(Received for publication, October 17, 1994; and in revised form, December 14, 1994)
From the
The Alzheimer amyloid precursor protein (APP) undergoes complex
processing resulting in the production of a 4-kDa amyloid peptide
(A) which has been implicated in the pathogenesis of
Alzheimer's disease. Recent studies have shown that cells can
secrete carboxyl terminus truncated APP derivatives (APP-S) in response
to physiological stimulus. We have used human central nervous system
neurons (NT2N) derived from a teratocarcinoma cell line (NT2) to study
the signal transduction pathways involved in APP-S secretion and A
production. Muscarinic receptors (m2 and m3) as well as the
heterotrimeric GTP-binding protein G
and the
1 isoform
of phospholipase C were present in NT2N neurons. Stimulation of the
muscarinic receptor with carbachol resulted in phospholipase C
activation as shown by a transient increase in the second messengers
1,2-diacyl-sn-glycerol and inositol 1,4,5-trisphosphate.
Carbachol also caused an increase in intracellular Ca
levels measured in single NT2N neurons. Under these conditions,
carbachol caused a time-dependent 2-fold increase in APP-S secretion
into the medium. In contrast, prolonged treatment with carbachol caused
a decrease in A
production into the medium. These results suggest
that APP-S secretion and A
production in NT2N neurons are
regulated by the muscarinic/phospholipase C signal transduction
pathway. Furthermore, activation of this pathway results in
dissociation of APP-S secretion and A
production.
Alzheimer's disease, is defined by specific pathological
lesions in the brain which include neurofibrillary tangles and
-amyloid (A
) (
)deposits affecting selected areas
of the brain(1, 2, 3) . This
neurodegenerative disease is associated with neuron loss and death
which affect many neuronal populations. In particular, the cholinergic
cells which arise in the basal forebrain and terminate in the
hippocampus and cerebral cortex are severely affected(1) . The
selective vulnerability of cholinergic neurons has been the focus of
intensive studies to elucidate its biochemical
mechanism(4, 5) . There is a loss of choline
acetyltransferase, the enzyme that synthesizes acetylcholine from
choline and acetyl-CoA. More recently, it has been shown that the high
affinity choline uptake is abnormally high in Alzheimer's
disease(6) . Thus, these observations suggest that
abnormalities in cholinergic function may be involved in the
pathogenesis of Alzheimer's disease.
Recent studies strongly
suggest that amyloid deposition is linked to the pathogenesis of
Alzheimer's disease(7, 8, 9) . The
A peptide is derived from amyloid precursor proteins (APP). APP is
an integral membrane, tyrosine-sulfated glycoprotein with one
membrane-spanning domain and an extracytoplasmic NH
terminus. APP exists in three major isoforms in the central
nervous system, which are encoded by the same gene on chromosome 21.
Alternative mRNA splicing generates 695- (APP
), 751-
(APP
), and 770-amino acid APP (APP
). The
brain is the richest source of APP, and in particular APP
is restricted almost exclusively to the central nervous system
and the peripheral nervous system(10, 11) .
In
recent years, there has been an intense effort to elucidate the
pathways whereby A is generated from APP. A
peptide is a
39-43 amino acid internal sequence that extends from within the
transmembrane domain into the extracytoplasmic domain of APP. There are
several pathways to process APP. In the constitutive secretory pathway
(or
-secretase), APP is cleaved, within the A
sequence at
residue 687 just outside the transmembrane domain, by the action of a
protease, to a large secreted NH
-terminal derivative
(APP-S) and a membrane-associated fragment, neither of which can
produce
-amyloid protein(12, 13) . The presence
of a
-secretase has also been inferred which cleaves APP precisely
at the amino terminus of A
(14) . In a third processing
pathway, APP is processed in the endosomal and lysosomal system, and
yields complex COOH-terminal derivatives, some of which are potentially
amyloidogenic(15, 16) . More recently, several groups
have shown that APP processing in the endosomal/lysosomal system
produces a 4-kDa
-amyloid protein that is essentially similar to
the deposited amyloid of Alzheimer's disease (reviewed in Refs.
17, 18).
It has recently been recognized that A is present in
human cerebrospinal fluid from normal and Alzheimer's disease
patients and that cultures of neuronal and non-neuronal cells
transfected with the APP gene secrete A
into the
medium(19, 20) . Primary fetal human neurons secrete
A
(20, 21) . Importantly, a unique human neuronal
cell line NT2N has been shown to secrete endogenous A
into the
culture medium(11) . Furthermore, intracellular A
can be
detected in the NT2N neurons. Collectively, these observations strongly
suggest that normal human neurons can generate the A
peptide.
