From the Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
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INTRODUCTION |
Alzheimer's disease is characterized by the deposition of
-amyloid (A
)1 into
senile plaques, the formation of neurofibrillary tangles, and neuronal
death. Senile plaques consist of a A
protein surrounded by
dystrophic neuritic processes, astrocytes, and microglia. The
-amyloid protein is a 39-43-amino acid cleavage product of
-amyloid precursor proteins (APP) (1-4). APP is a family of
transmembrane glycoproteins with a large extracytoplasmic domain, a
membrane-spanning domain containing the A
peptide, and a short
intracytoplasmic domain (4). APP exists as three alternatively spliced
isoforms, ranging from 695 to 770 amino acids in length and is
expressed in mammalian neuronal and nonneuronal cells and tissues (5). APP is most abundant in the brain and APP695 is the major
APP in the human neuronal cell line NTera 2/cl.D1 (NT2) (6). APP is
thought to be processed through various pathways. APP processed by a
constitutive secretory pathway (or
-secretase) results in a large
secreted form (APP-S
) excluding A
formation (7, 8).
Processing by a
-secretase or by an endosomal and lysosomal pathway
produces the 4-kDa A
protein (9, 10). Mutations in the APP gene have
been shown to result in the abnormal processing of APP, which may lead
to subsequent A
deposition and accumulation (11, 12). Recent
evidence for this was provided by a human familial Alzheimer's disease
mutant APP transgenic mouse that expresses high levels of APP, as well
as A
, which increases in an age-dependent manner. This
results in the formation of amyloid plaques, dendritic and synaptic
loss, and astrocytosis, as seen in Alzheimer's disease (13, 14).
Mutations in the presenilin 1 and 2 genes (responsible for many cases
of familial Alzheimer's disease) alter APP processing by increasing
A
generation (15-18). This supports the idea of a primary role for
APP and A
in the pathogenesis of Alzheimer's disease.
Many populations of neurons are affected in Alzheimer's disease, in
particular the cholinergic and glutamatergic neurons of the hippocampus
and cerebral cortex, therefore it is important to examine these
receptor signaling pathways. The m1 and m3 muscarinic receptors have
been shown previously to stimulate the secretion of APP, and recently
the activation of metabotropic glutamate receptors was shown to cause
an increase in APP secretion (19, 20). Metabotropic glutamate receptors
are seven transmembrane-spanning proteins that consist of eight
receptor subtypes. The mGluR1 and R5 subtypes are linked to
phosphatidylinositol (PI) hydrolysis, whereas the other six subtypes
are negatively coupled to adenylate cyclase. The degeneration and loss
of neurons seen in Alzheimer's disease and a recent study
demonstrating the involvement of metabotropic glutamate receptors in
the regulation of APP secretion suggest that a defect in the receptors
or in their signaling pathways might lead to abnormal processing of APP
and subsequent A
deposition. The regulatory mechanism of APP
processing and secretion is unknown; however, recent findings suggest
that the control is through neurotransmitter receptor coupling to
signal transduction pathways (21).
In order to examine the glutamatergic signaling pathway, the human
teratocarcinoma cell line NTera 2/cl.D1 (NT2N) was used. NT2N cells are
post-mitotic, terminally differentiated neurons that possess cell
surface markers consistent with neurons of the central nervous system.
Pure cultures of neurons are obtained through differentiation of the
stem cell population with retinoic acid and treatment of the replated
neuronal population with mitotic inhibitors to clear any contaminating
nonneuronal cells. NT2N neurons provide a strong model for examining
APP secretion because they endogenously express APP and secrete both
APP and A
in response to physiological stimuli. In contrast to
non-neuronal cells, NT2N neurons express APP695, the major
APP isoform expressed in the brain, and NT2N neurons process APP
differently (6, 22). For this study, NT2N neurons were used to
determine if glutamate is involved in the regulation of APP
secretion.
