Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

Article (PDF Available)inEMBO Reports 7(7):739-45 · August 2006with38 Reads
DOI: 10.1038/sj.embor.7400704 · Source: PubMed
Abstract
gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.
Regulated intramembrane proteolysis of amyloid
precursor protein and regulation of expression
of putative target genes
Se
´
bastien S. He
´
bert
1
, Lutgarde Serneels
1
,AlexandraTolia
1
, Katleen Craessaerts
1
,CarmenDerks
1
,
Mikhail A. Filippov
2,3
,UlrikeMu
¨
ller
2,3
& Bart De Strooper
1+
1
Neuronal Cell Biology and Gene Transfer, CME, Flanders Interuniversity Institute for Biotechnology (VIB4) and Katholieke
Universiteit Leuven, Leuven, Belgium,
2
Max-Planck Institute for Brain Research, Frankfurt, Germany, and
3
Institute for Pharmacy
and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
c-Secretase-dependent regulated intramembrane proteolysis of
amyloid precursor protein (APP) releases the APP intracellular
domain (AICD). The question of whether this domain, like the
Notch intracellular domain, is involved in nuclear signalling is
highly controversial. Although some reports suggest that AICD
regulates the expression of KAI1, glycogen synthase kinase-3b,
Neprilysin and APP, we found no consistent effects of c-secretase
inhibitors or of genetic deficiencies in the c-secretase complex
or the APP family on the expression levels of these genes in cells
and tissues. Finally, we demonstrate that Fe65, an important
AICD-binding protein, transactivates a wide variety of different
promoters, including the viral simian virus 40 promoter,
independent of AICD coexpression. Overall, the four currently
proposed target genes are at best indirectly and weakly
influenced by APP processing. Therefore, inhibition of APP
processing to decrease Ab generation in Alzheimer’s disease will
not interfere significantly with the function of these genes.
Keywords: Alzheimer’s disease; amyloid precursor protein;
regulated intramembrane proteolysis; secretase
EMBO reports (2006) 7, 739–745. doi:10.1038/sj.embor.7400704
INTRODUCTION
Alzheimer’s disease is characterized by the accumulation of
insoluble Ab deposits in the brain (Selkoe, 2001). Ab peptides are
produced by proteolytic processing of the amyloid precursor
protein (APP) by b- and g-secretases (Haass, 2004). Most
problematic for therapy, but very interesting from a fundamental
point of view, is the possibility that g-secretase cleavage of APP
activates a signalling pathway analagous to the Notch signalling
pathway (Annaert & De Strooper, 1999). Indeed, proteolytic
processing of APP by g-secretase releases, concomitantly with
the Ab peptides, the APP intracellular domain (AICD), which
has been proposed to function in gene transcription regulation
(Cao & Su
¨
dhof, 2001).
A series of candidate AICD target genes have been identified in
the past few years, including tetraspanin KAI1/CD82, APP,
glycogen synthase kinase-3b (GSK-3b) and Neprilysin (Baek
et al, 2002; Kim et al, 2003; von Rotz et al, 2004; Pardossi-
Piquard et al, 2005). In the present work, we raised the questions
of whether g-secretase inhibition would affect the expression of
these putative AICD target genes and whether AICD indeed has
a direct role in gene transcription regulation of these genes.
RESULTS
Pharmacological inhibition of AICD generation
To assess the risk of blocking AICD generation on gene
transcription events, we probed the effects of inhibiting
g-secretase activity on the expression of the putative AICD target
genes. Inhibition of g-secretase activity with L-685,458
(g-secretase inhibitor X) or DAPT (g-secretase inhibitor IX) did
not significantly affect endogenous (full-length) APP, KAI1, GSK-
3b or Neprilysin protein levels in any of the cell lines tested,
including murine embryonic fibroblasts (MEFs), HeLa, COS and
the neuronal cell type Neuro2A (Fig 1A). We confirmed the
presence of endogenous AICD, migrating below endogenous APP
carboxy-terminal fragments (CTFs) in MEFs (Fig 1B, left panel), and
its disappearance in the presence of g-secretase inhibitors (Fig 1B).
