Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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77176
r-Secretase in vitro test system
Technical field of the invention
The present invention relates to the field of methods of discovering specific
Y-secretase
inhibitors which can be used to treat neurodegenerative disorders,
particularly methods which
can be carried out in vitro. This invention further relates to a test kit with
which the process
according to the invention can be carried out, and the use of this test kit or
of the method of
discovering substances which specifically inhibit y-secretase. Another
embodiment relates to the
use of these substances for preparing a medicament for treating
neurodegenerative disorders and
pharmaceutical formulations which contain these substances.
Background to the invention
The aggregation and precipitation of proteins are implicated in the origins of
various
neurodegenerative disorders such as Alzheimer's, Parkinson's and St. Vitus'
dance ("Huntington's
~s Chorea "). In Alzheimer's disease the amyloid-p-peptide (A/3) aggregates
and leads to insoluble
senile plaques which constitute one of the pathological markers of the disease
(Selkoe et al,
1996). A(3 is formed by the proteolytic cleaving of a precursor protein,
amyloid precursor
protein (APP). Two methods of metabolising APP have been detected, the non-
amyloidogenic
method and the amyloidogenic method (Selkoe, 1991, 1994).
ao
In the non-amyloidogenic metabolism of APP, a-secretase cleaves within the Ap
region of the
APP and thus leads to the secretion of the soluble N-terminal region of the
protein (a-APPs)
and, after the y-secretase cutting has taken place, to the release of p3. By
contrast, the
amyloidogenic route leads to the formation of Ap, two proteases generating the
N-terminus ((3-
2s secretase) and the C-terminus (y-secretase), respectively, of A~i (Haass,
1993; Selkoe, 1994).
Ap can be detected in human plasma and cerebrospinal fluid in vivo. In cell
culture, too, secreted
A(3 can be detected in the cell culture supernatant of various types of cells
which express or
overexpress APP or fragments thereof endogenously. Both A(3 production and
hence the
3o formation of amyloid plaques are influenced by various genetic risk
factors. These include
mutations in the homologous proteins preseniline 1 and preseniline 2 as well
as in the APP itself.
Analysis of these mutations on fibroblasts of Alzheimer's patients with
Familiar Alzheimer's
Disease (FAD) showed the influence they have on the formation of A(3. This was
confirmed by
investigations on transfected cells and transgenic animals. All mutations
increase the production
3s of A(3 and in the case of the preseniline mutations lead to a selective
increase in the longer A(3
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2
variant, A~342 (Selkoe, 1996; Price, 1998). This peptide aggregates to a
greater extent than the
shorter form, Aj340, and is found in the diffuse plaques and together with
A~340 in the senile
plaques (Lemere et al., 1996; Mann et al., 1996). In addition to this
influence of the mutations,
there are indications that the wild-type forms of the presenilines also have a
fundamental
s function in the physiological production of A~3. In neurones of mouse
embryos in which the PS 1
gene (PS: preseniline) has been switched off by genetic engineering, a drastic
reduction in A[3 40
and A(3 42 was detected. In addition, the C-terminal fragments of the APP
accumulate in these
cells, leading to the assumption that the presenilines activate the y-
secretase or themselves have a
y-secretase activity (De Strooper et al., 1998; Sisodia et al., 1998). First
in vitro test systems
combined with mutation studies on conserved aspartates of preseniline 1 lead
one to assume that
the presenilines could be special autocatalytically active aspartate proteases
which are
responsible for the y-secretase cutting in the membrane (Wolfe et al., 1999).
The discussion of the identification of y-secretase as a crucial step in the
generation of Ap and
~s hence in the onset of Alzheimer's is not concluded to this day. Quite apart
from this, this
protease is an interesting target for intervening pharmacologically in the
process of A(3
formation by finding inhibitors which will selectively reduce its activity.
For this reason it is
important to develop in vitro test systems, in addition to animal models and
cell assays, which
make it possible to test specific active substances independently of transport
processes within the
cell .
Wolfe et al. (1999) showed an in vitro system for measuring the activity of y-
secretase. To
prepare the system, membranes from cells which stably express PS 1 are worked
up. They are
mixed with a plasmid coding the LC99 polypeptide and subjected to an in vitro
reaction of
2s transcription/translation in the presence of 35S-methionine, forming the y-
secretase substrate
C99. The mixture is then incubated under suitable conditions, during which
time the C99
fragment of APP is proteolytically cleaved from the y-secretase and the
breakdown products are
detected by gel electrophoresis after immunoprecipitation. LC99 within the
scope of the
invention denotes a fusion protein between the y-secretase substrate C99 and a
signal sequence
30 (L,: "leader"; Shoji et al., 1992).
Brief summary of the invention
The present invention falls within the scope of methods of discovering
specific y-secretase
inhibitors which can be used to treat neurodegenerative disorders,
particularly methods which
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CA 02381952 2002-02-12
can be carried out in vitro. This invention further relates to a test kit with
which the process
according to the invention can be carried out, and the use of this test kit or
of the method of
discovering substances which specifically inhibit y-secretase. Another
embodiment relates to the
use of these substances for preparing a medicament for treating
neurodegenerative disorders and
pharmaceutical formulations which contain these substances.
Figures
Fig. 1: Characterisation of the microsomal fraction SIII
The H4-ind/APP-LC99 cells were left to grow in the absence of doxycycline for
three days in
la order to induce the expression of LC99. Post Nuclear Supernatant (PNS) was
prepared as
described and further concentrated using sucrose step gradients (Taylor et al.
1997)
A. Concentration of the y-secretase substrate C99 in the microsomal fraction
SIII. Aliquots
(in each case 30~g) of the PNS and the microsomal fraction (SIII) were placed
on a 12% SDS-
polyacrylamide gel. For this purpose, PNS was prepared from H4-ind/APP-LC99
cells which
r5 had grown in the presence and absence of doxycycline. The proteins were
transferred to a
polyvinylidene difluoride (PVDF) membrane (Poly Screen, New Life Science) and
C99 was
detected with the polyclonal antibody 5818, which is directed against the last
20 amino acids of
the C99 (diluted 1:1000; alternative antibodies with the same activity:
Product Number SAD
3138, Labgen).
2o B. Concentration of the presenilines in the microsomal fraction SIII.
