Results ATRA treatment upregulated mRNA and protein levels of a-secretases We first looked for induced mRNA levels of ADAM9 and ADAM10 upon administration of ATRA by real-time PCR in the
Trang 1Anna Koryakina, Jessica Aeberhard, Sabine Kiefer, Matthias Hamburger and Peter Ku¨enzi
Institute of Pharmaceutical Biology, University of Basel, Switzerland
The importance of vitamin A and its active metabolite
retinoic acid (RA) for cellular growth, differentiation,
and death, as well as for embryonic development and
vision, is well documented [1] Growing evidence
points towards an additional role of retinoids in the
mature brain, with effects on sleep [2], synaptic
plastic-ity, learning, and memory [3]
Several publications have described a crucial role of
retinoid signalling in neurodegenerative diseases,
par-ticularly in Alzheimer’s disease (AD) [4,5] Although
fibril-destabilizing [6] and neuroprotective [7] features
of retinoids against amyloid beta (Ab)-induced toxicity
have been demonstrated, the underlying mechanisms
remain unknown
According to the amyloid hypothesis [8], Ab accu-mulation is one of the most important steps in AD pathology, and results from impaired amyloid precur-sor protein (APP) processing Therefore, some emerg-ing therapeutic approaches involve modulation of APP cleavage via b-secretase inhibition, or a-secretase acti-vation, by, for example, activation of protein kinase C (PKC) [9] Links between retinoids and PKC were con-vincingly demonstrated [10], and even pointed to direct binding between PKCs and RA receptors and control
of transcriptional activity [11]
However, besides activation of a-secretases, little is known about their effects on b-secretase and c-secre-tase Moreover, tumour-promoting activity limits the
Keywords
Alzheimer’s disease; PDBu
(phorbol-12,13-dibutyrate); PKC (protein kinase C);
retinoic acid; secretases
Correspondence
P Ku¨enzi, Institute of Pharmaceutical
Biology, University of Basel,
Klingelbergstrasse, 50, 4056 Basel,
Switzerland
Fax: +41 61 267 14 74
Tel: +41 61 267 15 44
E-mail: peter.kueenzi@unibas.ch
(Received 12 January 2009, revised 19
February 2009, accepted 4 March 2009)
doi:10.1111/j.1742-4658.2009.06992.x
One of the emerging approaches for the treatment of Alzheimer’s disease aims at reducing toxic levels of Ab-species through the modulation of secretases, namely by inducing a-secretase or inhibiting b-secretase and⁄ or c-secretase activities, or a combination of both Although there is increas-ing evidence for the involvement of retinoids in Alzheimer’s disease, their significance in the regulation of Ab-peptide production remains unresolved Our work concentrated on the regulation of all secretases mediated by all-trans-retinoic acid (ATRA), and supports the hypothesis that ATRA is capable of regulating them in an antiamyloidogenic sense at the levels of transcription, translation, and activation Apart from increased a-secretase activity, we show a complex chain of regulatory events, resulting in impaired b-secretase trafficking and membrane localization upon protein kinase C (PKC) activation by ATRA Furthermore, ATRA demonstrates substrate specificity for b-site amyloid precursor protein-cleaving enzyme (BACE) 1 over nonamyloidogenic BACE2 in b-secretase regulation, which probably promotes competition for amyloid precursor protein between ADAM17 and BACE1 Additionally, we report enhanced secretion of solu-ble amyloid precursor protein a after ATRA exposure, possibly due to PKC activation, as pretreatment with the PKC inhibitor Go¨6976 abolished all these events
Abbreviations
Ab, antibody; AD, Alzheimer’s disease; ADAM, a disintegrin and metalloprotease; APP, amyloid precursor protein; ATRA, all-trans-retinoic acid; Ab, amyloid beta; BACE, b-site amyloid precursor protein-cleaving enzyme; CTF, C-terminal fragment; DAG, diacylglycerol;
ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; PDBu, phorbol-12,13-dibutyrate; PKC, protein kinase C; PS, presenilin;
RA, retinoic acid; sAPPa, soluble amyloid precursor protein derived by a-cleavage; SE, standard error.
