sulfoxide).
Data Analysis—

-Actin was used in the Western blotting
analysis to account for any differences in loading. The levels of
various proteins, e.g. full-length APP, mature and immature
APP, C99, and C83 were normalized to the

-actin values from
the same lane or sample. sAPP
␣
levels were normalized to the
cell lysates protein concentration and then compared with full-
length APP levels from each sample. All results were demon-
strated as means ⫾S.E. from at least three independent exper-
iments. We used the two-tailed Student’s ttest, as appropriate,
to reveal the differences between the experimental groups.
The Bonferroni correction analysis was used to correct for mul-
tiple comparisons within a single experiment. pvalues ⬍0.05
were considered statistically significant.
RESULTS
Curcumin Decreases A

Levels and Attenuates APP Mat-
uration in Mouse Primary Cortical Neurons—We first tested
whether curcumin affects A

levels and/or APP processing
and metabolism in mouse primary neuronal cells. Mouse
primary neurons (E18) were prepared on poly-D-lysine-coated
plates and treated with curcumin (0, 1, 2.5, 7.5, 10, and 20
M).
Cells were harvested after 24 h of treatment. Conditioned
medium was applied to ELISA analysis to measure the A

40
and A

42 levels, which were normalized to cell number. Cur-
cumin treatment decreased both A

40 and A

42 levels in a
dose-dependent manner (Fig. 1, Aand B). For example, 20
M
curcumin decreased A

40 and A

42 levels by 38.4% (p⬍0.01),
and 43.9% (p⬍0.05), respectively, compared with the control
treatment (0
M) (Fig. 1, Aand B).
Next, we studied the effects of curcumin on APP metabolism
and processing. Cell lysates from the previous experiment were
subjected to quantitative Western blotting analysis with anti-
body, APP8717, targeted at the C terminus of APP.

-Actin was
used to normalize loading variation between gel lanes. We
Curcumin Modulates APP Processing
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assessed the effects of curcumin treatment on levels of total
APP (APPma and APPim), mature and immature APP, as
well as the APPma:APPim ratio. Curcumin did not significantly
alter the levels of mature APP but increased levels of immature
APP and total APP in comparison to control. For example, 10
Mcurcumin treatment increased immature APP levels by
59.7% and increased total APP levels by 41.6% versus control
(p⬍0.05; Fig. 1, Cand D). In addition, curcumin decreased
the ratio of mature APP to immature APP compared with
control. For example, 10
Mcurcumin treatment decreased
the ratio by 23.3% (p⬍0.05) (Fig. 1, Cand D). Overall, these
data suggest that curcumin decreases A