The pathways involved in APP processing under non-amyloidogenic
conditions have begun to be studied. Using human embryonic kidney cell
lines transfected with the genes for the human brain muscarinic
acetylcholine receptors, Nitsch et al. have shown that
stimulation of the m1 and m3 receptor subtypes with carbachol increases
the release of APP derivatives(22) . Buxbaum et al.(23) have also demonstrated that cholinergic agonists
stimulates APP secretion in human glioma and neuroblastoma cells as
well as in PC12 cells transfected with the m1 receptor. The biochemical
pathways underlying muscarinic stimulation of APP secretion are not
well understood, although protein kinase C activation has been
implicated (24, 25, 26) . Recently, it has
been shown that A production is regulated by the muscarinic
pathway(27) .
Human neurons express mainly the APP isoform(11) . In addition to astrocytes, microglia, and
vascular cells, neurons are a likely source of A
deposited in
amyloid in Alzheimer's disease: it is therefore important to
study APP expression, processing, and regulation in neurons.
Furthermore, since Alzheimer's disease and related
neurodegenerative diseases only occurs in humans and primates (17, 28) and there is no rodent animal model which
recapitulates the development of this disease, these studies should
ideally be performed in human neurons. Postmitotic mature human
neurons, however, are difficult to isolate and maintain in culture.
Because of these limitations we have used a unique culture model of
human neurons, the NT2N neuronal cells.
NT2N neurons are derived
from a human teratocarcinoma cell line, Ntera 2/c1.D1 (NT2), that is
induced by treatment with retinoic acid to commit irreversibly to a
neuronal phenotype(29, 30) . Prior extensive studies
have shown that NT2N neurons express many cytoskeletal markers,
cell-surface antigens, and synaptic proteins typical of central nervous
system neurons(29) . They are permanently postmitotic and
develop functional dendrites and axons. They can be purified to yield
>99% pure postmitotic human neurons. The rationale for using NT2N
cells to study the regulation of APP-S secretion is that 1) different
cells process APP differently, 2) unlike non-neuronal cells, NT2N
neurons express predominantly APP, the major APP isoform
expressed in neurons in brain, 3) in NT2N cells, APP
can
be easily detected without transfection of the APP gene, and 4) NT2N
neurons constitutively generate intracellular A
peptide and
release it into the culture medium(11) . Thus, NT2N neurons
represent a unique and physiological human model system to study APP
processing and A
production which closely recapitulates events in
the normal human brain. In the present study, we have used NT2N neurons
to study muscarinic regulation of APP secretion and A
production
and to dissect the signal transduction pathways involved.
Figure 1: Identification of muscarinic receptor subtypes in NT2N neurons. Membranes from NT2N neurons (replate 3) were analyzed for muscarinic receptor subtypes with specific anti-muscarinic antibodies as described under ``Experimental Procedures.'' Results are expressed as the mean ± S.E. of receptor density (pmol/mg) from three experiments.
Figure 2:
Identification of phospholipase C isoforms
and GTP-binding protein subtypes in NT2N neurons. A,
phospholipase C isoforms. NT2 (lanes 1 and 3) and
NT2N neurons (lanes 2 and 4) lysates were purified by
SDS-PAGE, and immunoblotting was performed with specific monoclonal
antibodies. Lanes 1 and 2, 1 isoform; lanes
3 and 4,
1 isoform. B, GTP-binding protein
subtypes. The arrow indicates the 43-kDa
marker.
Figure 3:
Radiochromatogram of carbachol-induced DAG
accumulation in NT2N neurons. NT2N neurons were labeled with
[H]arachidonic acid for 24 h (1 µCi/100 mm
dish), washed in modified Krebs-HEPES buffer (25 mM HEPES pH
7.40, 115 mM NaCl, 24 mM NaHCO
, 5 mM KCl, 2.5 mM CaCl
, 1 mM MgCl
, 0.1% bovine serum albumin, 3 mMD-glucose), preincubated 30 min, and then incubated 0 to
30 min with medium ± 1 mM carbachol at 37 °C under
95% air, 5% CO
. Diacylglycerol (DAG) was
extracted, analyzed by TLC, and quantitated with a Berthold linear
analyzer. Equal amounts of radioactivity were loaded onto each
lane.