Trang 2use of several potential PKC activators, such as
phor-bol esters Therefore, an assessment of the action of
RA(s) in AD might be valuable for AD patients, as
retinoids have a long history of clinical use [11,12]
To elucidate the regulation of secretases, we
exam-ined the effect of all-trans-retinoic acid (ATRA) on
a-secretase [a disintegrin and metalloprotease
(ADAM) 9, ADAM10, and ADAM17], on b-secretase
[b-site amyloid precursor protein cleaving-enzyme
(BACE) 1 and BACE2], and on the components of the
c-secretase complex [presenilin (PS) 1 and PS2]
Results
ATRA treatment upregulated mRNA and protein
levels of a-secretases
We first looked for induced mRNA levels of ADAM9
and ADAM10 upon administration of ATRA by
real-time PCR in the human neuroblastoma cell line IMR-32
[13] Treatment with 5 lm ATRA for 2, 4, 6, 24 and
48 h increased the quantities of both ADAM9 and
ADAM10 mRNAs, peaking at 174% and 205%,
respec-tively, of the corresponding control levels (Fig 1A)
Enhanced ADAM9 and ADAM10 transcription
cor-related with increased protein amounts upon treatment
with 5 lm ATRA or 1 lm phorbol-12,13-dibutyrate
(PDBu) ATRA treatment led to maximal protein
levels of 127% and 168% for ADAM9 and ADAM10,
respectively (Fig 1B), whereas the expression of
ADAM17 remained largely unchanged or within the
range of experimental error in response to ATRA
treatment (Fig 1A,B)
Additionally, the localization of ADAM9, ADAM10
and ADAM17 rapidly changed in response to ATRA,
as shown by confocal immunofluorescence analysis: all
a-secretases showed strong translocation to the cellular
membrane (ADAM9 and ADAM10) or to perinuclear
compartments (ADAM17) after treatment with 5 lm
ATRA for the time periods indicated (Fig 1C)
ATRA activated PKCa and PKCbII, leading to
increased APP cleavage
To check whether ATRA induces PKC signalling, we
treated IMR-32 cells with different amounts of ATRA
(0, 1, 2, 5 and 10 lm) for the time periods indicated (0,
5, 10 and 15 min), and observed clear phosphorylation
of PKC with 5 lm ATRA using a pan-phospho-PKC
antibody (Fig 2A) To determine whether 5 lm ATRA
induced activation of classic PKCs for the indicated
time periods, we examined the expression, location and
phosphorylation of PKCa and PKCbII Both PKCa
and PKCbII showed increased phosphorylation (Fig 2C), and translocated to the cell membrane after
10 min of ATRA exposure (Fig 2B)
To study the effect of PKC stimulation on APP cleavage, IMR-32 cells were treated with 5 lm ATRA (6 h) or 1 lm PDBu (1 h) Proteins from the collected media were then analysed for soluble APPa (sAPPa)
by western blot, and elevated release was found after ATRA exposure, indicating increased a-secretase activ-ity (Fig 2D) The appearance of the soluble APP frag-ment derived by a-cleavage (sAPPa) was partly abolished by the addition of 1 lm Go¨6976, a known inhibitor of PKCs [14] Similarly, application of 1 lm PDBu for 30 min or 1 h resulted in appearance of the sAPPa fragment, an effect that was completely abol-ished by pretreatment with Go¨6976
As this effect might point towards the possibility that ATRA is capable of reducing Ab levels, we tried
to detect changes in intracellular and extracellular lev-els of total Ab and the amyloidogenic fragments Ab40 and Ab42, utilizing several approaches, such as ELISA
or immunoprecipitation Whereas extracellular Ab could not be detected at all, changes in intracellular levels remained negligible (not shown)
ATRA-induced signalling affects BACE1 but not BACE2
To study the effect of ATRA on BACE1 transcription,
we treated IMR-32 cells for 2, 4, 6, 24 and 48 h with
5 lm ATRA and performed real-time PCR BACE1 mRNA levels increased to a maximum of 168% of the control level after 4 h of ATRA treatment, and these levels persisted for up to 24 h (Fig 3A) This was con-sistent with increased protein amounts, which reached 143% of control levels after 24 h (Fig 3C), as shown
by fluorescence activated cell sorting (FACS) analysis Exposure to PDBu, however, only insignificantly increased the BACE1 protein quantity, to 106% (Fig 3C)
At the same time, neither ATRA nor PDBu led to any significant changes in the protein levels of BACE2, a homologue of BACE1, and the differences perceived remained within the range of experimental error (Fig 3C) Moreover, localization of BACE2 remained unaffected upon ATRA treatment (Fig 3B), whereas BACE1, which initially localized in the cytoplasm and cell membrane in untreated controls, showed a massively changed distribution 3 h post-treatment (Fig 3D)
As BACE1 is synthesized in the endoplasmic reticu-lum (ER), and increased BACE1 mRNA levels were observed in our experiments, we examined its distribu-tion by confocal immunofluorescence microscopy,
Trang 3looking particularly for BACE1 localization in the ER.