Aliquots (in each case 30pg) of the PNS and fractions of the sucrose step
gradient were loaded
onto a 12% SDS polyacrylamide gel and the proteins separated were transferred
to a PVDF
membrane (Poly Screen, New Life Science). In order to detect the two PS
proteins either the
monoclonal antibody BL3D7 (PS 1 CTF-specific; 1:3000; Steiner et al., 1999) or
the monoclonal
2s antibody BI-HFSC (PS2 CTF-specific; 1:3000; Steiner et al., 1999) was used.
It was shown that
CTFs of PS 1 and PS2 had been concentrated in the SIII fraction. It was not
possible to tell
whether the protein band observed at a molecular weight of about 46-50 kDa in
each case was
full length PS 1 or PS2.
C. Characterisation of the SIII fraction by the detection of marker proteins
which are
3o characteristic of certain compartments
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4
Aliquots (in each case 30~g) of the PNS and fractions of the sucrose step
gradient (9) were
loaded onto a 12% SDS polyacrylamide gel and the proteins separated were
transferred to a
PVDF membrane (Poly Screen, New Life Science).
To demonstrate the concentration of membranes of the endoplasmatic reticulum
in the SIII
fraction an anti-calreticulin antibody (Stressgen Biotechnologies; 1:2000) was
used. The
distribution of endosomal membranes in the gradient was shown with the aid of
anti-rab5
antibodies (Transduction Laboratories Inc.).
Fig. 2: Celt-free generation of A13 40 and A13 42 from isolated microsomes
A. De novo A!3 generation in a cell-free y-secretase test system with isolated
microsomes of H4
cells, stably transfected with APP-LC99. Microsomes were prepared as described
(Taylor et al.,
1997) (SIII-fraction) and incubated at 37°C or 4°C for 4 hours
under neutral conditions (pH
6.8). After incubation of the substrate C99 the products AJ340 and A1342 were
immunoprecipitated with the antibodies 6E10 and 4G8 (Senetek, Great Britain;
Galli et al.,
~s 1998). In this way the substrate/product ratio could be estimated. To
precipitate the A1340 and
A1342 peptides alone the specific antibodies BL40 and BL42 were used. The
precipitated proteins
were separated with a Tris-Bicine gel, transferred to a nitrocellulose
membrane and made visible
with the antibodies 6E10 and 4G8, using a highly sensitive Western Blot
procedure (Ida et al.,
1996). To determine the basal intracellular Al3 content, immunoprecipitations
were carried out
2o directly with the microsomal fraction stored at - 80°C (Lane c). The
A1340 and A1342 generated
in vitro migrates with the synthetic A13 peptides. Lower part: longer exposure
B. Time-dependency of the generation of A(i in vitro.
The SIII fraction was prepared as described and incubated for the specified
time at 37°C or 4°C
under neutral conditions (pH 6.8). A13 peptides were immunoprecipitated with
the specific
2s antibodies BL40 and BL42. The precipitated proteins were separated by Tris-
Bicine gel
electrophoresis (Klafki et al., 1996) and then detected using a highly
sensitive Western blot
procedure with the antibodies 6E10 and 4G8 (Ida et al., 1996). The synthetic
A1340 and A1342
peptides were used as the standard. After 3-4 hours' incubation at 37°C
the de novo generation
of A(3 reached a peak.
Fig. 3: The y-secretase cleavage product At3 is degraded by a Ca2+-dependent
protease
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CA 02381952 2002-02-12
The SIII fraction was produced as described and incubated under standard
conditions (37°C; pH
6.8; 4 hours) in the presence or absence of cation chelators such as EDTA or
BAPTA. As a
control the microsomes were incubated at 4 °C in the presence of EDTA.
A!3 peptides were
immunoprecipitated with the specific antibodies BL40 and BL42 and detected by
Western blot
s with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli et al.,
1998) as described. The
synthetic peptides Af340 and A1342 were used as standaxd. All the reactions
were carried out
three times. In the absence of a chelator the de novo Al3 production is
drastically reduced. Both
EDTA and BAPTA, which chelates only Ca2+ ions, result in a larger quantity of
A13 de novo.
This can be explained by the fact that Al3 is degraded again by a Caz+-
dependent protease
m immediately after production.
Fig. 4: y-Secretase is a transmembrane protease.
The SIII fraction was prepared as described and incubated under standard
conditions (pH 6.8; 5
mM EDTA; 4 hours) at 37°C or 4°C as a control. The microsomal
membranes were either
~s washed with a highly saline solution (1 M KCl) or extracted with Na2C03 in
order to remove
weakly bound proteins from the microsomal membranes. To do this the pelleted
membranes
were resuspended in KCl buffer (1 M KCI, 250 mM sucrose, 20 mM Hepes, pH 6.8)
and
incubated for 30 min at 4°C. For the NazC03 extraction the membranes
were homogenised in
100 mM Na2C03, pH 11,5, and incubated for 30 min at 0°C. The membranes
were incubated at
220.000 g for 1 hour at 4°C, carefully washed with cold water and
washed with 1 ml of reaction
buffer (9) during the homogenisation. The membranes were centrifuged as
described above and
resuspended in reaction buffer. Aliquots were frozen in liquid nitrogen. A(3
peptides were
immunoprecipitated with the specific antibodies BL40 and BL42 and detected by
Western blot
with the antibodies 6E10 and 4G8 as described. The synthetic peptides A(340
and AJ342 were
zs used as the standard. All the reactions were duplicated. Independently of
the pretreatment of the
membranes the content of AI3 generated de novo was virtually identical. These
data lead one to
assume that'y-secretase is a protease which is either fixedly bound to the
membrane or is an
integral membrane protein.
3o Fig. 5: The optimum pH for the y-secretase activity is between pH 6.8 and
7.4
The SIII fraction was prepared as described and incubated under standard
conditions (pH 6.8; 5
mM EDTA; 4 hours) at the pH values specified. A13-peptides were
immunoprecipitated with the
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CA 02381952 2002-02-12
6
specific antibodies BL40 and BL42 and detected by Western blot with the
antibodies 6E10 and
4G8 as described. All the reactions were duplicated. As a control the in vitro
reaction was
carned out at 4°C and pH 6.8. The optimum pH for the in vitro y-
secretase activity is in the
neutral range between pH 6.8 and pH 7.4. A sharply reduced y-secretase
activity was found both
under slightly acidic and under basic pH conditions.