Figure 4:
Time
course of carbachol-induced diacylglycerol accumulation in NT2N
neurons. NT2N neurons were labeled with
[H]arachidonic as in Fig. 3.
Diacylglycerol accumulation was normalized to percent of label
incorporated into the total phospholipid fraction. Results are shown as
the mean ± S.E. for control (solid squares, dashed
line) and 1 mM carbachol (solid circles, solid
line) from 4 to 16
observations/condition.
Since carbachol-induced
accumulation of DAG may reflect activation of other pathways in
addition to phosphatidylinositol-specific phospholipase C, we also
measured the muscarinic-induced accumulation of the second messenger
Ins(1,4,5)P which directly reflects phospholipase C
activation. NT2N neurons were labeled with
[
H]inositol and then stimulated for 2 and 5 min.
Inositol phosphates were extracted and separated by SAX-HPLC. Under
these conditions, carbachol caused a 2.7-fold increase in
Ins(1,4,5)P
levels at 2 min (from 18.9 ± 3.4
counts/min to 50.9 ± 17.0 counts/inm, n = 3). No
significant increase was noted at 5 min. Finally, carbachol had no
effect on the levels of the inactive isomer Ins(1,3,4)P
or
on Ins(1,3,4,5)P
(data not shown).
Ins(1,4,5)P mobilizes Ca
from intracellular stores. Since
carbachol activates phospholipase C with release of the second
messengers DAG and Ins(1,4,5)P
, we next examined whether
carbachol could affect intracellular Ca
levels in
NT2N neurons. In these experiments, NT2N neurons on coverslips were
loaded with fura 2 and then mounted on a microscope equipped for
epifluorescence. Single neuron cytosolic Ca
was
calculated from the ratio of fura 2 fluorescence at 340 and 380 nm.
Transient stimulation with carbachol caused a very rapid and
significant increase in intracellular Ca
levels (Fig. 5).
Figure 5:
Carbachol stimulation of intracellular
Ca in single NT2N neurons. NT2N neurons were loaded
with fura 2 and Ca
fluorescence from a single neuron
was quantitated as described under ``Experimental
Procedures.'' The addition of carbachol is indicated by the arrow. Representative of at least three
experiments.
Figure 6:
Time course of APP-S secretion from NT2N
neurons. NT2N neurons were pulse-labeled 30 min with
[S]methionine, washed, and then chased with 1
mM carbachol in modified Krebs-HEPES medium as described under
``Experimental Procedures.'' APP-S secretion into the
supernatant was measured after ammonium sulfate precipitation and
immunoprecipitation with the anti-APP KAREN antibody. Proteins were
separated by SDS/7.5% PAGE and analyzed with a PhosphorImager. Top
panel, representative gel of APP-S secretion. A and B, lysate; C and D, supernatant. The time
(0, 60, and 90 min) is indicated on the x axis. Bottom
panel, time course of APP-S secretion into the supernatant. APP-S
secretion was quantitated by PhosphorImager as the amount of
radioactivity in the 90 kDa band and is expressed as the ratio to the
condition within each experiment with the highest counts (90 min,
carbachol). Results are shown as the mean ± S.E. of APP-S
secretion from six separate experiments. Control, hatched
bars; carbachol, solid bars.
In order to assess A production,
slightly different labeling conditions were used. NT2N neurons were
labeled for 3 h with [
S]methionine, and then
stimulated with carbachol. As shown in Fig. 7(top
panel), a 4 kDa peptide band was detected. In most experiments,
the related 3-kDa peptide was not detected. There was a significant and
time-dependent accumulation of A
peptide over 8 h. Under 1 h,
A
levels were too low to quantitate. Stimulation with carbachol
caused a decrease in A
levels which represented a 42% decrease by
8 h (0.588 ± 0.079 relative units versus 1.000 for
control, p < 0.05).
Figure 7:
Time course of carbachol on A
production from NT2N neurons. NT2N neurons were pulse-labeled 3 h with
[
S]methionine, washed, and then chased with 1
mM carbachol in modified Krebs-HEPES medium as described under
``Experimental Procedures.'' A
production into the
supernatant was measured after ammonium sulfate precipitation and
immunoprecipitation with the anti-A
4G8 antibody. Proteins were
separated on 16.5% Tris-Tricine gels and analyzed with a
PhosphorImager. Top panel, representative gels of A
production. Bottom panel, time course of A
production
into the supernatant. A
secretion was quantitated by
PhosphorImager as the amount of radioactivity in the 4 kDa band and is
expressed as the ratio to the condition within each experiment with the
highest counts (8 h, control). Results are shown as the mean ±
S.E. of A
production from three to four separate experiments.