AICD target genes in c-secretase-deficient models
We next investigated whether genetic (continuous) inactivation of
g-secretase activity would affect the expression of any of the AICD
Received 14 February 2006; revised 11 April 2006; accepted 12 April 2006;
published online 19 May 2006
+
Corresponding author. Tel: þ 32 163 46227; Fax: þ 32 163 47181;
E-mail: bart.destrooper@med.kuleuven.ac.be
1
Neuronal Cell Biology and Gene Transfer, CME, Flanders Interuniversity Institute
for Biotechnology (VIB4) and Katholieke Universiteit Leuven, Herestraat 49,
Leuven 3000, Belgium
2
Max-Planck Institute for Brain Research, Deutschordenstrasse 46, 60598 Frankfurt,
Germany
3
Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg,
Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 7 | 2006
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scientific report
739
candidate target genes. The steady-state protein levels of APP,
KAI1, GSK-3b and Neprilysin were essentially unaffected in
g-secretase-deficient presenilin (PS) double-knockout (dKO) fibro-
blasts (Fig 2A, compare lanes 1 and 2). Likewise, the expression
levels of these genes were not significantly affected in other
g-secretase-deficient cells such as Aph-1A KO fibroblasts (Serneels
et al, 2005; Fig 2C). To further assess the functional relationship
between endogenous AICD levels and the expression of the target
genes, we reconstituted the PS dKO cells with various PS1
constructs. We chose three types of PS1 molecule: (i) functionally
active (that is, g-secretase protein and complex maturation rescue)
and catalytically active (wild type (WT), D1–81 and D303–372);
(ii) functionally but not catalytically active (P433L); or (iii) non-
functionally and non-catalytically active (D451–467). As shown in
Fig 2A, endogenous AICD levels did not correlate with the target
genes (compare, for example, lanes 3 and 7). For APP, GSK-3b
and Neprilysin, these observations were confirmed in vivo in PS1
KO embryo brain (Fig 2B) and in Aph-1A KO whole embryos
(Fig 2D; Serneels et al, 2005). KAI1 was below detection levels
in western blots of these samples (Fig 2B,D).
AICD target genes in APP-deficient models
We also investigated different MEF cell lines deficient for APP, the
APP family member APLP2 or both (Fig 3A; supplementary Fig 1A
online). As APLP1 is not expressed in fibroblasts (Herms et al,
2004), APP/APLP2 dKO MEFs provide a cellular model in which
all APP family members are lacking. We reconstituted, in
addition, APP expression in APP/APLP2 dKO MEFs using human
APP isoform 695 (see supplementary information online). As
observed in Fig 3A, GSK-3b and Neprilysin expressions were not
significantly affected by the presence or absence of APP.
However, we found, in the different MEF cell lines, large
variations in KAI1 expression that were not APP genotype
dependent (Fig 3A, compare lanes 3 and 4). No significant
changes in expression of KAI1 were detected at the mRNA level
by quantitative reverse transcription–PCR (RT–PCR; Fig 3B).
Deficiency of APP/APLP2 in the embryo brain did not significantly
affect GSK-3b or Neprilysin protein levels (Fig 3C). Likewise, we
could not observe any significant change in Neprilysin mRNA
levels in APP/APLP2 dKO and APP/APLP1/APLP2 triple-knockout
(tKO) embryonic cortices when compared with WT controls
(Fig 3D). By quantitative RT–PCR, we found a very slight
upregulation of KAI1 mRNA (1.5- to 1.9-fold) in APP/APLP2
dKO and APP/APLP1/APLP2 tKO embryonic cortices when
compared with WT controls (Fig 3D). However, we observed
no significant difference in KAI1 protein levels in the brain of
APP-deficient adult mice (supplementary Fig 1B online).