Fig. 6: Effect of a possible y-secretase inhibitor in the cell-free r-
secretase test system.
The SIII fraction was prepared as described and incubated at a pH of 6.8 under
standard
conditions in the presence or absence of different concentrations of compound
A (Fig.: A) (pH
l0 6.8; 5 mM EDTA; 4 hours). A13 peptides were immunoprecipitated with the
specific antibodies
BL40 and BL42 and detected by Western blot with the antibodies 6E10 and 4G8
(Senetek, Great
Britain; Galli et al., 1998) as described. All the reactions were performed in
duplicate or
triplicate. As a control the reactions were carried out at 4°C.
A. Compound A exhibited an inhibition of the in vitro generation of AI3
dependent on
rs concentration. The Af3 generated de novo was quantified with the
Chemiluminescence
Imaging System (Biorad) and this is shown in the lower part.
B. Compound A exhibited a concentration-dependent reduction in eztracellular
A~i40 and
A(342
Compound A was also active in a cellular test system in which the
extracellular content of A(340
zo and 42 secreted by the U373 cell line (U373: ATCC No. HTB 14) is
determined. This cell line
U373/APP751 is an Astrocytoma cell line which overexpresses human APP~51 and
secretes large
amounts of A(340 (~1000pg/ml/ 4 hours with SxlO' cells in 15 ml of medium) and
A(342
(~100pg/ml/ 4 hours with SxlO' cells in 15 ml of medium). The assay is done
using ELISA
(ELISA: "Enzyme linked immunosorbent assay" (Steiner et al., 1998). The A(340
secretion was
~s sharply reduced by compound A in a concentration-dependent manner, slightly
increasing the
secretion of A(342 at subinhibitory doses and then also inhibiting it at
higher doses.
Fig. 7: Effect of a y-secretase inhibitor as described in the cell-free y-
secretase test system.
The SIII fraction was prepared as described and incubated at a pH of 6.8 under
standard
3o conditions in the presence or absence of various concentrations of the
compound MG132
(Biomol Order No. : PI-102; De Strooper et al. 1999) (pH 6.8; 5 mM EDTA; 4
hours). A13
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peptides were immunoprecipitated with the specific antibodies BL40 and BL42
and detected by
Western blot with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli
et al., 1998) as
described. All the reactions were duplicated. As a control the reactions were
carried out at 4°C
or at 37°C without any inhibitor.
A) The compound MG132 displayed a concentration-dependent inhibition of the in
vitro
generation of Af3.
B) Quantifying the A13 generated de novo was done using the Chemiluminescence
Imaging
System (Biorad) and is shown in the lower part.
!o Fig. 8 : Characterisation of the microsomal fractionation of H4indLC99
cells
The H4-ind/APP-LC99 cells were allowed to grow for three days in the absence
of doxycycline
in order to induce the expression of LC99. The fractions were prepared as
described (Schroter et
al. 1999).
A) Detection of marker proteins which are characteristic of certain
compartments
~s Aliquots (in each case 30~g) of the fractions were loaded onto a 12% SDS-
polyacrylamide gel
and the separated proteins transferred onto a PVDF membrane (Poly Screen, New
Life Science).
To show the concentration of membranes of the endoplasmatic reticulum in the
Mi fraction an
Anti-PDI antibody (Stressgen Biotechnologies; 1:2000) was used. As a
comparison the SIII
fraction of the first microsomal fractionation was also added. PDI is
concentrated in the
2o microsomal fraction. The distribution of lysosomal membranes in the
fractions was shown using
Anti-CathepsinD antibodies (Transduction Laboratories Inc.; 1:1000). The
microsomal fraction
is free from lysosomal proteins. As a comparison, the PNS of the H4indLC99
cells was also
added.
B) Celt-free formation of AI3 in the microsomal fraction
2s Microsomes (Mi-fraction) were prepared as described and incubated at a pH
of 6.8 under
standard conditions (pH 6.8; 5 mM EDTA; 4 hours).
A13 peptides were with the specific antibodies BL40 and BL42
immunoprecipitated and detected
by Western blot with the antibodies 6E10 and 4G8 (Senetek, Great Britain;
Galli et al., 1998) as
described. The incubations at 37°C were carried out in duplicate. The
formation of A13 takes
3a place in the microsomal fraction, as well as in the endosomal fraction.
Detailed description of the invention
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8
The problem of providing an improved test system is solved by the present
invention within the
scope of the specification and claims. According to the invention, an in vitro
test system for
discovering substances which are capable of specifically inhibiting y-
secretase is provided. In
another embodiment of the invention a test kit for discovering substances
which are capable of
s specifically inhibiting y-secretase is disclosed. Another embodiment of the
invention relates to
the use of the process according to the invention or the test kit according to
the invention for
discovering substances which are capable of specifically inhibiting y-
secretase. Moreover,
substances are prepared which can be found using the process according to the
invention or the
test kit according to the invention . Another embodiment relates to the use of
these substances
for preparing a medicament for treating neurodegenerative disorders and
pharmaceutical
formulations which contain these substances.
The process according to the invention uses purified membranes which are
isolated from cells
which detectably express a substrate of y-secretase and have a y-secretase
activity. By y-secretase
is meant, within the scope of this invention, a protein which has the property
of proteolytically
~s cleaving APP or fragments thereof, particularly the C99 peptide (Shoji et
al., 1992), in p3 or
A(3. Thus, any cells in which the skilled man can detect a substrate of y-
secretase or the cleavage
products thereof by Western Blot and the use of specific antibodies are
suitable for the process
according to the invention. Suitable membranes are any membranes in which the
skilled man can
detect a substrate of y-secretase by Western Blot and the use of specific
antibodies, preferably
lysosomal and endosomal membranes, most preferably microsomal membranes.
Methods of
specifically purifying membranes are known to the skilled man from Methods in
Enzymology,
Vol. 219, title: Reconstitution of intracellular transport, and the book
"Biochemische
Arbeitsmethoden" [Biochemical working methods], T.G. Cooper, published by De
Gruyter,
1981. In order to carry out the process according to the invention, cells can
be transfected with a
2s DNA sequence (DNA = deoxyribonucleic acid) which codes for a substrate of y-
secretase, as
described in a non-limiting embodiment in the example. In one embodiment by
way of example,
the expression of this substrate can be induced by the removal of doxycycline.