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EXPERIMENTAL PROCEDURES |
Materials--
The media and serum used to maintain the NT2N
culture were from Life Technologies, Inc., and all of the mitotic
inhibitors and antibiotics used in the cell culture were purchased from
Sigma. Matrigel was from Collaborative Research (Bedford, MA). The DAG kinase and lipase inhibitors were from Biomol (Plymouth Meeting, PA),
and the glutamate receptor agonists and antagonists were purchased from
Tocris Cookson (Ballwin, MO). The phorbol ester PMA was from Sigma and
the calcium ionophore A23187 was from Boehringer Mannheim.
[35S]Methionine (1000-1500 Ci/mmol) was purchased from
ICN Biomedicals (Costa Mesa, CA) and
myo-[3H]inositol (84 Ci/mmol),
[3H]inositol 1-phosphate (10 Ci/mmol),
[3H]inositol 1,4-bisphosphate (10 Ci/mmol),
[3H]inositol 1,4,5-trisphosphate (21 Ci/mmol), and
[3H]inositol 1,3,4,5-tetrakisphosphate (21 Ci/mmol) were
from NEN Life Science Products. Trichloroacetic acid,
trichlorotrifluorethane, and trioctylamine used in the inositol
extraction procedure were also from Sigma. SAX Amprep minicolumns were
obtained from Amersham Pharmacia Biotech. RNA isolation kit was from
CLONTECH, RT-PCR reagents were from Promega
(Madison, WI), and the mGluR1 primers were from the Wistar Institute
(University of Pennsylvania, Philadelphia, PA), and mGluR5 primers were
from National Biosciences Inc. (Beverly, MA).
Cell Culture of NT2N Neurons--
The human teratocarcinoma
NTera2/c1.D1 (NT2) cells were maintained in Opti-MEM supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin (23). 2.3 × 106 cells were seeded into a 75-cm2 flask
and treated with 10 µM retinoic acid (in DMEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin) twice a week
for 5 weeks. Cells were then trypsinized and replated (Replate 2) onto
poly-D-lysine/Matrigel (Collaborative Research)-coated 10-cm dishes or six-well plates. Cells were fed once a week with DMEM
medium supplemented with 5% fetal bovine serum, 1%
penicillin/streptomycin, and mitotic inhibitors (1 mM
cytosine arabinoside, 10 mM fluorodeoxyuridine, 10 mM uridine) for 4 weeks. This yielded a >95% pure culture
of differentiated human neurons (NT2N) that could be maintained in culture for 7-8 weeks.
Pharmacological Treatment of NT2N Neurons--
NT2N 10-cm dishes
or six-well plates (240-480 ng of DNA) were washed three times in
Krebs-Hepes buffer (25 mM Hepes, pH 7.4, 115 mM
NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% bovine serum albumin, 3 mM D-glucose),
preincubated 30 min under an atmosphere of 95% O2/5%
CO2 at 37 °C, and then incubated for the appropriate
time with agonists (glutamate, ACPD, quisqualate, CCG-I, PMA, A23187,
OAG). To examine the effect of the glutamate receptor antagonists
(CNQX, AP5, AIDA, MPPG, MSOPPE) and DAG lipase and kinase inhibitors
(RG80267, R59949), the cells were pretreated with the agents for 30 min
at 37 °C and then incubated for an additional 30 min alone or in
presence of glutamate.
Measurement of APP-S Secretion by Metabolic Labeling and
Immunoprecipitation--
In order to metabolically label APP, NT2N
cells were serum-starved for 20 min in DMEM methionine-free medium and
then labeled with 100-400 µCi/ml [35S]methionine in
DMEM methionine-free medium (1% penicillin/streptomycin, 5% fetal
bovine serum) for 3 h. The cells were then treated as described
above. After treatment, the supernatant was removed and centrifuged for
15 min at 15,000 × g to remove any remaining cells.
The supernatant was precleared with protein A-Sepharose beads and then
immunoprecipitated overnight with 10-20 µg of an anti-APP-S
polyclonal antibody (Karen) and protein A-Sepharose beads as described
previously (6). Immunoprecipitates were analyzed on 7.5% SDS-PAGE
mini-gels, and the radioactivity quantitated using Image Quant software
on a Molecular Dynamics PhosphorImager. For data analysis, the control
was designated as 100% within each experiment due the differences in
radioactive counts between the controls in separate experiments. The
glutamate-induced APP-S secretion was determined to be specific for APP
after demonstrating that glutamate did not have an effect on the amount
of total secreted proteins (data not shown).