AICD is a poor gene transcription stimulator
To answer the question of whether AICD could directly modulate
gene transcription, we tested the transactivation properties of
AICD in a luciferase-based reporter assay using the endogenous
promoter regions of KAI and APP. We overexpressed two non-
tagged AICD constructs on the basis of the e- and g-secretase
cleavage sites of human APP (C50 and C60, respectively). In
contrast with the approach of other authors who used artificial
chimaeric AICD/DNA binding domain constructs (Cao & Su
¨
dhof,
2001), this strategy allowed a direct and quantitative measurement
of the intrinsic transactivation properties of AICD. Hes1, an
established target gene of the Notch intracellular domain (NICD)
signalling pathway, was analysed in parallel to benchmark our
reporter assays. Overexpression of C50, C60 and APP C99 (the
product of APP generated by b-secretase cleavage) resulted in a
weak (maximal B2-fold) activation of the KAI1 promoter (Fig 4A).
As expected, NICD expression resulted in a marked stimulation
(B20-fold) of Hes1 promoter-mediated luciferase expression
(Fig 4C). Although AICD overexpression per se can apparently
weakly activate a subset of gene promoters, this effect is minor
when compared with the effect of a protein fragment that is
really involved in gene transcription regulation.
D
M
SO
DAPT (10 µM
)
DAPT (10 µM
)
DAPT (10 µM
)
L-685,458 (1 µM
)
L-685,458 (1 µM
)
L-685,458 (1 µM
)
L-685,458 (1 µM
)
D
M
SO
D
M
SO
D
M
SO
D
APT (10 µM
)
APP FL
APP CTFs
KAI1
NEP
GSK-3α/β
β-Actin
N2A
COS
HeLa
MEF
D
M
SO
PS1–/–, PS2–/–
PS1–/–, PS2–/– Res PS
1
W
T
PS1–/–, PS2–/– Res PS
1
D257A
D
APT (10 µM
)
L-685,458 (1 µM
)
APP CTFs
APP CTFs
AICD
(longer exposure)
β-Actin
MEF
6
3
A
B
Fig 1
|
Pharmacological inhibition of g-secretase and effects on the
expression of AICD target genes protein. (A) MEF, HeLa, COS and
Neuro2A cells were treated with DAPT or L-685,458 for 16–18 h. Levels
of endogenous proteins are shown. APP CTF accumulation demonstrates
the activity of the inhibitors. (B) Control western blot analysis of MEFs
showing endogenous AICD migrating below APP CTFs, downregulated in
the presence of g-secretase inhibitors (left panel). The presence of
endogenous AICD is dependent on catalytically active PS (right panel).
AICD, amyloid precursor protein (APP) intracellular domain; APP CTFs,
APP carboxy-terminal fragments; APP FL, APP full-length; DMSO,
dimethylsulphoxide; GSK-3a/b, glycogen synthase kinase-3a/b; MEFs,
murine embryonic fibroblasts; NEP, Neprilysin; WT, wild type.
AICD in gene regulation
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bert et al
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We next tested the transactivation properties of AICD in
the presence of the putative scaffolding protein Fe65. Fe65
can effectively bind to APP and APLP intracellular domains
and is necessary to drive the expression of reporter genes by
the APP–DNA binding domain fusion proteins (Cao & Su
¨
dhof,
2001). Coexpression of Fe65 with C50 moderately stimulated
KAI1 and APP expression (B5- to B8-fold, respectively;
Fig 4A,B). Interestingly, coexpression of Fe65 with NICD
resulted also in a similar B4-fold increase of Hes1 expression
(from B20- to B80-fold; Fig 4C). In addition and surprisingly,
C50 could also stimulate Hes1 promoter activity when Fe65
was present (B4-fold; Fig 4C), and coexpression of NICD
with Fe65 resulted in a significant increase in APP and KAI1
promoter activity (comparable to C50/Fe65 induction of
activity; Fig 4D,E). Finally, and consistent with a general role
of Fe65 in gene transcription activation (Duilio et al, 1991;
Yang et al, 2006), coexpression of Fe65 with AICD or NICD
stimulated the expression of a control simian virus 40 (SV40)
promoter (Fig 4F).
DISCUSSION
Since the demonstration that APP and Notch are proteolytically
processed by PS to release their intracellular domain, it has been
speculated, by analogy with the Notch signalling pathway, that
AICD could function directly in gene transcription regulation
(Annaert & De Strooper, 1999). Since then, many conflicting data
and models concerning the role of AICD in promoting gene
transcription have emerged in which AICD could function inside
(Cao & Su
¨
dhof, 2001; Cupers et al, 2001; Kimberly et al, 2001) or
outside (Cao & Su
¨
dhof, 2004; Hass & Yankner, 2005) the nucleus.