The cells can be
opened and a post nuclear supernatant (PNS) can be prepared, which is worked
up further in
order to isolate the microsomal fraction, for example. This fraction can be
incubated under
3o suitable conditions and the formation of the product of the reaction of y-
secretase with a suitable
substrate can be determined by immunoprecipitation and subsequent detection
using suitable
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9
antibodies by the Western Blot method. The y-secretase substrate was found in
the PNS and in a
higher concentration in the microsomal fraction (Fig. 1A). The C-terminal
fragments (CTF) of
PS1 and PS2 were also concentrated in the SIII fraction (Fig. 1B). The
microsomal fraction
contained no endosomal membranes, but ER and Golgi compartments were
concentrated (Fig.
s 1C). A~i generated de novo was found in the microsomal fractions which had
been incubated at
37 °C (Fig. 2A). Incubation at 4 °C prevented the de novo
formation of A(3. Small amounts of
A~3 were the result of the existence of intracellular A(3 in the freshly
prepared microsomal
fractions (Fig. 2A, trace c). The de novo formation of A~i was dependent on
time and reached its
peak after 3 to 4 hours' incubation (Fig. 2B). However, an evaluation of the
substrate/ product
equation showed that the in vitro generation of A(3 was not an efficient
proteolytic reaction (Fig.
2A). The incubation of the microsomal membranes in the absence of EDTA led to
a dramatic
reduction in the in vitro generation of A(3 (Fig. 3). The calcium chelator
BAPTA had the same
effect. This leads to the assumption that a Ca2+-dependent protease present in
the same fraction
breaks down A(3. The treatment of the microsomal membranes with 1 M KCl in the
buffer or
~s extraction of these membranes with 0.1 M NazC03 pH 11.5 before the y-
secretase test system is
carried out shows that the y-secretase has to be located on the membrane or at
least firmly bound
to the membrane (Fig. 4). In contrast to data published previously (Wolfe et
al., 1999) the
optimum pH determined for the y-secretase activity using this test system is
between 6.8 and 7.4
(Fig. 5). There was no in vitro generation of A(3 either at a more basic pH
(pH 8.0 to 8.5)or at a
?o more acid pH (pH 6.0 to 6.4).
In one embodiment according to the invention, the purified membranes
described, particularly
microsomal membranes, are mixed with a suitable reaction buffer and a test
substance. By test
substance is meant, for the purposes of the invention, any substance which is
to be tested to find
out whether it might have an inhibitory effect on the y-secretase activity.
Then the mixture is
2s incubated under conditions in which the substrate of the y-secretase is
cleaved in the absence of
the test substance. The quantity of the cleavage product formed is then
determined and the value
obtained is compared with the value obtained in the absence of the test
substance. If the amount
of cleavage product formed in the presence of the test substance is less than
in the absence of the
test substance, the test substance inhibits the formation of the cleavage
product and the test
3o substance is an inhibitor of y-secretase. In the prior art, compounds are
identified, inter alia,
which are referred to as specific y-secretase inhibitors and had an inhibitory
effect on the
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CA 02381952 2002-02-12
secretion of A[340 and A[i42 without influencing the formation of A(3. The y-
secretase-test
system according to the invention can now advantageously be used to identify
and validate
specific y-secretase inhibitors and distinguish them from inhibitors that
prevent the secretion of
A(340 and A(342. A compound was tested using the test system according to the
invention as
s described in the Example. The compound A ([aS-(aR*, yR*, 8R*)] N-butyl-y-
hydroxy-oc-(1-
methylethyl)-8-[(4-methylpentyl)amino]cyclohexane hexanamide-hydrochloride;
cf. Example 20
of EP 778 266 A1 ) was prepared as described in Example 20 of European Patent
Application EP
778 266 A1 and exhibited a concentration-dependent inhibition of A[3
generation with an IC50
value of about 6 p,M (Fig. 6A). Similar results were obtained in a test system
which measures
secreted A(340 and A(342 in a quantitative ELISA, which is specific to both
A(i peptides (Steiner
et al., 1998). Here, an Astrocytoma cell line (U373) is used which
overexpresses wild-type
APP751 and secretes detectable amounts of both species of A(3. A concentration-
dependent
reduction in the amount of extracellular A(340 and A(342 was observed after
overnight treatment
with compound A (Fig. 6B).
~s In one embodiment of the invention, cells are cultivated which express an
endogenous
polypeptide which is a substrate of y-secretase. The term endogenous within
the scope of this
invention means that this cell or cell line expresses the polypeptide referred
to in a sufficient
quantity without any need for further manipulation, e.g. by genetic
engineering methods .
Within the scope of the invention a sufficient quantity of substrate of y-
secretase denotes a
quantity which will produce a detectable signal standing out from the
background in an
established biochemical method of detection (e.g. ELISA, Western Blot) using
specific
antibodies.
In a particularly preferred embodiment of the invention cells are cultivated
which express an
exogenous polypeptide which is a substrate of y-secretase. The term exogenous
within the scope
25 of this invention means that this cell or cell line is manipulated by
genetic engineering methods
so that it expresses the y-secretase substrate. If the cell or cell line
contains the Y-secretase
substrate even without these manipulations, this term means that the quantity
of y-secretase
substrate is measurably increased compared with the value without any
manipulation. In order to
prepare a cell which exogenously expresses the y-secretase substrate a
nucleotide sequence
3o which codes for the amino acid sequence of the y-secretase substrate can be
inserted in a suitable
expression cassette of a euka.ryotic expression vector. Suitable expression
cassettes have a
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CA 02381952 2002-02-12
11
promoter which is functional in eukaryotic hosts such as, for example, the
cytomegalovirus
promoter (CMV promoter) and a functional polyadenylation signal, e.g. from the
SV40 virus
(Simian Virus; abbr. SV). Suitable expression vectors are vectors which can
replicate in
eukaryotic hosts, i.e. have a functional origin of replication. These
expression vectors may be
present episomally after the transfection or may be integrated into the genome
if they carry
suitable sequences that make integration possible.