levels by retarding
APP maturation.
Curcumin Significantly Alters APP Maturation and Process-
ing in Other Cell Types—Next, we asked whether curcumin
impairs APP maturation in other cell types. For this purpose,
we used stably transfected rat neuroblastoma B104-APP751
cells, which were treated with different concentrations of cur-
cumin for 24 h and then collected for Western blotting analysis
with the antibody APP8717. In addition, the media was probed
with antibody 6E10 to measure sAPP
␣
levels. Curcumin signif-
icantly increased levels of mature APP at doses of 5 and 10
M
(by 60.0%; p⬍0.05, compared with control) but revealed a
trend toward decreased levels at 15 and 20
Mcompared with
control (Fig. 2, Aand C). The level of immature APP was
increased significantly by curcumin treatment (e.g. increased by
279.1% at 20
M;p⬍0.01). Additionally, curcumin treatment
increased markedly the ratio of APPma/APPim at lower con-
centrations, but significantly decreased the ratio with increas-
ing concentrations (Fig. 2, Aand C). Specifically, the doses of 5
and 10
Mcurcumin decreased the ratio by 56.3% (p⬍0.01)
and 82.1% (p⬍0.01), respectively, compared with the control,
whereas 15 and 20
Mcurcumin markedly decreased the
APPma/APPim ratio by 55.8% (p⬍0.01) and 84.4% (p⬍0.01),
respectively, compared with the control. Finally, curcumin
treatment significantly increased total APP levels in compari-
son to control treatment (e.g. increased by 137.7% at 20
M;p⬍
0.01) (Fig. 2C). In contrast to what was observed in the primary
neurons, lower doses of curcumin, e.g. 5 and 10
M, increased
APP maturation. However, at higher doses, e.g. 15 and 20
M,
these data recapitulate the data from the primary neurons
showing that curcumin treatment retards APP maturation in
rat neuroblastoma B104-APP751 cells.
In this same experiment, we also assessed the effects of cur-
cumin on APP processing by measuring levels of the APP pro-
teolytic products, sAPP
␣
and C83, the products of
␣
-secretase
cleavage of APP. We observed a trend toward decreased levels
of C83 and sAPP
␣
(Fig. 2, Aand C–E) with curcumin treatment.
We also observed a significant decrease in the ratio of C83/total
APP as well as in the ratio of sAPP
␣
/total APP, compared with
the control treatment (Fig. 2, D–E), following curcumin treat-
ment. For example, 20
Mcurcumin decreased the ratio of C83/
APPtotal by 74.3% (p⬍0.01) (Fig. 2D) and decreased the ratio
of sAPP
␣
/total APP by 57.1% in comparison to control (p⬍
FIGURE 1. Curcumin significantly decreases A

levels and the ratio of APPma:APPim in mouse primary cortical neurons in a dose-dependent manner.
Aand B, curcumin significantly decreases both A

40 and A

42 levels. Mouse primary cortical neurons (E18) were treated with different concentrations of
curcumin and harvested after 24 h. Conditioned medium was used in ELISA analysis to detect the A

40 and A

42 levels, which were normalized to cell
numbers. Cand D, the curcumin treatment altered APP levels and decreased the ratio of APPma:APPim. In the Western blotting analysis, cell lysates were
probed with the APP8717 antibody to reveal APP.

-Actin was used as the loading control. C, a representative gel showing full-length APP and

-actin.
D, densitometry of C(n⫽3 for each treatment group). Mean ⫾S.E. *, p⬍0.05; **, p⬍0.01.
Curcumin Modulates APP Processing
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0.05) (Fig. 2E). Collectively, these data show that curcumin
treatment attenuates
␣
-secretase processing of APP in rat neu-
roblastoma B104-APP751 cells, consistent with attenuation of
APP maturation.
Next, we tested the effects of curcumin on APP metabolism
and processing in human H4 neuroglioma cells stably trans-
fected with APP751 (H4-APP751) and in Chinese hamster
ovary cells stably transfected with APP751 (CHO-APP751). As
observed with the rat neuroblastoma B104-APP751 cells, cur-
cumin treatment led to an increase (trend) and then a decrease
in the level of mature APP with increasing concentrations (p⬎
0.05) (Fig. 3A) in H4-APP751 cells. Curcumin treatment also
significantly increased levels of immature APP compared with
the control treatment (0
M) (Fig. 3B). 20
Mcurcumin mark-
edly increased immature APP levels by 79.1% (p⬍0.01; versus
control) (Fig. 3B). Additionally, curcumin significantly de-
creased the ratio of mature APP to immature APP with in-
creasing doses, compared with control. 20
Mcurcumin mark-
edly decreased the ratio of APPma:APPim by 42.2% (p⬍0.01;
compared with control). Curcumin treatment significantly
FIGURE 2. Curcumin significantly modulates APP processing in B104-APP751 cells in a dose-dependent manner. Stable rat neuroblastoma B104-APP751
cells were treated with different concentrations of curcumin for 24 h and were collected for Western blotting analysis. Cell lysates were probed with the
APP8717 antibody to reveal APP.