In addition, several authors have described changes in gene
transcription or protein expression of KAI, APP, GSK-3b,
Neprilysin and other genes (von Rotz et al, 2004; Ryan
& Pimplikar, 2005) due to APP or AICD overexpression or
g-secretase inhibition. However, the overall reported changes
have always been weak, and in many cases additional controls to
benchmark the reported observations were lacking. Obviously,
it is very important to know whether the reported effects of AICD
on gene transcription are biologically significant, as inhibition of
ABCD
PS1 holo
PS1 NTF
PS1 CTF
Nct
APP FL
APP FL
APP CTFs
NEP
KAI1
APP CTFs
AICD
(longer exposure)
KAI1
MEF
6
3
NEP
E14.5
brain
β-Actin
β-Actin
GSK-3α/β
GSK-3α/β
APP FL
APP FL
APP CTFs
Nicastrin
Aph1A
L
Aph1A
L
NEP
KAI1
KAI1
β-Actin
GSK-3α/β
NEP
β-Actin
GSK-3α/β
C
ontrol
C
ontrol
PS1/, PS2/
PS1/
PS1/, PS2/ Res PS
1
W
T
C
ontrol
C
ontrol
Aph1A/
Aph1A
+/
A
ph1A/
Aph1A/ Res A
ph1A
WT
PS1/, PS2/ Res PS
1
1-81
PS1/, PS2/ Res PS
1
303-372
PS1/, PS2/ R
es PS
1
451-467
PS1/, PS2/ Res PS
1
P433L
E9.5
embryo
MEF
Fig 2
|
Expression levels of AICD target genes in various biological models lacking g-secretase activity. (A) Western blot analysis of PS1 and PS2 dKO
MEF cells. PS dKO MEFs were reconstituted with human PS1 or with PS1 functional mutants. (B) Effects on protein expression levels of target genes
in the absence of PS1 in vivo. No KAI1 expression is observed at this embryonic stage. (C) Western blot analysis of Aph-1A KO MEFs. In the third
lane, Aph-1A KO MEFs were reconstituted with mouse WT Aph-1AL. (D) Effects on protein expression levels of target genes in the absence of Aph-1A
in vivo. Note that KAI1 protein levels are below detection at this embryonic stage. AICD, amyloid precursor protein (APP) intracellular domain; APP
CTFs, APP carboxy-terminal fragments; APP FL, APP full-length; dKO, double knockout; E, embryonic day; GSK-3a/b, glycogen synthase kinase-3a/b;
MEFs, murine embryonic fibroblasts; NEP, Neprilysin; PS, presenilin; PS1 CTF, PS1 C-terminal fragment; PS1 NTF, PS1 amino-terminal fragment;
WT, wild type.
AICD in gene regulation
S.S. He
´
bert et al
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Ab generation by g-secretase inhibitors would affect such a
putative signalling pathway.