In a preferred embodiment, an expression system is used that makes it possible
to induce the
expression of the exogenous polypeptide; various systems may be used here such
as e.g. the
"Tet-on"- or "Tet-ofd' system (US-Patent 5,464,758; Gossen and Bujard, 1992,
1995, marketed
by Clontech, Heidelberg) or the LacSwitch system (see U.S. Patent. 5,589,392;
sold by
Stratagene). In a particularly preferred embodiment of the invention the
expression of the
y-secretase substrate is induced by the removal of tetracycline or doxycycline
("Tet-OfF'
system). To do this the nucleotide sequence which codes the amino acid
sequence of the
!s y-secretase substrate is cloned behind at least one binding site of the
tetracycline repressor. In
addition, another nucleotide sequence can be found on the same plasmid,
another plasmid or
chromosomally integrated, coding for a fusion protein between the Tet
repressor and an acidic,
activating domain which is constitutively expressible. By an acidic,
activating domain is meant a
protein domain which has a high proportion of acidic amino acids and has the
capacity to
mediate the transcription of a gene when the domain is placed in a suitable
position in the
transcription complex located in front of the gene. This capacity can be
determined by the
known "one-hybrid assay". In this method, a DNA-binding domain is fused with a
domain which
is to be investigated, the DNA-binding domain binding to a sequence located in
front of a
reporter protein, and the activity of said reporter protein being measured
(Clontech, Heidelberg).
z5 In the case described above the expression of the y-secretase substrate
will not take place if the
concentration of tetracycline or a tetracycline derivative such as e.g.
doxycycline in the cell
exceeds a certain level, as the tetracycline repressor binds tetracycline or
the derivative thereof,
and consequently does not bind to its binding site in the DNA and therefore
does not induce the
transcription of the gene inserted behind this binding site which codes for
the y-secretase
3o substrate. In the absence of tetracycline or a derivative thereof the
fusion protein between Tet
repressor and the acidic activating domain binds to its DNA binding site and
induces the
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12
transcription of the gene inserted behind it which codes for the y-secretase
substrate. This
ensures that controlled and targeted expression of the y-secretase substrate
takes place.
In one embodiment of the invention the y-secretase substrate is the amyloid
precursor protein
(APP) or a fragment thereof provided that it contains the y-secretase cutting
site. In a preferred
s embodiment of the invention the y-secretase substrate is the C99 fragment of
the amyloid
precursor protein (Shoji et al., 1992).
The y-secretase substrate may be generally membrane-associated, but is
preferably membrane-
based. By membrane-based is meant, within the scope of this invention, that
the substrate is an
integral part of the membrane. By membrane-associated is meant within the
scope of this
to invention that the substrate is bound to the surface of the membrane or to
integral membrane
proteins. This definition of membrane-associated substrates is also intended
to cover substrates
which interact with the hydrophobic part of the membrane via chemical
groupings added on by
post-translational modifications. Moreover, the term membrane-associated
substrate is also
intended to include substrates which interact with the hydrophobic part of the
membrane via
!s amino acid side chains, albeit to a lesser extent than the integral
membrane proteins.
Prostaglandin synthetase may be mentioned here by way of example.
In another embodiment of the invention the y-secretase substrate is a fusion
protein of a reporter
protein with the amyloid precursor protein or a fragment thereof provided that
it contains the y-
secretase cutting site. In a preferred embodiment the y-secretase substrate is
a fusion protein
2o between a reporter protein and the C99 fragment. For the purposes of this
invention a reporter
protein is a protein which has the ability to generate an easily detectable
signal and correlates the
quantity thereof with the quantity of the cleavage product in question. The
signal is generated
either by determining the enzymatic activity of the reporter protein with
easily detected
substrates or by measuring the intensity of the fluorescence of the reporter
protein. Examples of
~s reporter proteins are green fluorescent protein (GFP; c~ e.g. W095/07463)
or derivatives
thereof that fluoresce at different wavelengths or enzymes such as luciferase,
secretory alkaline
phosphatase or (3-galactosidase. Tn this embodiment of the invention the
fusion protein of the
cleavage product with the reporter protein is separated from the fusion
protein of the uncleaved
y-secretase substrate with the reporter protein by immunoprecipitation, for
example. This may be
3o carried out with antibodies that selectively recognise the cleavage
product. Then the quantity of
reporter protein is determined by the processes mentioned above which are
independent of its
m m~-r~ r
CA 02381952 2002-02-12
13
qualities. The fusion proteins referred to may be prepared by current genetic
engineering
methods (Sambrook et al., 1989) if the DNA coding for the reporter protein and
the y-secretase
substrate is available. The DNA which codes for the reporter proteins may be
obtained, for
example, from commercial suppliers such as Clontech of Heidelberg, and
inserted into the
s desired vectors by standard methods (Sambrook et al., 1989). The DNA which
codes for the y-
secretase substrate or for the C99 fragment can be obtained from suitable gene
banks using
standard methods (Sambrook et al., 1989).
The invention may be performed using any cells or cell lines known to the
skilled person,
particularly eukaryotic cells or cell lines. Cells or cell lines used in
neurological or
neurobiological research are preferred, e.g. mammalian cells or cell lines
such as H4, U373,
NT2, HEK 293, PC 12, COS, CHO, fibroblasts, myeloma cells, neuroblastoma
cells, hybridoma
cells, oocytes, embryonic stem cells. Also suitable are insect cell lines
(e.g. using Baculovirus
vectors such as pPbac or pMbac (Stratagene, La Jolla, CA)), yeast (e.g. using
yeast expression
vectors such as pYESHIS (Invitrogen, CA)), and fungi.
~s Cells or cell lines of neuronal or glial origin are particularly preferred.
In one particularly
preferred embodiment of the invention the cells used are H4 cells, human
neuroglioma cells
from the brain which are deposited under ATCC Number HTB-148 at the "American
Type
Culture Collection (ATCC)", in Manassas, Virginia, USA.
In a preferred embodiment of the invention purified cell membranes are used,
preferably
Zo intracellular membranes, most preferably purified lysosomal or endosomal
membranes.