RT-PCR Analysis of Metabotropic Glutamate Receptor Subtypes
mGluR1
a/
b and mGluR5
a in NT2N Cells--
60-90 µg of total
RNA was isolated from NT2N cells as described previously (24). Reverse
transcription was then performed as follows. For reverse transcription,
RNA was incubated at 65 °C for 5 min and then placed on ice for 10 min. For each reaction of 31 µl, less than 30 µg of total RNA was
added to the reaction mixture of: RNase inhibitor (40 units/ml), first
strand cDNA 5 × buffer, Moloney murine leukemia virus reverse
transcriptase (200 units/ml), bovine serum albumin (1 mg/ml), 0.1 M dithiothreitol, and oligo(dT) (1.5 µg). The reverse
transcription reaction was performed in a thermal cycler for 60 min at
42 °C, followed by a 10-min incubation at 72 °C. The cDNA
generated was then amplified in a 50-µl PCR using 8.5 µl of the
cDNA, 10 × PCR buffer, 1 µl of dNTP mix (10 mM
each of dATP, dCTP, dGTP, dTTP), 0.25 µl of Taq polymerase
(5 units/µl), and 2.5 µl of each specific primer (20 µM each) (designed from the published sequence of human
mGluR1 (5' CTGCATGTTCACTCCCAAGATGTACAT, 3' CACGCGCCTGTGCACCACCATGGAAG) and mGluR5 (5' TGTGCCCAGCTAGTGATTGC, 3' TGCTCTTCTCATTCTGGGC) (25, 26). The reaction mixtures were layered with mineral oil, placed in the
thermal cycler, and programmed for 2 min at 94 °C, followed by 40 cycles at 94 °C for 1 min, 60 °C for 2 min, 72 °C for 3 min,
and then a final 7 min at 72 °C. The tubes were placed at 4 °C
until the PCR products were analyzed for purity and size on a 1.5%
agarose gels. The products were excised from the gels and purified with
the Geneclean II kit and the purified products subjected to a
restriction enzyme digest with EcoRI for the identification of mGluR1
/
and BglI for mGluR5
.
Measurement of IP3 Accumulation--
NT2N cells were
labeled for 48 h with 10 µCi/dish of
myo-[3H]inositol, washed three times with
Krebs-Hepes buffer (25 mM Hepes, pH 7.4, 115 mM
NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2,
0.1% bovine serum albumin, 3 mM D-glucose, 10 mM LiCl), preincubated 30 min under an atmosphere of 95%
O2, 5% CO2 at 37 °C, and then incubated for
2 min with fresh Krebs-Hepes buffer ± 1 mM glutamate.
Inositol phosphates were extracted with 0.5 ml of a 1:5 trichloroacetic
acid solution and then neutralized with 1 ml of a 3:1 solution of
trichlorotrifluoroethane/trioctylamine. The aqueous layers, as well as
commercially available inositol phosphates for standardization of the
columns, were loaded onto pre-equilibrated Amprep SAX minicolumns and
the inositol phosphate adducts (inositol 1-phosphate (IP), inositol
1,4-bisphosphate (IP2), inositol 1,4,5-trisphosphate
(IP3), inositol 1,3,4,5-tetrakisphosphate (IP4)) eluted with a step gradient of 5 ml each of 0.05, 0.10, 0.16, and 0.17 M potassium bicarbonate
(KHCO3). The fractions were collected and counted in a
Wallac scintillation counter.