BACE1 was initially localized in both membrane and
ER, and colocalized to some extent with calnexin, a
widely used ER marker protein ATRA exposure for
3 h resulted in increased BACE1 colocalization with calnexin (Fig 3D)
IMR-32
6
24
ADAM10 ADAM9
ADAM10 ADAM9
*
*
*
**
**
ADAM17
* *
ADAM17
A
B
C
5 µ M ATRA (h)
* P < 0.05
** P < 0.01
Fig 1 ATRA activated a-secretases in
IMR-32 cells (A) mRNA levels of ADAM9 and
ADAM10 increased in response to ATRA
treatment, as shown by real-time PCR,
whereas changes in ADAM17 levels
remained within the standard error (SE) (B)
As assessed by FACS analysis, ADAM9 and
ADAM10 protein levels were increased by
both 5 l M ATRA or 1 l M PDBu, whereas
that of ADAM17 remained largely
unchanged Error bars: mean ± SE (C)
Rep-resentative confocal fluorescence images of
ADAM9 (first row), ADAM10 (second row)
and ADAM17 (third row) translocation in
response to 5 l M ATRA addition are shown.
ADAM9 and ADAM10 demonstrated
time-dependent translocation to the cellular
membrane, whereas ADAM17 translocation
to the cytoplasm was seen after 3 h of
treatment, and continually increased over
the 24 h of testing.
Trang 4However, further incubation of IMR-32 cells with
5 lm ATRA (6 h and longer) provoked translocation of
BACE1 to the plasma membrane (Fig 3D) Analogous
treatment with 1 lm PDBu also resulted in BACE1
trans-location to the membrane; this effect was completely
abolished by the PKC inhibitor Go¨6976 (Fig 3E)
ATRA affected the activity, transcription and
localization of PS1
As APP cleavage is sequential and modulated by
ATRA at the a⁄ b-secretase level, we also investigated
possible modulation of c-secretase-dependent cleavage
We focused on PS1 and PS2, which are homologous transmembrane proteins forming the functional core of the c-secretase [15]
RT-PCR experiments revealed slightly increased PS1 mRNA levels after treatment with 5 lm ATRA for 24 h (Fig 4A) This was accompanied by the appearance of the full-length PS1 and its active C-terminal fragment (CTF) in protein samples from cells treated with ATRA or PDBu (Fig 4B) This effect was efficiently blocked by addition of 1 lm Go¨6976 PS2 levels remained
C
D
Fig 2 ATRA treatment activated PKCs and caused increased secretion of sAPPa in IMR-32 cells (A) Phosphorylation of PKC was induced by application of various con-centrations of ATRA, and reached a maxi-mum upon treatment with 5 l M ATRA (B) Exposure to 5 l M ATRA for 10 min induced translocation of PKCa and PKCbII to the cellular membrane, as shown by confocal microscopy (C) PKCa and PKCbII were immunoprecipitated using specific antibod-ies, separated by SDS ⁄ PAGE, and subse-quently probed with PKCa, PKCbII and phospho-PKC antibodies The protein levels
of PKCa and PKCbII remained similar, but their phosphorylation levels increased upon ATRA treatment Densitometric analysis is shown for phosphorylation of PKCa and PKCbII, based on basal expression of PKCa and PKCbII, respectively, from three inde-pendent experiments (D) Treatment with
5 l M ATRA for 3, 6 and 24 h, or 1 l M PDBu for 0.5 and 1 h, induced sAPPa secretion into the cell culture media This effect was partly abolished by addition of the classic PKC inhibitor Go¨6976, as shown by immu-noblot with concentrated media Represen-tative results obtained in at least three experiments based on cell counts are shown, as well as densitometric analysis of three independent experiments.