Particularly preferably, microsomal membranes are used which are purified from
the cells used
by lysing the cells, removing the cell nuclei, purifying using sucrose density
gradients,
re-sedimenting by ultracentrifugation, homogenising, re-sedimenting by
ultracentrifugation and
re-homogenising. Standard methods of purifying membranes are described in
Methods in
25 Enzymology, Vol. 219, and in the book "Biochemische
Arbeitsmethoden"[Biochemical
Working Methods], T.G. Cooper, published by De Gruyter, 1981. Theoretically,
ultracentrifugation with gradients of sucrose, metrizamide, ficoll and
iodoxanol is suitable for
purifying the membrane.
In a preferred embodiment of the present invention the reaction buffer has a
pH value in the
3o range from 5-10, preferably in the range from 6.0 to 8.0, particularly
preferably from 6.8 to 7.4
and also contains at least one membrane stabiliser. By membrane stabiliser is
meant within the
scope of this invention a substance which prevents the aggregation of the
membranes. By
m m~-rc:n
CA 02381952 2002-02-12
14
membrane aggregation is meant within the scope of this invention the clumping
together or
aggregation and possible subsequent fusion of vesicles or liposomes or
multilamellar structures.
This may be determined by the experimentally measurable increase in turbidity
or light
diffraction of a solution or suspension being investigated, while the
properties of a substance in
s terms of its membrane-stabilising activity may also be determined in this
way. The membrane
stabiliser within the scope of this invention is preferably sucrose or
sorbitol, although the skilled
person may replace these with substances of equivalent activity. Preferably,
the concentration of
the membrane stabiliser in the reaction buffer is between 200 and 1000 mM,
preferably between
200 and 500 mM, most preferably between 200 and 300 mM.
to In a particularly preferred embodiment of the process according to the
invention the reaction
buffer additionally contains a complexing agent, preferably for divalent ions.
This may be, for
example, ethylene-diamine-tetraacetic acid (EDTA) or a salt thereof, 1,2-bis(o-
aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA) or a salt thereof or
ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) or a salt
thereof. The
m complexing agent is preferably in a concentration of 0.1 to 20 mM,
preferably 5 to 10 mM. The
term complexing agent denotes compounds which as ligands are capable of
forming complexes,
i.e. particularly complexing and masking metals, particularly divalent metal
ions. The term is
often used as a synonym for chelating agents. In the process according to the
invention other
complexing agents may also be used.
In one embodiment of the invention an ATP-regenerating system is also added to
the reaction
mixture. This system contains adenosine triphosphate (ATP), guanosine
triphosphate (GTP),
phosphocreatine, creative phosphokinase, preferably in a concentration of 1 mM
ATP, 0.1 mM
GTP, 8 mM phosphocreatine, 31 mM creatinephosphokinase at a pH value in the
neutral range,
preferably between 6.8 and 7.2, most preferably at a pH of 7Ø
?s In a preferred embodiment of the invention the quantity of the cleavage
product is determined by
immunoprecipitation or Western Blot, preferably by a combination of
immunoprecipitation and
Western Blot. The immunoprecipitation and the Western Blot are carried out as
described by Ida
et al. (1996). By Western Blot is meant a method in which proteins are
separated by gel
electrophoresis, usually polyacrylamide gel electrophoresis, according to
their charge in their
3o native or usually in their denatured state, transferred onto carriers such
as e.g. nitrocellulose or
polyvinylidene difluoride and then detected using antibodies. In another
embodiment of the
1/ 11 U1-Y(_;1
CA 02381952 2002-02-12
invention the quantity of the cleavage product is determined by enzyme
immunoassay (enzyme-
linked immunosorbent assay; abbr.: ELISA) (Steiner et al., 1999) or by mass
spectrometry.
In another embodiment of the invention the quantity of the cleavage product is
determined by
measuring the quantity of reporter protein fused to the cleavage product,
after it has been
purified of the fusion protein between the uncleaved y-secretase substrate and
reporter protein.
The quantity of luciferase fused to the cleavage product, secretory alkaline
phosphata.se or [3-
galactosidase is determined by measuring the enzymatic activity of the
reporter protein after the
addition of substrate. In another embodiment of the invention the amount of
green fluorescent
protein or a derivative thereof is determined by measuring the intensity of
the fluorescent light.
A preferred embodiment of the invention is a test kit according to the
invention for finding
substances which are capable of specifically inhibiting y-secretase. A test
kit is a collection of all
the components for the process according to the invention. Some far from
exhaustive examples
of other elements for carrying out the process according to the invention are
containers such as,
1s for example, 96-well plates or microtitre plates, test tubes, other
suitable vessels, surfaces and
substrates, membranes such as nitrocellulose filters, washing reagents and
buffers or the like.
Similarly, a test kit may contain reagents which may have bound antibodies
such as e.g. labelled
secondary antibodies, chromophores, enzymes (e.g. conjugated to antibodies)
and the substrates
thereof or other substances capable of binding antibodies. The test kit
according to the invention
2o contains at least purified cell membranes from cells which exhibit y-
secretase activity and
contain a substrate of y-secretase, and reaction buffer. In a preferred
embodiment the membranes
are purified intracellular membranes, preferably lysosomal or endosomal, most
preferably
microsomal membranes. Most preferably, the membranes have been purified from
cells which
exogenously express a substrate of y-secretase. In a preferred embodiment of
the invention the
~s reaction buffer has a pH in the range from 5-10, preferably in the range
from 6.0-8.0, most
preferably in the range from 6.8 to 7.4, and contains as other components at
least one membrane
stabiliser according to the invention as described above. Preferably, the
concentration of the
membrane stabiliser, which is preferably sucrose or sorbitol, in the reaction
buffer is between
200 and 1000 mM, preferably between 200 and 500 mM, most preferably between
200 and 300
so mM.
Most preferably, the reaction buffer additionally contains a complexing agent
according to the
invention, preferably for divalent ions. This may be as described above, e.g.
EDTA, BAPTA or
m m~-rc.; r
CA 02381952 2002-02-12
16
EGTA or a salt thereof. The complexing agent is present in a concentration of
0.1 to 20 mM,
preferably 5 to 10 mM.