We therefore checked the expression levels of APP, GSK-3b,
KAI1 or Neprilysin, four putative AICD-regulated genes, in cell
lines treated with g-secretase inhibitors or in biological models
genetically deficient for AICD generation. The significant decrease
or even absence of endogenous AICD production was not
reflected in systematic changes in the levels of expression of
three out of four putative AICD target genes, namely APP itself and
GSK-3b and Neprilysin. For KAI1, the situation is slightly more
complex. On the one hand, in all AICD-deficient cell lines tested
(that is, PS KO, Aph-1A KO and APP/APLP2 dKO) and in adult
A
B
D
C
APP
non-rescued
APP
rescued
C
ontrol (1)
C
ontrol (2)
APP/, APLP2/ (1.1)
APP/, APLP2/
APP/, APLP2/ (1.4)
APP/, APLP2/
APP/, APLP1/,
APLP2/
A2
A3
A4
A5
A7
A25
1B
2
3B
2
(clonal cell lines)
APP FL
KAI1
NEP
NEP
NS
GSK-3α/β
β-Actin
APP FL
NEP
GSK-3α/β
β-Actin
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Rel. expression (n-fold)
NS
NS
P =0.452
KAI1
KAI1
APP rescued versus
APP/,
APLP2/
APP
non-rescued
C
ontrol
E15.5
embryo
2.5
2.0
1.5
1.0
0.5
0
Rel. expression (n-fold)
0.026
0.3215
NS
0.08
0.0115
Fig 3
|
Expression levels of AICD target genes in various biological models lacking APP and its family members. (A) Western blot analysis of MEFs
deficient for APP and APLP2 (two independent cell lines are shown). The APP/APLP dKO MEF (1.4) cell line was reconstituted with human N-Myc-
tagged APP695 WT using the retroviral vector FUW (see supplementary information online). Clonal cell lines resistant to puromycin selection were
categorized as ‘APP rescued’ or ‘APP non-rescued’ cell lines. Western blot analysis of four independent cell lines positive or negative for APP
expression was performed. Here, KAI1, GSK-3b and Neprilysin protein levels are shown. Considerable variations in KAI1 expression in the different
cell lines were observed but were not dependent on the presence or absence of APP expression. (B) Quantitative RT–PCR analysis showed no
significant difference in KAI1 expression between ‘APP rescued’ cell lines (n ¼ 4) and APP/APLP2-deficient cell lines (APP/, APLP2/; n ¼ 2, line
1.1 and 1.4 in A). Likewise, quantitative RT–PCR analysis of ‘APP rescued’ (n ¼ 4) versus ‘APP non-rescued’ (n ¼ 4) cell lines showed no significant
difference in KAI1 messenger RNA expression. Bars represent standard errors; P-values were calculated by pairwise fixed reallocation randomization
test. (C) Western blot analysis of AICD candidate target genes in APP/APLP2 dKO embryos (lethal combination). GSK-3b and Neprilysin protein
levels from two independent age-matched (E15.5) controls and knockout mice are shown. KAI1 was below detection levels (data not shown).
(D) Quantitative RT–PCR analysis of KAI1 and Neprilysin expression in embryonic brain. mRNA expression of KAI1 or Neprilysin was analysed in the
cortices of E15.5 dKO (APP/ APLP2/, open boxes, n ¼ 4) or tKO (APP/ APLP1/ APLP2/ , filled boxes, n ¼ 4) mice relative to WT
controls (n ¼ 4). Note that no significant difference in expression was found for Neprilysin, whereas KAI1 was slightly upregulated in dKO and tKO
brains. Bars represent standard errors; P-values (shown above SE bars) were calculated by pairwise fixed reallocation randomization test. AICD,
amyloid precursor protein (APP) intracellular domain; APP FL, APP full-length; dKO, double knockout; E, embryonic day; GSK-3a/b, glycogen
synthase kinase-3a/b; MEFs, murine embryonic fibroblasts; NEP, Neprilysin; RT–PCR, reverse transcription–PCR; tKO, triple knockout; WT, wild type.
AICD in gene regulation
S.S. He
´
bert et al
EMBO reports VOL 7 | NO 7 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
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742
mice haploinsufficient for APP (that is, APP/, APLP1/,
APLP2 þ / and APP/, APLP1 þ /, APLP2 þ /), KAI1 expres-
sion was never correlated with the presence or absence of AICD
(thus, in some cell lines, high AICD was correlated with low KAI1
expression, and vice versa in other experiments). Only a slight
increase (1.4- to 1.9-fold) in KAI1 message was demonstrated in
embryos deficient for APP and its family members (Fig 3D), but the
significance of this observation and whether this really supports
the role of AICD in KAI1 regulation is at least doubtful, the more
so because APP or AICD was suggested to cause upregulation of
KAI1 (Baek et al, 2002; Ryan & Pimplikar, 2005). Taken together,
our data show that the role of AICD in KAI1 expression cannot be
considered as established.
We finally moved to an overexpression paradigm, analysing
the effects of transfected AICD on the activity of the promoter
regions of KAI1 (and APP) in a luciferase-based reporter assay.