Cytosolic Free Ca2+ Measurement--
Cells were
loaded with fura-2 during a 40-min incubation at 37 °C in 2 ml of
Krebs-Hepes buffer (115 mM NaCl, 24 mM
NaHCO3, 5 mM KCl, 1 mM
MgCl2, 2.5 mM CaCl2, 25 mM glucose, and 25 mM Hepes, pH 7.40)
supplemented with 2.5 µM fura-2 acetoxymethylester
(Molecular Probes, Eugene, OR) and 0.2 mg/ml pluronic F-127 (Molecular
Probes), which was used to increase the loading. The coverslip with the loaded cells was then mounted in a perifusion chamber placed on the
homeothermic platform of an inverted Zeiss microscope. The cells were
superfused with Krebs-Hepes buffer at 37 °C at a flow rate of 1.5 ml/min. The microscope was used with a × 40 oil objective. Fura-2
was successively excited at 334 and 380 nm by means of two narrow
band-pass filters. The emitted fluorescence was filtered through a
520-nm filter, captured with an Attofluor CCD video camera at a
resolution of 512 × 480 pixels, digitized into 256 gray levels,
and analyzed with version 6.00 of the Attofluor RatioVision software
(Atto Instrument, Rockville, MD). The concentration of Ca2+
was calculated by comparing the ratio of fluorescence at each pixel to
an in vitro 2-point calibration curve. The Ca2+
concentration is presented by averaging the values of all pixels in the
body of a differentiated NT2N cell (neuron soma), excluding the
Ca2+ data of neurites. Data points were collected at an
interval of 4.5 s.
Data Analysis--
Student's t test was performed
when two groups were compared. Analysis of variance was used, followed
by the Student-Newman-Keuls method when multiple groups were compared.
In cases where the data did not have a normal distribution,
Kruskal-Wallis one-way analysis of variance on ranks was used, followed
by Dunn's method of multiple comparison. Differences were considered
significant for p < 0.05.
 |
RESULTS |
Glutamate Stimulates APP-S Secretion from NT2N Neurons in a Time-
and Dose-dependent Manner--
NT2N neurons were
metabolically labeled with [35S]methionine and then
stimulated with 1 mM glutamate for 0-90 min or various concentrations of glutamate for 30 min. APP-S was immunoprecipitated from the medium and the proteins separated on 7.5% SDS-PAGE gels. Fig.
1 (top panel) shows the
resulting APP-S bands. The lower APP-S band is the 695 form, while the
higher APP-S band is the 751 and/or 770 form, the result of any
remaining non-neuronal cells within the culture. Glutamate treatment
resulted in a 2-fold increase in APP-s secretion between 5 and 30 min,
with maximal secretion at 30 min (p < 0.05 versus control) (Fig. 1, bottom panel). Between
30 and 90 min, glutamate-induced secretion decreased to the control
level of secretion. Glutamate-induced APP-S secretion was
dose-dependent (Fig. 2,
bottom panel). Fig. 2 (top panel) shows a
representative gel depicting an increase in APP-S695
secretion with increasing concentrations of glutamate. Glutamate also
caused a rapid and sustained increase in cytosolic Ca2+
(Fig. 3, top panel). Likewise,
the glutamate-induced increase in cytosolic Ca2+ was also
dose-dependent (Fig. 3, bottom panel).

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Fig. 1.
Effect of time on glutamate-induced APP-S
secretion from NT2N neurons. Cells were labeled for 3 h with
[35S]methionine and then treated with ± 1 mM glutamate for varying times. APP was immunoprecipitated
from the medium and the proteins separated on 7.5% SDS-PAGE gels. The
gels were dried and quantitated on a PhosphorImager. Top
panel, representative gel showing the 110-kDa APP-S695
band after treatment with ± 1 mM glutamate.
Bottom panel, APP-S secretion after treatment with ± 1 mM glutamate for 5-90 min. APP-S secretion was quantitated
by PhosphorImager as the amount of radioactivity in the APP-S band and
is expressed as a percentage of the control within each experiment.
Results are shown as the mean ± S.E. of APP-s secretion from two
to four separate observations/condition.
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Fig. 2.
Effect of glutamate concentration on APP-S
secretion in NT2N neurons. Cells were labeled for 3 h with
[35S]methionine as in Fig. 1 and stimulated with
increasing concentrations of glutamate for 30 min. Top
panel, representative gel showing the 110-kDa APP-S band after
treatment with glutamate. Bottom panel, APP-S secretion
after treatment with 0, 100 nM, 1 µM, 10 µM, 100 µM, and 1 mM glutamate.