In one embodiment of the invention the test kit additionally contains an ATP-
regenerating
system according to the invention as described above, which can be added to
the reaction
s mixture. In another embodiment of the invention the test kit contains
antibodies which make it
possible to determine the quantity of cleavage product by immunoprecipitation
or Western Blot,
preferably by a combination of immunoprecipitation and Western Blot.
In a preferred embodiment of the invention the process according to the
invention or the test kit
according to the invention is used to find substances which are capable of
specifically inhibiting
y-secretase. In another embodiment a substance is prepared which can be found
using the
process according to the invention or the test kit according to the invention
and which
specifically inhibits the proteolytic cleaving of a y-secretase substrate. The
substance according
to the invention may be used for preparing a medicament for treating
neurodegenerative
disorders, particularly Alzheimer's disease. In addition, pharmaceutical
formulations are
~s prepared which contain a substance according to the invention and a
pharmaceutically acceptable
carrier.
A pharmaceutically acceptable carrier may contain physiologically acceptable
compounds which
increase the stability or absorption of the substance according to the
invention, for example.
2o Such physiologically acceptable compounds contain e.g. carbohydrates such
as glucose, sucrose
or dextrane, antioxidants such as ascorbate or glutathione, chelating agents,
proteins with a low
molecular weight or other stabilisers (cf. e.g. Remington's Pharmaceutical
Sciences (1990)).
Anyone skilled in the art knows that the choice of a pharmaceutically
acceptable carrier
including a physiologically acceptable compound depends for example on the
route of
~s administration.
a i m~-rc: n
CA 02381952 2002-02-12
17
Example 1- Using the test system:
1.1 Preparation of a suitable cell line
H4 Neuroglioma cells (Accession number HTB 148 at the "American Type Culture
Collection",
Manassas, Virginia, USA) were transfected under standard conditions with the
regulator plasmid
pUHDlS-lneo (pUHDlS-1 with neomycin resistance gene), which carries the gene
for a
tetracycline repressible transactivator (Gossen and Bujard, 1992, 1995). By
transient transfection
experiments an individual clone was selected for the second stable
transfection with the APP-
LC99 construct, having a strictly regulated and strongly inducible transient
expression of a
m reporter gene (pUHDlO-3/ SEAP; SEAP: secretory alkaline phosphatase). To
prepare the APP-
LC99 construct a sequence which contains the N-terminal signal sequence and
the last 99 amino
acids of the APP (Shoji et al., 1992), was cloned into the tetracycline-
controlled expression
vector pUHD 10-3 via BamHI and SacII restriction cutting sites. This construct
was called
pUHDlO-3/APP-LC99. The cell clone obtained as described above was co-
transfected with 10
~s pg of pUHD 10-3/ APP-LC99 and 1 ~,g of pTK-Hyg (Clontech, Heidelberg; Gene
bank
accession number U40398) and selection was carried out using the Fugene
transfection system
made by Boehringer Mannheim in accordance with the manufacturer's
instructions. Individual
hygromycin-resistant cell clones were investigated for the inducible
expression of LC99 by the
removal of doxycycline and subsequent immunofluorescence and/or Western Blot
with an APP-
2o CTF-specific antibody. The selected clone was called H4-ind/ APP-LC99.
1.2 Working up a microsome fraction from H4 LC99 cells
The H4-ind/ APP-LC99 cells were grown until confluent on 15 cm Petri dishes at
37 °C at 5%
COZ with DMEM medium (DMEM: "Dulbecco's Modified Eagle Medium", sold by
~s BioWittacker) and 10% foetal calf serum (FCS), 1% glutamine, 1% penicillin
and streptomycin
in the absence of doxycycline. By the removal of doxycycline the expression of
the fusion
protein was induced. All the steps of preparing the postnuclear supernatant
were carried out on
ice or at 4°C. After the addition of 2 ml of PBS per Petri dish the
cells were removed from the
Petri dishes with a cell scraper. After centrifugation at 500 g for 10 min the
cells were carefully
3o resuspended in HIS buffer (250 mM sucrose, 5 mM imidazole, 10 mM HEPES pH
6.8),
centrifuged again at 1400 g for 10 min and then resuspended in 300 p1 of HIS+
buffer (HIS
buffer with S mM EDTA) per Petri dish. The homogenised cell material was
forced through a 22
m m~-rc: n
CA 02381952 2002-02-12
18
gauge needle using a 1 ml syringe and the lysing of the cells was monitored by
phase contrast
microscopy. The cell lysate was centrifuged at 2500 g for 10 min in order to
separate the
supernatant from the intact cells and the cell debris.
The PNS was worked up further with a sucrose step gradient (Taylor et al.,
1997), first adjusting
s it to 100 mM KHZP04/ KZHPO4, pH 6.8. All the sucrose solutions contained 100
mM KHzP04/
KzHP04, pH 6.8, 5 mM MgCl2 and the protease inhibitors leupeptin (10 ~g/ml)
and aprotinin
(10 ~g/ml). The gradient contained steps of 1.3 M, 0.86 M and 0.5 M sucrose,
which were
overlaid with the PNS and then centrifuged at 100,000 g for 1.5 hours at 2
°C. The intermediate
layer between 1.3 M and 0.86 M sucrose is the microsomal fraction SIII. In
order to pellet the
m membranes, the SIII fraction was adjusted to 250 mM sucrose with 100 mM
KHZP04/ KZHP04,
pH 6.8, and centrifuged at 220,000 g for 20 min at 4°C. The membranes
were washed with
reaction buffer (250 mM sucrose, 50 mM KCI, 2,5 mM magnesium acetate, 20 mM
HEPES, pH
6.8, 5 mM EDTA), centrifuged as described above and resuspended in 1 ml
reaction buffer until
homogeneous. Small aliquots of the microsomal fraction were frozen in liquid
nitrogen and
~s stored at -80 °C.
1.3 Using the y-secretase inhibitor test system
The frozen microsomal fraction of the induced H4-ind/APP-LC99 cells was thawed
on ice and
~l aliquots were used for each cell-free reaction . The samples were diluted
to 30 p,1 with
reaction buffer and incubated at specific temperatures and pH values and for
specific times.