-Actin was used as the loading control. The cell medium was probed with 6E10 to reveal sAPP
␣
.Aand B, a representative gel
showing full-length APP, APP-C83, and sAPP
␣
.C, densitometry of A. Curcumin significantly increased the levels of immature APP and total APP. It also markedly
increased and then decreased the level of mature APP, as well as the ratio of APPma:APPim with increasing curcumin concentration. D, curcumin treatment
significantly decreased the ratio of C83/APPtotal. E, curcumin treatment significantly decreased the ratio of sAPP
␣
/APPtotal compared with control (n⫽3 for
each treatment group). Mean ⫾S.E.; *, p⬍0.05; **, p⬍0.01.
Curcumin Modulates APP Processing
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increased levels of total APP (APPma and APPim) with
increased concentration. 20
Mcurcumin markedly increased
total APP levels by 44.3% (p⬍0.01; compared with control)
(Fig. 3B). Curcumin treatment significantly increased levels of
mature APP and immature APP, as well as total APP compared
with the control in CHO-APP751 cells (Fig. 3, C–D). 20
M
curcumin elevated the level of immature APP by 65.0% (p⬍
0.05) and increased the levels of total APP by 59.2% (p⬍0.05).
Curcumin treatment also revealed a trend toward decrease in
the ratio of APPma:APPim compared with control (p⬎0.05)
(Fig. 3D). We also measured the A

levels from curcumin
treated samples in these three cell models. We found that cur-
cumin significantly decreased both A

40 and A

42 levels in all
cell models (supplemental Fig. 1, A–C). Collectively, these data
recapitulated the effects of curcumin treatment on APP metab-
olism and processing in several different cell types.
Curcumin Alters the Turnover of Mature and Immature
APP—In the next step, we assessed the effects of curcumin
on the rate of APP turnover using cycloheximide treatment.
H4-APP751 cells were treated with 20
Mcurcumin for 24 h
and then treated with 40
g/ml cycloheximide for various time
intervals (0, 0.5, 1.5, and 3 h). Cell lysates were collected and
utilized for quantitative Western blotting analysis. Curcumin
markedly decreased the half-life of mature APP from 2.03 to
1.14 h (changing by 43.4%), increased the half-life of imma-
ture APP from 1.86 to 2.36 h (changing by 26.0%), and short-
ened total APP half-life from 1.94 to 1.87 h (Fig. 4, A–D).
Curcumin also markedly decreased the ratio of APPma/APPim
(e.g. decreased by 67.6% at 0.5 h; p⬍0.05) (Fig. 4E). Thus,
curcumin increased the stability of immature APP while
decreasing the stability of mature APP, consistent with attenu-
ated APP maturation.
Curcumin Increases the Level of Plasma Membrane APP—
Next, we tested the effects of curcumin on plasma membrane
levels of APP. Stable H4-APP751 and CHO-APP751 cells were
treated with 20
Mcurcumin for 24 h and then subjected to
biotinylation analysis to assess the levels of cell surface APP.
Cell lysates were collected and utilized for Western blotting
analysis. Curcumin significantly increased the levels of cell sur-
face APP by 28.1% in H4-APP751 cells (p⬍0.05) (Fig. 5, Aand
B) and markedly increased the level of cell surface APP by
133.7% in CHO-APP751 cells (p⬍0.01) (Fig. 5, Cand D), in
comparison with control. We also studied whether curcumin
may alter plasma membrane levels of APLP2, an APP homo-
logue protein. We did not find any differences in the levels of
APLP2 (amyloid precursor-like protein 2) between cells treated
with curcumin versus control samples.
3
In combination with
prior observations of attenuated maturation of APP following
treatment with curcumin, these data suggest that curcumin
treatment may also lead to decreased endocytosis of APP, con-
sistent with decreased A

levels.
3
C. Zhang, A. Browne, D. Child, and R. E. Tanzi, unpublished data.
FIGURE 3. Curcumin treatment alters APP metabolism in H4-APP751 cells and CHO-APP751 cells in a dose-dependent manner. Various cell models were
treated with different concentrations of curcumin for 24 h and collected for Western blotting analysis. Cell lysates were probed with the APP8717 antibody to
reveal APP.