Control, hatched bars; carbachol, solid
bars.
We have shown that NT2N neurons express m2 and m3 muscarinic
receptors, and upon muscarinic stimulation of normal human NT2N neurons
there is: 1) activation of phospholipase C with release of the second
messengers Ins(1,4,5)P and DAG, 2) increased intracellular
Ca
levels, and 3) time-dependent secretion of APP-S
associated with decreased A
production. These studies represent
the first demonstration in non-transfected human neurons of muscarinic
regulation of APP-S secretion and A
production, and extend
previous studies which have shown muscarinic regulation of A
production(27) .
The signal transduction pathway involved in
muscarinic-induced APP-S secretion has several components. The
muscarinic acetylcholine receptor family consists of five cloned and
expressed receptor genes designated m1 through m5 and is part of the
large family of seven transmembrane receptors(51) . These
receptors work by activation of heterotrimeric GTP-binding proteins:
m1, m3, and m5 are coupled to stimulation of phospholipase C, while m2
and m4 inhibit adenylate cyclase(51) . Our study identified the
m3 receptor as the most prominent subtype in NT2N neurons. The m3
subtype is known to be coupled to the heterotrimeric GTP-binding
protein G in other systems(52, 53) .
Interestingly, we did not find any significant levels of the m1
muscarinic receptor subtype which has been implicated in APP secretion
in human embryonic kidney cell lines transfected with the genes for the
m1 subtype(22) . Although significant levels of m2 receptors
were also found in NT2N cells, this and previous studies implicate
activation of the m1/m3-coupled phospholipase C signal transduction
pathway in APP-S secretion(22, 23) .
Among the
various heterotrimeric GTP-binding protein subunits which were
screened, we demonstrated the presence of the - and
-subunits. There are four members (G
,
G
G
, and G
)
of the G
class of
-subunits(54) . There is
overwhelming evidence demonstrating that G
, a 42-kDa
protein, directly regulates phospholipase C-
(54) . Thus,
purified bovine brain phospholipase C-
was shown to be markedly
stimulated by brain G
(55) . The identification
of G
in NT2N neurons is an important link in the
muscarinic receptor/phospholipase C signal transduction cascade. The
presence of the
-subunit also may have functional
implications since it has recently been shown that
stimulates various isoforms of phospholipase C-
, although it
stimulates more the
3 isoform than the
1
isoform(56) .
G was also identified in NT2N
neurons. In most cells, the
-subunit, a pertussis
toxin-sensitive G protein, is thought to inhibit Ca
channel activity(57) . Recently, however, it has been
proposed that APP itself may function as a membrane receptor coupled to
G
(58) . The cytoplasmic APP sequence
His
-Lys
had a specific G
activating function and was necessary to form a APP
G
complex. Although this observation was demonstrated in
vitro, its physiological significance and role in the pathogenesis
of Alzheimer's disease are unclear at the present time.
Two
main isoforms (1,
1) of phospholipase C were present in NT2N
neurons. In other cells, activation of phospholipase C-
1 is
typically the result of growth factor occupancy of the growth factor
receptor and autophosphorylation by a receptor-tyrosine
kinase(59) . Activation by epidermal growth factor and
platelet-derived growth factor causes translocation of phospholipase C
to the membrane(60) . Whether these growth factors have any
role in regulating APP secretion remains to be determined. However,
since activation of phospholipase C-
1 results in hydrolysis of
polyphosphoinositides and accumulation of the second messengers
Ins(1,4,5)P
and DAG, it is also conceivable that these
agonists will regulate APP secretion. Phospholipase C-
1 is the
phospholipase C isoform which is known to be regulated by
heterotrimeric GTP-binding proteins(61) . In particular, the
muscarinic receptor, m3, is most likely an activator of phospholipase
C-
1(62) . Based on post-mortem studies, brain
phosphoinositide metabolism appears abnormal in Alzheimer's
disease as reflected by decreased levels of phosphoinositides and
aberrant accumulation of phospholipase C-
in the temporal cortex
and hippocampus(63) . Indeed, it has been postulated that these
abnormalities may be related to the characteristic cellular pathology
of Alzheimer's disease(63) , although there are
well-established changes in other classes of phospholipids such as
phosphatidylcholine (64) . Since activation of phospholipase C
stimulates APP secretion and decreases A
production, this signal
transduction pathway may be a relevant target for the development of
Alzheimer's disease.