After incubation the samples were adjusted to 2 % SDS and heated to 95
°C for 5 minutes. 1 ml
IP buffer (150 mM NaCI, 10 mM Tris pH 7.4, 1 mM EDTA, 0.2 % NP40 and the
protease
inhibitors aprotinin (10 ~,g/ ml), leupeptin (10 ~,g/ ml), 5 ~g/ml pepstatin,
1 mM Pefabloc) and
in each case 6 p,g/ ml of the specific antibodies BL40 and BL42 (alternative
antibodies with the
~s same effect can be obtained from QCB, Quality Control Biochemicals, Inc.,
Hopkinton, USA;
catalogue numbers 44-348 and 44-344) were added to the samples. After one hour
at 4°C , 20 ~,1
of prewashed Gammabind-Sepharose G beads (Pharmacia Biotech) were added and
incubated
overnight at 4°C. The sepharose immunocomplex was washed with IP buffer
and precipitated
proteins were eluted with 20 ml of Tris-Bicine (Klafki et al., 1996) sample
buffer. The samples
3o were separated by Tris-Bicine polyacrylamide gel electrophoresis as
described (Klafki et al.,
1996). The highly sensitive Western Blot method described earlier was used
with the antibodies
1I 11 Ul-Yl: l
CA 02381952 2002-02-12
19
6E10 and 4G8 (Product numbers mAb 200-10 and mAb 300-10, Senetek, Great
Britain; Galli et
al., 1998) in order to detect the immunoprecipitated A(3 species (Ida et al.,
1996).
Chemiluminescence was detected with the Western Star substrate (Tropix) and
quantified with a
chemiluminescence detection system supplied by BioRad.
1.4 Obtaining the cytosol from guinea pig liver cells
A postnuclear supernatant (PNS) is obtained from the liver of a guinea pig by
homogenisation
and centrifugation as described (Taylor et al., 1997). This supernatant is
applied to a sucrose
step gradient (Taylor et al., 1997) and centrifuged at 100,000 g and
4°C for 1.5 h. The fraction
with 500 mM sucrose is diluted with 1 x KPi buffer on 250 mM sucrose and
centrifuged at
200,000 g and 4°C for 20 minutes. The supernatant is the cytosol (Jones
et al., 1998).
1.5 Alternative method of the y-secretase inhibitor test system with an ATP-
regenerating system
A purified microsomal fraction of these recombinant H4 cells is incubated at
37 °C with a
suitable buffer system (50-150 mM KCI, 1.5-5 mM of magnesium acetate, 250 mM
sucrose, 20
mM Hepes pH 6.8), an ATP regenerating system ( 1 mM ATP, 0.1 mM GTP pH 7.0; 8
mM
phosphocreatine, 31 mM creatine phosphokinase) and cytosol, which has been
worked up as
described in 1.2, and then the y-secretase activity is measured by detecting
the product A13 by
Western Blot as described above.
Zo
Example - Using the test system
1.6 Alternative working up of a microsome fraction from H4 LC99 cells
The same cell line (H4 neuroglioma cell clone with APP-LC99 construct) as
described in 1. l
2s was used.
The H4-ind/ APP-LC99 cells were grown until confluent on 15 cm Petri dishes at
37 °C at 5%
COZ with DMEM medium (DMEM: "Dulbecco's Modified Eagle Medium", sold by
BioWittacker) and 10% foetal calf serum (FCS), 1% glutamine, 1% penicillin and
streptomycin
in the absence of doxycycline. By the removal of doxycycline the expression of
the fusion
3o protein was induced for three days. All the steps of preparing the
postnuclear supernatant were
carried out on ice or at 4°C. For a preparative batch 10 times 15 cm
Petri dishes were processed
together. After the addition of 2 ml of ice-cold PBS per Petri dish the cells
were removed from
iii m~-ran
CA 02381952 2002-02-12
the Petri dishes with a cell scraper. All the following steps were carried out
as described in
Schroter et al. After centrifugation at S00 g for 10 min the cells were
carefully resuspended in
ST buffer (250 mM sucrose, 10 mM Tris pH 7.4), centrifuged again at 1400xg for
10 min and
then all the cells were resuspended in 5 ml of ST buffer. The cells were
homogenised using a S
s ml Potter (Braun, Melsungen) at 500 rpm and the lysing of the cells was
monitored by phase
contrast microscopy. The cell lysate was first centrifuged at 2000xg for 2 min
to precipitate any
intact cells and large cell debris. Then the supernatant was centrifuged at
4000xg for 2 min to
separate offthe cell membranes and cell nuclei (fraction PN). The sediment
consisting of cell
nuclei and plasma membranes were washed twice with ST buffer and centrifuged
as described
above. The supernatants were combined and centrifuged in a new container at
100,000xg for 2
min in order to eliminate mitochondria, lysosomes and endosomes (fraction EL).
Finally, in
order to sediment the purified microsomes, the supernatant was centrifuged at
400,OOOxg. In
order to separate out the lysosomes and the endosomes the EL fraction was
processed further.
The lysosomes were burst open by a 10 minute hypotonic lysis on ice (Bohley et
al. 1969) and
~s then the intact endosomes were sedimented at 100,000xg for 2 min.
(Lysosomes = fraction L;
endosomes = fraction E).
To characterise the separation, 30~g of total protein from each fraction were
placed on a
polyacrylamide gel and the distribution of various marker proteins of
individual compartments
in the fractions was detected by the Western blot method.
2o The endosomal and microsomal membranes were taken up with reaction buffer
(250 mM
sucrose, 50 mM KCI, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, S mM EDTA)
and
resuspended in 1 ml reaction buffer until homogeneous. Small aliquots of the
different fractions
were frozen in liquid nitrogen and stored at -80 °C.
Then the y-secretase inhibitor test system was carried out exactly as
described in 1.3.
m m~-r~ n
Literature:
CA 02381952 2002-02-12
21
Bohley et al. (1969), FEBS Lett. 5,233-236
De Strooper.et al. (1998). Nature 391, 387-390.
s De Strooper et al. (1999). Nature 398, 518-522.
Galli et al. (1998). Proc. Natl. Acad. Sci. USA, 95, 1247-1252.
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Gossen, M., and Bujard, H. (1995). Biotechniques 19, 213-216.
Haass, C., and Selkoe, D.J. (1993). Cell 75, 1039-1042.
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~s Price, D., and Sisodoa, S. (1998). Ann. Rev. Neuroscience 21, 479-505.
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