Trang 5unchanged upon ATRA treatment, and the active
PS2 form, a CTF of 23 kDa, remained undetectable
(not shown)
Both PSs partly colocalized with calnexin in
the ER and nucleus in control cells, as assessed
by DNA counterstaining with DRAQ5 in
confocal immunofluorescence microscopy Whereas
PS1 displayed a weak increase in nuclear
distribution (Fig 4C), PS2 localization remained
unchanged after 6 and 24 h of ATRA treatment (not
shown)
ATRA-dependent regulation of secretases
in other cell lines Additional experiments, performed in the murine neuroblastoma cell line N2a and the human embryonic kidney cell line HEK293, basically confirmed the results obtained with IMR-32 cells
We observed similar increases in PKCa and PKCbII phosphorylation (Fig 5A,B), as well as translocation
to the cellular membrane, upon ATRA treatment (Fig S1)
–
3
Pearson’s coefficient
Overlap coefficient
6
1
IMR-32
1
+ 1µM Gö6976 pre-treatment
E
–
B
BACE 2
C
BACE 2 BACE 1
A
BACE 1
*
Fig 3 ATRA-induced regulation of BACE1 was partly dependent on PKC activation (A) BACE1 mRNA levels increased in IMR-32 cells in response to ATRA treatment for the times indicated, as shown by real-time PCR analysis Results from four independent experiments are given Error bars: mean ± SE (C) FACS analysis showed increased BACE1 protein levels in IMR-32 cells after 24 h of ATRA treatment, whereas those of BACE2 remained within the range of experimental error Results from at least three independent experiments are given Error bars: mean ± SE (B) BACE2 was localized in the outer membrane, and this remained unchanged upon exposure to 5 l M ATRA Repre-sentative images are shown (D, E) Localization of BACE1 in IMR-32 cells in response to 5 l M ATRA (D) or 1 l M PDBu (E) was assessed by confocal microscopy Colocalization analysis between BACE1 and calnexin was performed using IMAGEJ software Colocalized areas are shown in white, and Pearson’s and overlap coefficients are provided for each merged image (D) ATRA treatment for 3 h affected the BACE1 cytoplasmic distribution and increased BACE1 colocalization with the ER marker calnexin in IMR-32 cells Prolonged ATRA exposure (6 h and longer) resulted in BACE1 translocation towards the cellular membrane The images shown are based on visibility and not protein amount (E) PDBu treatment (1 l M ) led to BACE1 translocation, similar to that induced by ATRA (first row), but this was abolished by cotreatment with 1 l M Go¨6976 (second row).
Trang 6Levels of secreted sAPPa increased in response to
5 lm ATRA (Fig S2E) and 1 lm PDBu (not shown)
treatments Cotreatment with the PKC inhibitor
Go¨6976 partially diminished sAPPa secretion into cell
media to control levels (Fig S2E) However, changes
in intracellular and extracellular levels of total Ab,
Ab40 and Ab42 remained irrelevant or below the limit
of detection
Changes in mRNA and protein levels of ADAM9
and ADAM10 in N2a and HEK293 cells in response
to ATRA treatment matched the observations seen in
IMR-32 cells ADAM9 and ADAM10 displayed
increased mRNA and protein quantities in N2a and in
HEK293 cells (Fig S2) Additionally, translocation to
the cellular membrane (ADAM9 and ADAM10) or to
perinuclear compartments (ADAM17) upon treatment
with 5 lm ATRA were observed (Fig S3)
Enhanced transcription of PS1 corresponded
with increased protein expression in all cell lines
(Fig S4A,B) This was accompanied either by stable
expression of full-length protein in N2a cells or by
enhanced cleavage of PS1 in HEK293 cells (Fig 5C)
Addition of 1 lm Go¨6976 abolished this effect
(Fig S4B) All cell lines displayed a weak increase in
PS1 nuclear distribution (Fig S4C,D)
Increased mRNA levels (Figs S5 and S6A) and
protein levels of BACE1 (Figs S5 and S6C) were
accompanied by its impaired trafficking and late
translocation to the cellular membrane due to ATRA
(Figs S5 and S6D) and PDBu treatment (Figs S5 and
S6E) in all cell lines ATRA distinguished equally
between BACE1 and BACE2, and influenced nei-ther the expression (Figs S5 and S6C) nor localization (Figs S5 and S6B) of BACE2 in any of the cell lines tested
Discussion One strategy in AD treatment is aimed at protecting neurons from the production of toxic Ab species [16] Reduction of Ab40⁄ 42 levels is mainly achieved by modulation of secretases, namely by the induction of a-secretase activity, by inhibition of b-secretases and⁄ or c-secretases, or by a combination of both This study provided evidence that ATRA regulates all secretases at the levels of transcription, expression, and activation
PKC activators upregulate a-secretases, eventually promoting the antiamyloidogenic pathway [17] Pub-lished data on positive and⁄ or negative PKC modula-tion by ATRA are controversial, which may be explained by a biphasic effect of ATRA on PKC activity [18] We observed increased phosphorylation
of PKCa and PKCbII in response to 5 lm ATRA treatment in all examined cell lines