-Actin was used as the loading control. Aand B, the effects of curcumin treatment on H4-APP751 cells. Curcumin treatment had a trend to increase
the level of mature APP, significantly increased the levels of immature APP and total APP, and markedly decreased the ratio of APPma/APPim. Cand D, the
effects of curcumin on CHO-APP751 cells. Curcumin significantly increased the levels of mature APP, immature APP, and total APP and had a trend to decrease
the ratio of APPma:APPim with increasing concentration (n⫽3 for each treatment group). Mean ⫾S.E.; *, p⬍0.05; **, p⬍0.01.
Curcumin Modulates APP Processing
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Curcumin Decreases the Levels of Intermediate APP Induced
by Brefeldin A—Next, we investigated the mechanism by which
curcumin may increase immature APP levels. Previous reports
have shown that curcumin is a sarcoplasmic/endoplasmic
reticulum calcium-ATPase inhibitor (31). It potentially may
affect the functions of ER lumen chaperones, which are calcium
binding proteins (32). We hypothesized that curcumin may
affect APP metabolism at the level of the endoplasmic reticu-
lum. BFA is an agent that disassembles the Golgi complex and
redistributes proteins into the ER. BFA treatment has been
shown to induce the buildup of an intermediate APP isoform
(33). Thus, if curcumin decreased levels of intermediate APP
induced by BFA, it would strongly suggest that the curcumin
acts before the Golgi complex, likely at the ER.
First, H4-APP751 cells were treated with 5
g/ml BFA for 5
or 30 min and then harvested and subjected to Western blotting
analysis. As expected, 5
g/ml and 10
g/ml BFA for 30 min
induced the generation of intermediate APP (Fig. 6A). Then,
H4-APP751 cells were treated with 20
Mcurcumin ⫾5
g/ml
BFA for 0.5 or 3 h. BFA markedly induced the generation of the
intermediate APP at both 0.5 and 3 h, compared with control
(Fig. 6, B–C). BFA-induced generation of intermediate APP was
attenuated significantly in the presence of curcumin (at both
0.5 and 3 h; Fig. 6, Band C). These data suggest that curcumin
affects APP metabolism in the secretory pathway at the level of
the endoplasmic reticulum.
Curcumin Does Not Alter APLP2 Levels—To assess whether
the effects of curcumin are specific to APP, we next measured
FIGURE 4. Curcumin treatment alters the turnover rate of both mature and immature APP in H4-APP751 cells. H4-APP751 cells were treated with 20
M
curcumin for 24 h and then treated with 40
g/ml cycloheximide for different time (0, 0.5, 1.5, and 3 h). Cell lysates were collected and utilized for Western
blotting analysis. Cell lysates were probed with the APP8717 antibody to reveal APP.

-Actin was used as the loading control. A, a representative gel revealing
the cycloheximide treatment of different time points with or without curcumin treatment. Band C, quantitative Western blot analysis for mature (B) and
immature (C) APP levels. Immature APP levels in curcumin treatment were significantly higher at 0.5, 1.5, and 3 h, compared with the corresponding control
treatment. D, quantitative Western blot analysis for total APP levels. E, the ratio of APPma:APPim was decreased in curcumin treatment compared with control
at cycloheximide treatment of 0, 0.5, 1.5, and3h(n⫽3 for each treatment group). Mean ⫾S.E. *, p⬍0.05; **, p⬍0.01.
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the levels of APP homologue APLP2
following curcumin treatment. Sta-
ble B104-APP751 cells and H4-
APP751 cells were treated with dif-
ferent concentrations of curcumin
for 24 h, and cell lysates were col-
lected and utilized for Western blot-
ting analysis. APLP2 antibody has
been reported previously(26, 27).