Although AICD was able to stimulate (less than twofold) KAI1 and
APP gene expression, these effects turned out to be minimal when
compared with the positive control experiment with NICD, which
induced in a similar assay the activity of the Hes1 promoter more
than 20-fold (Fig 4C). As AICD has a very relaxed tertiary
conformation and is probably undergoing an ‘induced fit’ when
binding its various binding partners (a phenomenon called
‘binding promiscuity’; discussed by Reinhard et al, 2005), over-
expression of AICD could lead to rather unspecific protein–protein
interactions. Therefore, the subtle alterations of gene transcription
induced by C50 (and C60) overexpression have to be interpreted
with caution. Interestingly, in the same luciferase reporter
assays, coexpression of Fe65 with AICD led consistently to a
more pronounced increase in gene transcription, in line with
previously published observations (Cao & Su
¨
dhof, 2001).
However, and importantly, we show here that this stimulating
effect is not specific for AICD, as Fe65 when coexpressed
with NICD resulted in a similar activation of the APP and KAI1
promoter (Fig 4D,E) and also of the Hes1 promoter (Fig 4C).
This suggested that Fe65 has a general, not AICD-dependent,
weak stimulating effect on different promoters, enhancing their
activity with a factor of B3–4. This conclusion is strikingly
confirmed with the viral SV40 promoter, on which FE65 has
a similar effect.
Regulated intramembrane proteolysis is a novel type of cell
signalling mechanism (Annaert & De Strooper, 1999; Brown et al,
2000). Apart from Notch family members, most evidence
regarding the role of AICD or other intracellular domains in
gene transcription regulation was deduced from experiments
in vitro and relies in most cases on artificial overexpression
approaches. The results presented here suggest, for APP at
least, that the intracellular fragment generated by g-secretase
cleavage is not necessarily directly involved in a nuclear
signalling pathway. We suggest that in the case of APP, the
main role of g-secretase cleavage is the removal of the
membrane-bound CTF after ectodomain shedding (Kopan &
Ilagan, 2004).
In conclusion, and from a more practical perspective, we
demonstrate here that the selective inhibition of g-secretase and
Ab production does not cause significant effects on the protein
levels of genes that were suggested to be regulated by AICD.
Although this work cannot exclude the possibility that other yet
unidentified genes are controlled by AICD-mediated signalling,
we dare to conclude that deregulation of the proposed AICD target
6
5
4
3
2
1
0
Fold increase
Fold increase
Fold increase
14
12
10
6
8
4
2
0
Fold increase
1.0
1.9
1.8
1.4
1.1
4.7
3.9
2.9
KAI1
KAI1Hes1
APP
APP
SV40
Fe65
Fe65
+Fe65 Fe65 +Fe65
+Fe65 Fe65 + Fe65 Fe65 +Fe65 Fe65 +Fe65
Mock
C50
C60
C99
FL
Mock
Mock
C50
NICD
NICD
NICD
NICD
Mock
NICD
NICD
C50
Mock
C50
NICD
NICD
C50
C50
C99
FL
C50
*
Mock
C50
C60
C99
FL
C50
C99
FL
C50
*
1.0
1.5
1.6
1.4
1.1
8.7
5.0
3.6
100
80
60
40
20
0
1.0
1.0
1.0
1.0
1.4
1.1
2.7
2.8
1.9
1.5
6.1
4.5
82.0
22.6
1.1
4.1
6
5
4
3
2
1
0
Fold increase
Fold increase
6
8
4
2
0
4
3
2
1
0
AB
CDEF
Fig 4
|
Non-specific activation of gene transcription by exogenous Fe65 in luciferase-based reporter assays. Transactivation assays performed with the
endogenous promoter regions of KAI (A,D), APP (B,E), Hes1 (C) or SV40 (F) coupled to luciferase. The different APP constructs (C50, C60, C99 or
full-length 695 isoform) or NICD were expressed alone or in combination with Fe65 in HeLa cells. Control experiments were performed with pGL3-
luciferase empty vector (that is, no promoter; indicated with an asterisk). All assays were performed at least three times in duplicate. Similar results
were obtained in COS cells (data not shown). APP, amyloid precursor protein; NICD, Notch intracellular domain; SV40, simian virus 40.
AICD in gene regulation
S.S. He
´
bert et al
&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 7 | 2006
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743
genes is not an important issue when contemplating g-secretase
inhibitors as a therapy in Alzheimer’s disease.