It is generally accepted that classic and novel PKCs become activated by diacylglycerol (DAG), triggering localization to the cellular membrane Endogenous DAG levels differ in various cell lines, and determine the PKC activation profile The classic model for PKC activation involves its phosphorylation and transloca-tion from the cytosol to the binding domain on
5 µM ATRA (h) – 6 24
PS1/FL
PS1/CTF
5 µM ATRA (h) – 24
GADPH
PS1
50 kDa
25 kDa
1 µM PDBu (h) – 1 1
5 µM ATRA (h) – 6 6
Presenilin 1
–
+
DRAQ5™ Merged
0.171
0.227
A
B
coefficient
Fig 4 Modulation of PS1 upon activation of PKC (A) Increased mRNA levels of PS1 were observed upon PKC activation by ATRA treat-ment, as shown by RT-PCR analysis (B) In IMR-32 cells, immunoblot analysis revealed increased levels of PS1 after exposure to 5 l M ATRA
or 1 l M PDBu Inhibition of PKC by cotreatment with 1 l M Go¨6976 blocked this increase in IMR-32 cells (C) Representative confocal fluo-rescence images of PS1 in IMR-32 cells showed slightly increased colocalization with the DNA counterstain DRAQ5 Colocalization analysis was performed using IMAGEJ software Colocalized areas are highlighted in white, and Pearson’s and overlap coefficients are provided for each merged image GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Trang 7cellular membranes, a translocation that we observed
in all cell lines This event correlated with positive
modulation of a-secretases
In contrast to other findings, we observed increased
mRNA and protein levels of ADAM9 Notably, recent
findings suggest that ADAM9 acts as an important regulator upstream of ADAM10 by shedding and releasing its catalytically active ectodomain [19] These findings are consistent with transcriptional and transla-tional upregulation of ADAM10, and the enhanced APP cleavage seen in our experiments The observed translocation of ADAM9 and ADAM10 to the cyto-plasmic membrane further supports the idea of APP cleavage at the membrane by these secretases
Our experiments showed a strong correlation between PKC activation, translocation of ADAM17 into the perinuclear space and sAPPa secretion into the extracellular space As PKCa and PKCd – both classic PKC isoforms – are located in the ER [20], we believe that other APP cleavage sites, at the Golgi and
in the ER, are also impaired as a consequence of PKC activation b-Secretase cleaves APP within endosomes during APP recycling from the plasma membrane, as well as in the Golgi and ER [21,22] As release of active ADAM17 ultimately occurs in the same com-partments, namely the Golgi and ER [23], we deduce that ADAM17 is the main BACE1 competitor for intracellular APP cleavage
We further explored the effects of ATRA on BACE1
at the level of transcription, translation, and activity, and found ATRA-dependent upregulation of its mRNA and⁄ or protein levels Poor correlation between increased BACE1 transcription and b-secretase activity has been previously reported, leading to the ideas of control at the level of translation [24] or its localization
by phosphorylation [25] Interestingly, BACE1 increas-ingly colocalized with the ER marker calnexin upon ATRA treatment in our experiments As pro-BACE1 is predominantly located within the ER [26], this suggests that addition of ATRA leads to BACE1 accumulation within the ER by obstructing its maturation
After long-term treatment, BACE1 was mainly detected at the cellular membrane This localization might further impair BACE1-derived APP cleavage, which typically occurs intracellularly, owing to its requirement for an acidic pH Moreover, we believe that membranous BACE1 mainly consists of the fully matured form, as transportation of BACE1 is initiated
by phosphorylation on its cytoplasmic tail, which occurs exclusively after full maturation only [25] BACE2, a structural homologue of BACE1, was not affected by ATRA, despite its sequence homology BACE2 processes APP within the Ab domain between Phe19 and Phe20, close to the a-secretase site [27], and has distinct transcriptional regulation and function [28] BACE2 localization at the cellular membrane remained unchanged in any of the cell lines tested, which is possibly of interest for the antiamyloidogenic
5 10
5 µ M ATRA (min)
IP PKC βII
p-PKC
PKCβII
HEK 293
IP PKC α
p-PKC
PKCα
IP PKC α
p-PKC
PKCα
5 µ M ATRA (min)
IP PKC βII
p-PKC
PKC βII
N2a
HEK 293
PS1
CTF
PS1
CTF
N2a
A
B
C
Fig 5 Cell line-specific differences in response to PKC activation
in N2a and HEK293 cells (A, B) Immunoprecipitation followed by
immunoblot analysis showed similar PKCa and PKCbII protein
lev-els upon ATRA treatment, and both proteins showed increased
phosphorylation in N2a (A) and HEK293 (B) cells (C) ATRA
treat-ment slightly increased the expression of both full-length PS1 and
its active CTF domain in N2a cells (first line), but enhanced the
cleavage of full-length PS1 to its active CTF form in HEK293 cells
(second line), as shown by immunoblot.