-Actin was used as the loading
control. Curcumin treatment did
not alter the levels of full length
APLP2 in either B104-APP751 cells
(p⬎0.05) (Fig. 7, Aand B)or
H4-APP751 cells (p⬎0.05) and did
not alter levels of mature versus
immature forms of APLP2 (Fig. 7, C
and D). In addition, we found that
curcumin treatment does not alter
the levels of ADAM10 in mouse pri-
mary cortical neurons (supplemen-
tal Fig. 2). Thus, these data suggest
that the effects of curcumin on APP
levels and maturation are specific.
Curcumin Does Not Decrease Cell
Viability—Finally, we confirmed that
the treatment of curcumin did not
decrease cell viability, which could,
otherwise, alter A

levels via apo-
ptotic pathways (34). H4-APP751
FIGURE 6. Curcumin treatment affects APP metabolism at the endoplasmic reticulum. A, BFA disrupts APP
maturation process at the Golgi complex and induces the generation of the intermediate APP in H4-APP751
cells. H4-APP751 cells were treated with or without 5 and 10
g/ml BFA for 5 or 30 min. Cell lysates were
collected and prepared for Western blot analysis. Band C, curcumin treatment markedly decreased the level of
intermediate APP in the presence of BFA. H4-APP751 cells were treated with 5
g/ml BFA in the presence or
absence of 20
Mcurcumin for 5 or 30 min (n⫽3 for each treatment group). Cell lysates were collected and
prepared for Western blot analysis, as described under “Experimental Procedures.”
FIGURE 5. Curcumin treatment significantly increases cell surface APP levels in both H4-APP751 and CHO-APP751 cells. Cells were treated with 20
M
curcumin for 24 h and then subjected to biotinylation analysis to assess cell surface APP. Cell lysates were collected and utilized for Western blotting analysis. Aand B,
curcumin treatment markedly increased the level of cell surface APP in H4-APP751 cells compared with control treatment. Cand D, curcumin treatment significantly
increased the level of cell surface APP in CHO-APP751 cells compared with control (n⫽3 for each treatment group). Mean ⫾S.E. *, p⬍0.05; **, p⬍0.01.
Curcumin Modulates APP Processing
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cells and mouse primary cortical neurons were treated with
varying concentrations of curcumin for 24 h and were then
treated with the alamarBlue for 6 h. Curcumin did not decrease
cell viability in either cell type compared with control (0
M)
(Fig. 8, Aand B). In fact, 2.5
Mcurcumin significantly
increased cell viability by 29.3% in H4-APP751 cells compared
with control (0
M)(p⬍0.05; Fig. 8A).
DISCUSSION
We have described a possible cellular mechanism underlying in
vivo observations of decreased A

deposition and plaque burden
in AD transgenic mice following treatment of curcumin (14, 15, 18,
19). We have shown that curcumin decreases A

levels by retard-
ing APP maturation and possibly, impairing endocytosis from the
plasma membrane. Immature APP (or N-APP) is N-glycosylated
in the ER, and a fraction of these molecules exit the ER and
undergo O-glycosylation in the Golgi complex to become mature
APP (or N,O-APP) (35). Mature APP is then sorted onto the
plasma membrane after which it can undergo endocytosis via
clathrin-coated pits (36). Disruption of APP trafficking and sorting
has been proposed to underlie the pathogenesis of AD (33, 35, 37).
Our data show that curcumin significantly alters the ratio of
APPma/APPim and markedly decreases the level of intermediate
APP induced by BFA, an agent that disrupts the Golgi complex.
Additionally, curcumin has been shown to be an inhibitor of the
sarcoplasmic/endoplasmic reticulum calcium ATPase pump (31,
32). Collectively, these findings suggest that curcumin may affect
APP metabolism at the level of the ER. Though the precise under-
lying mechanism requires further elucidation, curcumin may
delay the exit of immature APP from the ER, thereby increasing
the stability of immature APP at the ER. Curcumin also may affect
the endocytosis of APP from the cell surface. The cumulative effect
would be a significant decrease in
both A