APP-S secretion was quantitated by PhosphorImager analysis as the
amount of radioactivity in the APP-S band and is expressed as a
percentage of the control within each experiment. Results are shown as
the mean ± S.E. of APP-S secretion from three separate
observations/condition.
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Fig. 3.
Time course and dose dependence of glutamate
effects on cytosolic Ca2+ in NT2N neurons. NT2N
neurons were loaded with fura-2 before they were perifused with
Krebs-Hepes buffer. The cells were excited by dual wavelength (334 and
380 nm), and the emission light was filtered at 520 nm before it was
captured by a CCD camera. Ca2+ concentrations are
calculated from the ratio of the intensities obtained at the two
excitation wavelengths. Results are shown as the mean ± S.E. from
at least 30 cells in three different experiments. Top panel,
time course of glutamate; bottom panel, dose curve of
glutamate.
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Metabotropic Glutamate Receptor Agonists Stimulate APP-S Secretion
from NT2N Neurons--
Glutamate receptors can be categorized into two
distinct groups, ionotropic and metabotropic receptors (27, 28).
Ionotropic receptors are selectively permeable ion channels and are
subdivided into three groups according to agonist selectivity, NMDA,
kainate, and AMPA receptors. The metabotropic receptors are seven
transmembrane-spanning proteins coupled to intracellular second
messengers. The metabotropic receptor family consists of at least eight
different subtypes that are subdivided into three groups according to
sequence similarities, agonist selectivity, and intracellular signaling
machinery. Therefore, the effects of both metabotropic and ionotropic
glutamate receptor agonists and antagonists were examined in NT2N
cells. The effects of both metabotropic and ionotropic glutamate
receptor agonists on APP-S secretion are shown in Table
I. The ionotropic glutamate receptor
agonist NMDA (10 µM) did not have any effect on APP-S secretion; however, the non-NMDA glutamate receptor agonists AMPA (100 µM) and kainate (100 µM) caused a 1.5-fold
and a 2-fold increase, respectively, in APP-S secretion. AMPA and
kainate also caused an increase in intracellular Ca2+ (Fig.
4). The nonspecific glutamate receptor
agonist quisqualate (100 µM) had a slight effect on APP-S
secretion; however, the specific metabotropic glutamate receptor group
I and II agonists, ACPD (100 µM) and CCG-I (100 µM) both resulted in a 1.5-fold increase in APP-S
secretion, suggesting the glutamate-induced APP-S secretion is mediated
in part by metabotropic glutamate receptors.
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Table I
Effect of glutamate receptor agonists on APP-S secretion in NT2N cells
Cells were labeled for 3 h with [35S]methionine as in
Fig. 1 and then treated with various of glutamate receptor agonists for
30 min. APP-S secretion was quantitated by PhosphorImager analysis as
the amount of radioactivity in the APP-S band and is expressed as a
percentage of the control within each experiment. Results are shown as
the mean ± S.E. of APP-S secretion from 4 to 13 separate
observations/condition.
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Fig. 4.
Effect of glutamate receptor agonists on
cytosolic Ca2+ in NT2N neurons. Cytosolic
Ca2+ was measured as in Fig. 3.
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Metabotropic Glutamate Receptor Antagonists Reduce
Glutamate-induced APP-S Secretion in NT2N Cells--
The metabotropic
glutamate receptor antagonists AIDA (100 µM, Group I),
MSOPPE (100 µM, Group II), and MPPG (100 µM, Group III) decreased glutamate-induced APP-S
secretion by 17, 27, and 24%, respectively, while NMDA receptor
antagonists, CNQX (10 µM) and AP5 (10 µM),
did not have any effect on glutamate-induced APP-S secretion (Table
II) (29-31). These results suggest that glutamate-induced APP-S secretion is mediated in part by metabotropic glutamate receptors.