Trang 8processing of APP, as constitutive a-cleavage occurs at
the membrane [22]
To investigate whether PKC activation affects
further steps associated with c-secretase cleavage,
we studied the effects of ATRA and PDBu on PS1
and PS2 participation in the formation of the
macromolecular c-secretase complex [15] We could
not identify any changes in the level of expression of
PS2, which was mainly detected as a full-length protein
of 52 kDa at any of the time points tested Moreover,
wild-type PS2 was weakly expressed in all cell lines
examined, and ATRA had no effect whatsoever
PS1, on the other hand, showed delicate
ATRA-dependent modification, and displayed slightly
enhanced nuclear localization, with the most
pro-nounced effect being observed after 24 h PS1 could be
detected both as full-length protein and as active
endo-proteolytic CTF, and expression of both forms
increased after exposure to ATRA at 6 and 24 h
Interestingly, PDBu treatment had only minor effects
on full-length protein levels, but led to the appearance
of substantial amounts of the endoproteolytic
frag-ment This effect was abolished by cotreatment with
the PKC inhibitor Go¨6976 Intriguingly, Walter et al
[29] reported processed PS1 CTF as an in vivo
sub-strate for PKC, which indicates that the physiological
and⁄ or pathological properties of the active PS1 form
might be regulated by activated PKC
Overall, the human cell lines (IMR-32 and HEK293)
displayed faster and stronger responses to PKC
stimu-lation, and showed more stable phosphorystimu-lation, than
N2a, a cell line of murine origin This might depend
on variations in endogenous DAG levels, determining
the PKC activation profile We observed no marked
differences in either the incubation time required for
PKC stimulation and secretase activation, in the
tran-scription⁄ translation ratio, or in translocation of
secre-tases between tested cell lines
In conclusion, ATRA treatment specifically shifts
secretase-dependent APP cleavage towards the
antiam-yloidogenic, owing to activation of PKCa and
PKCbII Both subsequently affect various steps and
players involved in APP processing However,
ATRA-induced alterations appear to be modest in nature, and
further research is therefore needed to assess their
physiological significance
Experimental procedures
Cell culture and treatment
The human neuroblastoma IMR-32 cell line was
main-tained in DMEM⁄ F12 (1 : 1) (Invitrogen, Basel,
Switzer-land), and the murine neuroblastoma N2a and human embryonic kidney HEK293 cell lines were maintained in DMEM (Sigma-Aldrich, Buchs, Switzerland) Media were supplemented with 10% heat-inactivated fetal bovine serum (Amimed, Basel, Switzerland), 100 UÆmL)1penicillin⁄ strep-tomycin (Invitrogen), and 2 mm l-glutamine (Invitrogen) All cell types were grown in a humified atmosphere con-taining 5% CO2
ATRA, Go¨6976 and PDBu were dissolved in dimethyl-sulfoxide and directly added to the medium for the times indicated Go¨6976 was added 30 min prior to ATRA or PDBu treatment, unless indicated otherwise During pro-longed treatment, medium was exchanged every 2 days
Preparation of protein extracts and media samples
Cells were collected, washed with ice-cold NaCl⁄ Pi(pH 7.4), and lysed in a hypotonic buffer (10 mm Hepes, pH 7.9,
60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.5% NP-40) containing protease inhibitors [1 mm phenylmethanesulfonyl fluoride, 1· Complete Protease Inhibitors (Roche Diagnos-tics, Rotkreuz, Switzerland)] Cytoplasmic extracts were collected, and cleared by centrifugation at 16 100 g for
30 min Protein concentrations of extracts were measured using Coomassie Protein Assay Reagent (Sigma-Aldrich) Media were collected, snap frozen and stored at)80 C Before use, the media were thawed overnight at 4C, and then applied to Ultrafree MC filters (cut-off 30 kDa) (Milli-pore Corporation, Bedford, MA, USA) The samples were concentrated to 200 lL by centrifugation at 2300 g for
30 min, and the protein concentrations were measured as described above
Western blotting
Cell lysates were separated by SDS⁄ PAGE and blotted onto nitrocellulose membranes using standard procedures Membranes were blocked and incubated overnight at 4C with specific primary antibodies (Abs), diluted in blocking buffer: PKCa, 1 : 1000; PKCbII, 1 : 1000; anti-actin, 1 : 4000 (all Santa Cruz, CA, USA); anti-phospho-PKC, 1 : 1000; anti-PS1, 1 : 500; anti-PS2, 1 : 1000 (all Cell Signaling Technology, Beverly, MA, USA); and 6E10,