40 and A

42 levels.
Curcumin has been reported
to modulate trafficking and matu-
ration of other proteins, MLC1
(megalencephalic leukoencepha-
lopathy with subcortical cysts) (38)
and ⌬F508 cystic fibrosis trans-
membrane conductance regulator
(31). MLC1 encodes a plasma mem-
brane protein, MLC1, which is
responsible for a severe autosomal
recessive clinical disorder, MLC,
characterized by macrocephaly, de-
terioration in motor functions, cer-
ebellar ataxia, and mental decline.
Levels of MLC1 have been shown to
be significantly decreased in cells
expressing the mutant form of the
gene.
Curcumin has been shown to tar-
get dozens of proteins (39), and it
may act on different biological path-
ways with distinct mechanisms at
different dosages. It has been shown
FIGURE 7. Curcumin treatment does not alter APLP2 protein levels. B104-APP751 and H4-APP751 cells were
treated with different concentrations of curcumin for 24 h, and cell lysates were collected and utilized for
Western blot analysis. Cell lysates were probed with the APLP2 antibody to reveal APLP2.

-Actin was used as
the loading control. Aand B, the effects of curcumin treatment on APLP2 in B104-APP751 cells. Curcumin
treatment did not alter full-length APLP2 levels and did not alter levels of mature versus immature forms of
APLP2. Cand D, the effects of curcumin treatment on APLP2 in H4-APP751 cells. Curcumin treatment did not
alter full-length APLP2 levels (n⫽3 for each treatment group). Mean ⫾S.E. p⬎0.05.
FIGURE 8. Curcumin treatment does not decrease cell viability. Aand B, H4-APP751 cells (A) or mouse primary cortical neurons (B) were treated with different
concentrations of curcumin for 24 h and then subjected to alamarBlue analysis as described under “Experimental Procedures.” Curcumin treatment did not
decrease cell viability in either cell type, compared with control (0
M). The dose of 2.5
Mcurcumin significantly increased cell viability by 29.3% in H4-APP751
cells compared with control (A)(p⬍0.05) (n⫽3 in each treatment group). Mean ⫾S.E. *, p⬍0.05.
Curcumin Modulates APP Processing
SEPTEMBER 10, 2010•VOLUME 285• NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 28479
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that curcumin has a dose-dependent effect on A

levels and
inflammatory reactions in vivo (14, 15). Treatment with curcu-
min in vivo for 5–6 months potently attenuated proteins oxi-
dization and interleukin-1

generation in the brain (15, 16) and
reduced A

levels and plaque burden (14, 15). Here, we treated
mouse primary cortical neurons with different concentrations
of curcumin (1–20
M) for 24 h and found that both A

40 and
A

42 levels significantly decreased compared with control.
Collectively, our data suggest that the cellular mechanism by
which curcumin decreases A

levels is via the modulation of
APP levels in the secretory and, possibly, endocytic pathways.
Curcumin treatment significantly increased the retention of
immature APP in the ER and simultaneously attenuated APP
endocytosis from the plasma membrane. Collectively, our data,
together with the previously published in vivo data, suggest that
curcumin and its derivatives may prove useful in the search for
small molecule pharmacological agents for the effective treat-
ment and prevention of AD-related

-amyloid pathology.
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Curcumin Modulates APP Processing
28480 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 37• SEPTEMBER 10, 2010
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Can Zhang, Andrew Browne, Daniel Child and Rudolph E. Tanzi
Precursor ProteinβAmyloid-
Peptide Levels by Attenuating the Maturation ofβCurcumin Decreases Amyloid-
doi: 10.1074/jbc.M110.133520 originally published online July 9, 2010
2010, 285:28472-28480.J. Biol. Chem.
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