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Table II
Effect of glutamate receptor antagonists on APP-S secretion in NT2N
cells
Cells were labeled for 3 h with [35S]methionine as in
Fig. 1 and then pretreated for 30 min with various glutamate receptor
antagonists. The cells were then treated for 30 min with the control, 1 mM glutamate, the antagonist alone, or 1 mM
glutamate + the antagonist. APP-S secretion was quantitated by
PhosphorImager analysis as the amount of radioactivity in the APP-S
band and is expressed as a percentage of the control within each
experiment. Results are shown as the mean ± S.E. of APP-S
secretion from 3 to 14 separate observations/condition.
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Identification of Metabotropic Glutamate Receptor Subtypes
mGluR1
/
and mGluR5
in NT2N Neurons--
The metabotropic
glutamate receptor subtypes, mGluR1
/
and mGluR5
, were
identified in NT2N cells by RT-PCR analysis. For mGluR1
/
, RT-PCR
yielded the expected products of 463 and 547 bp, respectively (Fig.
5), while for mGluR5
, a PCR product of the expected size of 600 bp was obtained (Fig. 5). The products were
excised from the gels, purified with the Geneclean II kit, and
subjected to a restriction enzyme digest with EcoRI for the identification of mGluR1
/
and BglI for mGluR5
.
Restriction enzyme analysis yielded the expected size products for
mGluR1
/
(463, 356, and 191 bp) and mGluR5
(357 and 243 bp)
(data not shown). These results indicate that the metabotropic
glutamate receptor subtypes mGluR1
/
and mGluR5
are present in
NT2N neurons.

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Fig. 5.
Identification of metabotropic glutamate
receptors in NT2N neurons. RT-PCR was performed with specific
primers designed for the amplification of mGluR1 and mGluR5. PCR
products were analyzed on a 1.5% agarose gel. Lane 1, DNA
ladder; lane 2, 463- and 548-bp PCR products for mGluR1
and , respectively, lane 3, DNA ladder; lane
4, 604-bp PCR product for mGluR5 .
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Glutamate Stimulates the Rapid Accumulation of Inositol
1,4,5-Trisphosphate in NT2N Neurons--
NT2N neurons were labeled for
48 h with myo-[3H]inositol and then
stimulated ± 1 mM glutamate for 2 min. The inositol
phosphates were extracted and purified by strong anion exchange
chromatography. Glutamate caused an increase in inositol
1,4,5-trisphosphate at 2 min in NT2N neurons (p < 0.05 versus control) (Fig. 6). This suggests that glutamate activates the PI-linked metabotropic glutamate receptors resulting in the activation of phospholipase C and the subsequent increase in IP3.

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Fig. 6.
Effect of glutamate on IP3
accumulation in NT2N neurons. NT2N neurons were labeled for
48 h with myo-[3H]inositol and then
treated with ± 1 mM glutamate. Inositol phosphates
were extracted and purified by strong anion exchange chromatography.
[3H]IP3 fractions were quantitated.
IP3 accumulation was expressed as a percentage of the
control within each experiment. Results are shown as the mean ± S.E. of IP3 accumulation from nine separate
observations/ condition.
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Increases in Calcium and Diacylglycerol Increase APP-S
Secretion--
Because glutamate-induced APP-S secretion involves the
phospholipase C pathway, the contribution of downstream signaling
events was next examined. NT2N neurons were treated with the phorbol ester PMA, the cell-permeable DAG analog OAG, the calcium ionophore A23187, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, the DAG kinase inhibitor R59949, or the DAG
lipase inhibitor RG80267. Both PMA and OAG caused a robust increase in
APP-S secretion (p < 0.05 versus control),
comparable with that of glutamate (Fig. 7). These results suggest that protein
kinase C is involved in the secretion of APP-S. Furthermore, the DAG
kinase inhibitor R59949, which in other systems increases DAG levels,
caused a large increase in the secretion of APP (Table
III) (32). However, DAG metabolism does
not seem to be involved in APP-S secretion, since the DAG lipase
inhibitor RG80267 did not have any effect on glutamate-induced APP-S
secretion (Table III). The calcium ionophore A23187 also caused an
increase in APP-S secretion (3.5-fold), and thapsigargin caused a
slight decrease in glutamate-induced APP-S secretion. These results
suggest that both calcium and DAG activation of protein kinase C
mediate glutamate-induced APP-S secretion in NT2N neurons.