1 : 1000 (Signet Laboratories, Dedham, MA, USA) Specific bands were tagged using horseradish peroxidase-conjugated secondary Abs, and detected with the ECL Plus System (Amersham Pharmacia Biotech, Little Chalfont, UK)
Immunoprecipitation
Immunoprecipitation was performed according to standard procedures Briefly, cells were grown in 75 cm2 flasks to
Trang 980% confluency, starved overnight, and subsequently
trea-ted with 5 lm ATRA for the times indicatrea-ted Cells were
harvested, washed with ice-cold NaCl⁄ Pi, extracted in
100 lL of lysis buffer (20 mm Tris⁄ HCl, pH 7.4, 25 mm
MgCl2, 0.05% NP-40, 1 mm dithiothreitol, 1· protease
inhibitors), and cleared by centrifugation at 16 100 g for
2 min
Three hundred micrograms of total protein was
incu-bated with 1 lg of PKCa or PKCbII (both from Santa
Cruz) antibody in 500 lL of lysis buffer for 90 min at 4C
Protein complexes were precipitated by adding 40 lL of a
50% slurry of protein G Sepharose beads (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) for 90 min at 4C,
washed four times with wash buffer (20 mm Tris⁄ HCl,
pH 7.4, 25 mm MgCl2, 0.05% NP-40, 1 mm dithiothreitol,
120 mm NaCl), and dissolved by boiling with 30 lL of 1.5·
Laemmli buffer for 3 min at 95C Samples were resolved
by SDS⁄ PAGE and transferred to nitrocellulose
mem-branes Filters were blocked, and analysed using antibodies
against phospho-PKC, anti-PKCa and anti-PKCbII (all
from Cell Signaling Technology)
RNA extraction, real-time PCR, and sequencing
Total RNA was extracted using TRIZOL (Invitrogen),
according to the manufacturer’s instructions, and
tran-scribed to cDNA by a reverse transcriptase reaction using
Moloney murine leukemia virus reverse transcriptase
(Invitrogen)
Real-time PCR using SYBR Green PCR Master Mix
(Applied Biosystems, Foster City, CA, USA) was
per-formed for ADAM9, ADAM10 and BACE1, using the
ABI PRISM 7700 System (Applied Biosystems) b-Actin
was used as an endogenous reference to normalize the
quantification of target mRNAs Reactions were performed
in triplicate, and threshold cycle (Ct) values were
normal-ized automatically by the software Following reverse
transcription, the cDNAs for b-actin, ADAM9, ADAM10
and BACE1 were amplified under these conditions: one
cycle of 52C for 2 min, one cycle of 95 C for 10 min, 40
cycles at 95C for 15 s and 60 C for 1 min, and melting
curve analysis at 60–95C
The following primers were used: human b-actin forward,
murine b-actin reverse, 5¢-CTCTCAGCTGTGGTGGTG
AA-3¢; human ADAM9 forward, 5¢-GAATGAATCACG
ATGATGGGAG-3¢; human ADAM9 reverse, 5¢-CCAGC
GTCCACCAACTTATTAC-3¢; murine ADAM9 forward,
5¢-CTTAACATCCCGAAGCCTGAC-3¢; murine ADAM9
A-3¢; human ADAM10 reverse, 5¢-TCCTGGTGTGCAC
TCTGTTC-3¢; murine ADAM10 forward, 5¢-AGCAACAT
CTGGGGACAAAC-3¢; murine ADAM10 reverse, 5¢-TTG CACTGGTCACTGTAGCC-3¢; human ADAM17 forward,
reverse, 5¢-CCAGGACAGACCCAA-3¢; human BACE1
murine BACE1 forward, 5¢-CACCATCCTTCCTCAGCAA TAC-3¢; murine BACE1 reverse, 5¢-GTAACAAACGGACC TTCCACTG-3¢; human PS1 forward, 5¢-GTTACCTGCA CCGTTGTCCT-3¢; human PS1 reverse, 5¢-CTCATCTTGC TCCACCACCT-3¢; murine PS1 forward, 5¢-CTCGCCAT TTTCAAGAAAGC-3¢; murine PS1 reverse, 5¢-CAGT GCGGGTAAATCTCCAT-3¢
Nested PCR amplifications were carried out in individual
50 lL reactions in a Perkin Elmer Thermocycler Gene-Amp 9700 (Applied Biosystems) All amplicons were checked by sequencing (performed by Microsynth, Balgach, Switzerland)
Immunofluorescence microscopy and data analysis
IMR-32, N2a and HEK 293 cells were fixed in 4% formal-dehyde in NaCl⁄ Pifor a minimum of 15 min at 4C, per-meabilized using 0.2% Triton-X (prepared in NaCl⁄ Pi
containing 10% heat-inactivated fetal bovine serum), and then incubated with primary Abs The Abs were diluted in NaCl⁄ Pi containing 10% heat-inactivated fetal bovine serum as follows: anti-PKCa, 1 : 50; anti-PKCbII, 1 : 50 (both Santa Cruz, CA, USA); anti-PS1, 1 : 100; anti-PS2,
1 : 100 (both Cell Signaling Technology); anti-ADAM9,
1 : 50; anti-BACE2, 1 : 100 (both AbD Serotec, Du¨sseldorf, Germany); anti-ADAM10, 1 : 50; anti-ADAM17, 1 : 50 (both Chemicon Europe Ltd, Chandlers Ford, UK); anti-BACE1, 1 : 100 (Merck Chemicals Ltd, Beeston, UK; cat