Constitutive secretion of APP-S was
clearly detected in the supernatant of NT2N neurons consistent with our
previous results which have shown that NT2N neurons mainly secrete
APP-S(11) . Muscarinic stimulation of NT2N
neurons results in regulated APP-S secretion which is measured over the
background of constitutive secretion, and which most likely reflects
activation of the putative
-secretase. These studies are important
since they directly demonstrate for the first time that activation of
the muscarinic/phospholipase C signal transduction pathway results in
APP-S secretion in normal human neurons with normal levels of
muscarinic receptors and endogenous levels of APP as opposed to
non-neuronal or neuronal cell lines over-expressing muscarinic
receptors and transfected with the APP gene(22, 23) .
DAG, the product of phospholipase C hydrolysis of polyphosphoinositides
is an endogenous activator of protein kinase C, a Ca
-
and phospholipid-dependent protein kinase which has been implicated in
regulated APP secretion. Phorbol esters have been used as pharmacologic
probes to activate protein kinase C and APP secretion in cells
overexpressing the APP gene such as PC12 cells, Chinese hamster ovary
cells, 293 cells, human umbilical vein endothelial cells, and COS cells (23, 24, 27, 65) . In one study with
Swiss 3T3 fibroblasts overexpressing protein kinase C
, the
specific isoform of protein kinase C which mediates phorbol
ester-induced APP secretion was identified as protein kinase C
,
although the contribution of other isoforms of protein kinase C in APP
secretion cannot be excluded, specially as neurons are known to express
several isoforms(25, 66) . Ins(1,4,5)P
, a
second messenger generated from phospholipase C hydrolysis of
phosphatidylinositol 4,5-bisphosphate, mobilizes calcium from
intracellular stores resulting in increased cytoplasmic Ca
levels(67) . Recently, it has been suggested that
increases in intracellular Ca
levels in Chinese
hamster ovary cells transfected with cDNA encoding the m1 or m3
muscarinic receptor and APP
results in APP secretion
independently of protein kinase C(26) . Our study demonstrates
that the muscarinic-induced transient increase in DAG,
Ins(1,4,5)P
, and Ca
levels in NT2N
neurons correlates with APP-S secretion. Of note is the fact that
muscarinic-induced increase in these second messengers is transient
(2-5 min) whereas the increase in APP-S secretion is observed
over 90 min, suggesting that intermediate steps, such as protein
phosphorylation and/or
synthesis(24, 26, 27, 65) , are
distally involved in the regulated secretion of APP-S.
A
production from NT2N neurons was decreased following cholinergic
stimulation. Thus, activation of the muscarinic/phospholipase C pathway
results in opposite effects on APP-S secretion and A
production.
Similar results were observed in a variety of cells transfected with
the gene for APP
or
APP
(24, 26, 27, 65) .
However, one recent study showed that in the human neuroblastoma cell
line SY5Y transfected with cDNA encoding APP
, phorbol
esters caused an increase in A
production(68) . Our
studies, performed in human neurons expressing endogenous levels of
APP, strongly suggest that the pathways of APP-S secretion and A
production can be dissociated following stimulation of the
muscarinic/phospholipase C signal transduction pathway. Interestingly,
muscarinic-induced decrease in A
production was only observed
after prolonged stimulation (8 h) with carbachol which suggest that APP
levels have to be substantially depleted (by increased secretion of
APP-S) before A
production is decreased. Alternatively, this may
reflect differences in the release kinetics of APP-S and A
and the
difficulty in quantitating low levels of A
. Previously, we have
shown that A
can be recovered from cell lysates in NT2N neurons (11) . We did not examine the effect of carbachol treatment on
intracellular A
because the recovered levels were low and did not
allow for sufficient quantification.
In summary, we have shown that
the muscarinic agonist carbachol stimulates the
muscarinic/phospholipase C signal transduction pathway in normal human
neurons resulting in a transient increase in the second messengers DAG,
Ins(1,4,5)P, Ca
, and a sustained increase
in APP-S secretion with a subsequent decrease in A
production.