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Fig. 7.
Effect of phorbol ester PMA and
diacylglycerol analog OAG on APP-S secretion in NT2N neurons.
Neurons were labeled for 3 h with [35S]methionine as
in Fig. 1 and treated for 30 min with the control, 10 µM
PMA, or 200 µM OAG. Top panel, representative
gel showing the 110-kDa APP-S band. Bottom panel, APP-S
secretion after treatment with PMA, OAG, and glutamate. APP-S secretion
was quantitated by PhosphorImager as the amount of radioactivity in the
APP-S band and is expressed as a percentage of the control within each
experiment. Results are shown as the mean ± S.E. of APP-S
secretion from five to ten separate observations/condition.
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Table III
Effect of calcium and diacylglycerol metabolism on APP-S secretion in
NT2N cells
Cells were labeled for 3 h with [35S]methionine as in
Fig. 1 and then pretreated for 30 min with thapsigargin, RG80267, or
R59949. The cells were then treated for 30 min with the control, 1 mM glutamate, A23187, the inhibitor alone, or 1 mM glutamate + the inhibitor. APP-S secretion was
quantitated by PhosphorImager analysis as the amount of radioactivity
in the APP-S band and is expressed as a percentage of the control
within each experiment. Results are shown as the mean ± S.E. of
APP-S secretion from 4 to 13 separate observations/condition.
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 |
DISCUSSION |
We have shown that the metabotropic glutamate receptors mGluR1 and
mGluR5 are present in NT2N cells and that upon stimulation with
glutamate, increase the accumulation of the second messenger IP3 and the secretion of APP-S in a time- and
dose-dependent manner. This is a novel demonstration of
glutamatergic regulation of APP-S secretion in a nontransfected human
neuronal cell line.
The regulation of receptor-mediated APP-S secretion has been studied
extensively. However, a complete model of the signaling pathway has yet
to be developed. APP-S secretion has previously been shown to be
regulated by the muscarinic receptor signaling pathway (19). Muscarinic
receptor activation results in the activation of a phospholipase
C-linked pathway, as seen by increases in intracellular calcium, DAG,
and IP3, and subsequent secretion of APP-S (19). The
stimulation of APP-S secretion by glutamate receptors has also been
shown recently (20, 33-35). Glutamate, quisqualate, and ACPD were
shown to increase APP-S secretion from rat hippocampal neurons, as well
as from rat cortical and hippocampal brain slices. Glutamate,
quisqualate, and ACPD have also been shown to increase intracellular
IP3 accumulation in cultured rat hippocampal neurons,
cortical and hippocampal rat brain slices, and astrocytes (20, 34, 35).
Ionotropic glutamate receptor agonists NMDA, AMPA, and kainate did not
have any effect on APP-S secretion from rat hippocampal neurons or from
rat cortical and hippocampal brain slices. However, it is not known
which glutamate receptors are directly involved in the stimulation of
APP-S secretion. It has been demonstrated that metabotropic glutamate
receptors are involved in glutamate-mediated APP-S secretion. These
findings support our data and conclusion that metabotropic glutamate
receptors are involved in the regulation of APP-S secretion.