METHODS
Chemicals and antibodies. L-685,458 and DAPT were from
Calbiochem (VWR, Leuven, Belgium). The PS1 CTF, Aph-1AL
and APP (C-terminal) antibodies (Hebert et al, 2004), and the PS1
amino-terminal fragment (SB129) and Nicastrin antibodies were
described elsewhere (De Jonghe et al, 1999; Esselens et al, 2004).
The Neprilysin/CD10 (sc-9149), KAI1/CD82 (sc-1087) and GSK-
3a/b (sc-7291) antibodies were from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA).
Cells and cell culture. MEF, HeLa, COS and Neuro2A cells were
cultured in Dulbecco’s modified Eagle’s medium supplemented
with serum and antibiotics. The generation of the PS1/2 dKO MEFs
reconstituted with human PS1 WT, the catalytically inactive PS1
D257A, was described elsewhere (Nyabi et al, 2003). The
construction of the PS1 functional mutants will be described
elsewhere, and details are available on request. The Aph-1A KO
MEF cells were described previously (Serneels et al, 2005).
Mice. PS1 (De Strooper et al, 1998), Aph-1A (Serneels et al,
2005), APP/APLPs (Herms et al, 2004) KO mice were described
before. We used embryonic day (E)9.5 (Aph-1A), E14.5 (PS1),
E15.5 (APP/APLP1/APLP2) or 12- to 16-month-old (APP/APLPs)
mice in our experiments. Tissue samples were extracted in 1%
Triton X-100 lysis buffer and subjected to western blot analysis.
Complementary DNA cloning and plasmids. The regulatory
promoter region of APP (bp B2,100 to B þ 100; Quitschke
& Goldgaber, 1992) was amplified by PCR from purified human
chromosomal DNA and cloned into the pGL3-luciferase vector
(Promega, Leiden, The Netherlands). The KAI1 promoter (Baek
et al, 2002) was subcloned into the pGL3-luciferase vector
(Promega). The murine HES-1 promoter (bp 7–251; Takebayashi
et al, 1994) was generated by PCR from the pGL2-Hes1-Luc
template ( Jarriault et al, 1995). The coding sequence of the
luciferase gene from the pGL2 basic vector (Promega) was
subcloned downstream of the HES-1 promoter giving rise to the
pHes1-Luc construct. The APP-C50 (e49 position) and APP-C60
(g59 position) constructs were generated by PCR. These fragments
start at Leu 49 and Ile 41, respectively, amino acid 1 being the
methionine of the Ab sequence. Both constructs are preceded
by an ATG codon. The sequences of all PCR primers are available
on request. The APP pCS2-C99-6Myc-tagged construct was
a generous gift from A.M. Goate (St Louis, MO, USA).
Preparation of cell protein lysates and immunoblotting. Cells
were lysed in cell lysis buffer (1% Triton X-100, 50 mM HEPES pH
7.6, 150 mM NaCl, 1 mM EDTA and complete protease inhibi-
tors). Crude protein lysates (20 or 60 mg for AICD detection) were
immunoblotted and detected using the Renaissance chemilumi-
nescence detection system (Perkin-Elmer, Massachusetts, USA).
RNA preparation and quantitative PCR. Preparation of MEFs and
brain samples for quantitative PCR as well as RT–PCR procedures
including primer sequences are available as supplementary
information online.
Luciferase assays and transfections. HeLa cells (60–80% con-
fluent) were transfected with 0.5 mg of the appropriate cDNAs
(pGL3-Luc (that is, no promoter), pGL3-SV40-Luc, pGL3-APP-Luc,
pGL3-KAI1-Luc, pHES1-Luc, pSG5-APP C50, pSG5-APP C60,
pSG5-APP full-length, pCS2-APP C99-6Myc-tagged, pSG5-NICD)
using Fugene (Roche, Mannheim, Germany). At 26–28 h after
transfection, the luciferase assays were performed (Promega).
Supplementary information is available at EMBO reports online
(http://www.emboreports.org).