no 195111); and anti-calnexin, 1 : 100 (BD Biosciences, Basel, Switzerland) The immunogen in antibodies against BACE1 is a synthetic peptide (CLRQQHDDFADDISLLK) corresponding to amino acids 485–501 at the C-terminus of BACE1
Cells were then washed three times with NaCl⁄ Pi and incubated for 1 h with affinity-purified Alexa-Fluor 488 goat [rabbit IgG (H + L)], Alexa-Fluor 488 goat anti-[mouse IgG (H + L)] or anti-rabbit Texas Red (all Invitro-gen, Molecular Probes, Basel, Switzerland; diluted 1 : 1500
in NaCl⁄ Pi) Nuclei were stained with DRAQ5 (Alexis; diluted 1 : 3000 in NaCl⁄ Pi), and visualized with a Leica TCS SP scanning confocal microscope Identical exposure times were used across conditions Series of optical sections were taken at 1 lm intervals in line average mode with a picture size of 512· 512 pixels, using Leica confocal soft-ware, version 2.5 (Leica Microsystems, Heidelberg GmbH), and analysed with imagej 1.37t software (http://rsb.info nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA)
Trang 10For colocalization analysis, pictures were converted to
eight-bit grey scale images at a 0 < 255 fluorescence
inten-sity range, and the threshold for each channel was
deter-mined by colocalization threshold plug-in These
automatically determined threshold values were used in the
next step of colocalization analysis, performed with jacop
plug-in[21], and Pearson’s correlation and overlap
coeffi-cients are shown (for details, see http://rsbweb.nih.gov/ij/
plugins/track/jacop.html) Merged images with white areas
displaying the colocalization between BACE1 and calnexin
or PS1 and DRAQ5 (DNA counterstaining) were generated
using imagej colocalization finder plug-in
FACS
IMR-32, N2a and HEK293 cells were fixed in 2%
parafor-maldehyde in NaCl⁄ Pi for 10 min at 37C, permeabilized
using 90% ice-cold methanol, and then incubated with
pri-mary Abs overnight at 4C The Abs were diluted in
NaCl⁄ Pi containing 1% BSA as follows: anti-ADAM9,
1 : 50; BACE2, 1 : 100 (both AbD Serotec);
anti-ADAM10, 1 : 100; anti-ADAM17, 1 : 100 (both from
Chemicon Europe Ltd, Chandlers Ford, UK); anti-BACE1,
1 : 100 (Merck Chemicals Ltd, Beeston, UK); anti-Ab40,
1 : 50; anti-Ab42, 1 : 50 (both The Genetics Company Inc.,
Schlieren, Switzerland); and 6E10 Abs, 1 : 100 (Signet
Laboratories)
Cells were washed twice with 1% BSA⁄ NaCl ⁄ Piand
incu-bated for 30 min with affinity-purified Alexa-Fluor 488 goat
[rabbit IgG (H + L)] or Alexa-Fluor 488 goat
anti-[mouse IgG (H + L)], diluted 1 : 1500 in 1% BSA⁄
NaCl⁄ Pi) (Invitrogen-Molecular Probes), and analysed on
a Dako CyAn ADP LX 7 using summit 4.3 software
(DakoCytomation, Fort Collins, CO, USA)
Statistical analyses
Real-time PCR data were quantified by applying the DDCt
model, according to the equation ratio = (Etarget)DCt (target)⁄
amplifica-tion efficiency of a particular pair of primers The
amplification efficiency of each primer pair was determined
experimentally, as previously described [30] Additionally,
the Ct values were normalized within the logarithmic
phase with the highest PCR amplification efficiency by
abi prism7000 software For statistical analysis by
unpaired t-test, we assumed that both treatment and
con-trol groups have a Gaussian distribution of DCt values, as
well as equal variances
FACS data were quantified as described in manual for
summitV4.3 software The original method was published
by Overton [31] Briefly, FACS data were plotted on the
side scatter versus forward scatter histogram, and apoptotic
cells and cell debris were gated out For doublet
discrimina-tion, the main cell population was gated on the Lin pulse width histogram Data quantification was performed using the ‘subtraction histogram’ analysis tool in summit V4.3 Subtraction methods give a fluorescence difference between control and treated sample for a particular parameter (fluorescein isothiocyanate log) The Overton option was used for calculating this difference; this repre-sents a ‘true’ percentage of positively labelled cells Differ-ences between controls and treated samples were considered
to be significant with a P-value < 0.05 in Student’s t-test
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