For our model, the human neuronal NT2N cells were used. NT2N cells
provide a unique model system because they are human cells that
endogenously express APP and A
. These cells also express the
muscarinic receptor subtypes m2 and m3 and have been previously shown
to express ionotropic glutamate receptors (36, 37). We currently have
demonstrated the presence of the metabotropic glutamate receptor
subtypes mGluR1 and mGluR5 in NT2N cells. There are at least eight
different subtypes of metabotropic glutamate receptors, and these are
subdivided into three groups according to sequence similarities,
agonist selectivity, and intracellular signaling machinery. The
metabotropic glutamate receptors mGluR1 and mGluR5 are the group I
receptors, coupled to PI hydrolysis, and upon stimulation with specific
receptor agonists resulted in an increase in secretion of APP-S from
these cells. Specific metabotropic glutamate receptor antagonists
inhibited receptor-mediated APP-S secretion, suggesting that the
agonists were specific for the metabotropic receptors. In addition, but
contrary to previous reports in non-neuronal cells and rat neurons, the
ionotropic glutamate receptor agonists AMPA and kainate also caused an
increase in the secretion of APP-S from NT2N cells. This we believe is the result of the increase in intracellular calcium levels upon activation of these receptors (Fig. 4). The calcium ionophore A23187,
which results in an increase in intracellular calcium concentrations,
also caused an increase in the secretion of APP-S, suggesting that
calcium is a potentiator of APP-S secretion. Furthermore, stimulation
of these cells with glutamate resulted in the activation of a
phospholipase C-linked pathway, as seen by an increase in IP3 accumulation. We also examined the effect of OAG, a
cell-permeable DAG analog, and DAG lipase and kinase inhibitors to
determine if DAG metabolism or generation is involved in the
glutamate-induced APP-S secretion. The DAG lipase inhibitor RG80267 did
not have any effect on glutamate-induced APP-S secretion, suggesting
that DAG metabolism is not involved. However, both OAG and the DAG kinase inhibitor R59949, which in other systems has been shown to
increase DAG levels (38), caused an increase in glutamate-induced APP-S
secretion, suggesting that DAG is a downstream mediator in glutamate
receptor signaling. The effect of the protein kinase C activator PMA on
APP-S secretion mimicked the effect seen by glutamate, suggesting that
protein kinase C is also involved as a downstream mediator in the
regulation of APP-S secretion. Protein kinase C has extensively been
shown to be involved in the regulation of APP-S secretion (21, 39, 40);
however, it has not been demonstrated how protein kinase C is involved
or which isoform is involved in APP-S secretion.
Metabotropic glutamate receptors of Groups II and III are negatively
coupled to cAMP, and in astrocytes, forskolin and dibutyryl cyclic AMP
inhibited mGluR agonist-induced APP-S secretion and increased APP gene
expression and the cellular APP holoprotein. (35). These studies
demonstrated an inhibition of both glutamate- and phorbol
ester-stimulated APP-S secretion with forskolin, an activator of
adenylate cyclase, and dibutyryl cyclic AMP, suggesting that activation
of the cAMP pathway inhibits APP-S secretion. Additionally, activation
of adrenergic receptors coupled to cAMP also increased APP gene
expression and APP holoprotein in astrocytes (42). In C6 cells
transfected with APP751, forskolin, dibutyryl cyclic AMP,
and isoproterenol increased the concentration of cAMP, and forskolin
inhibited both the constitutive and phorbol ester-mediated secretion of
nexin II, the
-secretase cleavage product of APP751 (43). These studies also demonstrate an involvement of cAMP in APP-S
secretion. In contrast, Xu et al. (44) showed that forskolin
stimulated the secretion of APP-S from PC12 cells. Therefore, the
effect of the cAMP pathway on APP-S secretion still remains unclear and
may be cell-specific. In NT2N cells, preliminary experiments examining
the effect of forskolin on APP-S secretion did not demonstrate a change
in APP-S secretion after the addition of forskolin (APP-S secretion of
105% ± 11% of control in the presence of 10 µM
forskolin, n = 6). However, the metabotropic glutamate
receptor agonist CCG-I (a Group II agonist) did stimulate APP-S
secretion, although this could be due to its reported stimulation of
mGluR1 and phosphoinositide hydrolysis (41). In addition, Group II/III
antagonists partially inhibited glutamate-induced APP-S secretion
(Table II). These conflicting results suggest that activation of the
cAMP pathway may also be an important regulatory mechanism of APP-S
secretion in NT2N cells and will require further investigation.
In conclusion, we have demonstrated that glutamate stimulates the
metabotropic glutamate receptor signal transduction pathway in human
neurons causing an increase in the second messenger IP3 and
the secretion of APP-S.