ACKNOWLEDGEMENTS
Work in the laboratory was supported by a Freedom to Discover grant
from Bristol Myers Squib, a Pioneer award from the Alzheimer’s
Association, the Fund for Scientific Research, Flanders; Katholieke
Universiteit Leuven (GOA); European Union (APOPIS: LSHM-CT-2003-
503330); and Federal Office for Scientific Affairs, Belgium (IUAP P5/19).
M.A.F. and U.M. were supported by a grant from the Bundesministorium
fu
¨
r Forschung und Technologie (OIGS0469).
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&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 7 | 2006
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    • "APP Intracellular Domain (AICD) is released from APP by the presenilin-mediated cleavage that also causes extracellular release of Aβ. Numerous in vitro studies have shown that AICD regulates gene expression [13], alters cell-signaling pathways and causes deleterious effects (reviewed in [14, 15]). We previously showed that AICD-overexpressing transgenic mice (AICD-Tg) recapitulate AD-like features such as hyperphosphorylation of tau, non-convulsive seizures/aberrant EEGs, neural circuit re-organization, impaired memory and neurodegeneration in an age-dependent fashion [16][17][18][19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Amyloid precursor protein (APP) is cleaved by gamma-secretase to simultaneously generate amyloid beta (Aβ) and APP Intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer’s disease (AD) pathogenesis but recent studies suggest that amyloid-independent mechanisms also contribute to the disease. We previously showed that AICD transgenic mice (AICD-Tg) exhibit AD-like features such as tau pathology, aberrant neuronal activity, memory deficits and neurodegeneration in an age-dependent manner. Since AD is a tauopathy and tau has been shown to mediate Aβ–induced toxicity, we examined the role of tau in AICD-induced pathological features. We report that ablating endogenous tau protects AICD-Tg mice from deficits in adult neurogenesis, seizure severity, short-term memory deficits and neurodegeneration. Deletion of tau restored abnormal phosphorylation of NMDA receptors, which is likely to underlie hyperexcitability and associated excitotoxicity in AICD-Tg mice. Conversely, overexpression of wild-type human tau aggravated receptor phosphorylation, impaired adult neurogenesis, memory deficits and neurodegeneration. Our findings show that tau is essential for mediating the deleterious effects of AICD. Since tau also mediates Aβ-induced toxic effects, our findings suggest that tau is a common downstream factor in both amyloid-dependent and–independent pathogenic mechanisms and therefore could be a more effective drug target for therapeutic intervention in AD.
    Full-text · Article · Jul 2016
    • "Cleavage by α-secretase in the non-amyloidogenic pathway releases a secreted APP fragment (s-APP α) as well as a transmembrane α C-Terminal Fragment (CTF). Cleavage by β-secretase in the amyloidogenic pathway produces s-APP β and β CTF (for review see34567). APP CTFs can be further cleaved by β-secretase to produce p3 and APP Intracellular domain (AICD) in the non-amyloidogenic pathway or Aβ and AICD in the amyloidogenic pathway [5,789 . Aβ peptides can accumulate and form oligomers that eventually give rise to amyloid plaques [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: A central event in Alzheimer's disease is the accumulation of amyloid β (Aβ) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aβ generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region) at residues 3008-3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.
    Full-text · Article · Nov 2015
    • "In contrast, α-secretase inhibition did not cause alterations in NEP gene expression, further indicating that β-secretase processing generates transcriptionally active AICD peptides whereas αsecretase processing of APP seems to be not involved in the generation of AICD peptides active in nuclear signaling. This finding is substantiated by the fact that γ-secretase inhibition, which resulted in divergent outcomes in respect to the regulation of NEP in literature (Pardossi-Piquard et al., 2005; Hébert et al., 2006; Chen and Selkoe, 2007; Xu et al., 2011), also leads to a reduction of NEP expression to 80.7%. The importance of βsecretase processed APP in transcriptional regulation of NEP is further validated by the results obtained by stably expressing the β-secretase cleaved APP fragment β-CTF or the α-secretase cleaved APP fragment α-CTF. "
    [Show abstract] [Hide abstract] ABSTRACT: Alzheimer´s disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex (presenilin (PS) 1/2) or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity or protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9 and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD.
    Full-text · Article · May 2015
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