(Received for publication, September 14, 1995; and in revised form, December 5, 1995)
From the
Alzheimer's disease amyloid consists of amyloid
-peptides (A
) derived from the larger precursor amyloid
precursor protein (APP). Non-amyloidogenic APP processing involves
regulated cleavage within the A
domain followed by secretion of
the ectodomain (APPs). APPs secretion can be stimulated by muscarinic
acetylcholine receptors coupled to phospholipases and kinases. To
determine whether other receptor classes can regulate APP processing,
we examined the relation between serotonin receptors and APPs
secretion. Serotonin increased APPs release 3-4-fold in 3T3 cells
stably overexpressing 5-HT2aR or 5-HT2cR. The increase was
dose-dependent and was blocked by serotoninergic antagonists. Phorbol
esters also increased APPs secretion, but neither kinase inhibitors nor
down-regulation of PKC blocked the serotonin-induced increase in APPs
secretion. Thus PKC is not necessary to stimulate APPs secretion.
Phospholipase A
(PLA
) inhibitors blocked the
5-HT2aR-mediated increase in APPs secretion, suggesting a role of
PLA
in coupling 5-HT2aR to APP processing. In contrast,
coupling of 5-HT2cR to APPs secretion involved both PKC and
PLA
. Serotonin also stimulated the release of the APLP2
ectodomain, suggesting that additional members of the APP multigene
family are processed via similar regulated pathways. Inasmuch as
generation of APPs precludes the formation of amyloidogenic
derivatives, serotonin receptors provide a novel pharmacological target
to reduce these derivatives in Alzheimer's disease.
The amyloid precursor protein (APP) ()is an
ubiquitous membrane-spanning glycoprotein (Kang et al., 1987;
Weidemann et al., 1989) that is present at high levels in
brain cells. It is the biological precursor of the amyloid
-protein (A
), the principal proteinaceous component of
amyloid plaques in brains of Alzheimer's disease patients (for
review, see Selkoe(1994)). APP or its derivatives may be involved in
the pathogenesis of Alzheimer's disease because several familial
forms of the disease are linked to APP mutations within or close to the
-amyloid domain. In cell culture, these mutations cause
misprocessing of APP and related increases in the formation of A
(Cai et al., 1993; Citron et al., 1992, 1994) or the
generation of longer than normal A
molecules (Suzuki et al., 1994). Moreover, the overexpression of APP in transgenic mice
causes formation of brain amyloid plaques (Games et al., 1995)
that resemble those in Alzheimer's disease.
APP is a secretory
glycoprotein. The secreted APP ectodomain (APPs) has neurotrophic and
neuroprotective activities in selected experimental systems. It
promotes neurite outgrowth and branching in PC-12 cells (Milward et
al., 1992), mediates cell adhesion in cultured fibroblasts (Saitoh et al., 1989), and it can protect primary neuronal cultures
from excitotoxic damage (Barger et al., 1995; Mattson et
al., 1993). Furthermore, moderate overexpression of human APP in
mouse brain can increase synaptic density (Mucke et al. 1994),
and it protects neurons in vivo from neurotoxic events caused
by overexpression of the HIV-1 surface protein pg120 (Mucke et al., 1995b) or by excitotoxic damage (Mucke et al., 1995a). In
selected experimental systems, APPs activates cellular signaling
pathways, including p21 microtubule-associated
protein kinases (Greenberg et al., 1994), and cGMP-mediated
signaling in primary hippocampal neurons (Barger et al., 1995). Secretory APP processing involves either proteolytic
cleavage within the A
domain (Esch et al., 1990; Sisodia et al., 1990) or, alternatively, cleavage at both the N and C
termini of A
followed by rapid secretion of A
(Haass et
al., 1992; Shoji et al., 1992). Thus, APP can be
processed by at least two alternative secretory pathways to yield
either APPs and p3, a derivative of the cell-associated C terminus
produced by APPs secretion (Haass et al., 1993), or A
and
a C-terminally truncated form of APPs (Seubert et al., 1993).
Secretory APP processing and the formation of APPs can be readily
accelerated by an unusual mechanism coupled to a variety of extra- and
intracellular signals. These include muscarinic acetylcholine receptors
(Buxbaum et al., 1992; Nitsch et al., 1992; Wolf et al., 1995), metabotropic glutamate receptors (Lee et
al., 1995), protein kinase C (PKC) (Caporaso et al., 1992; Slack et al., 1993), tyrosine kinases (Slack et
al., 1995), and arachidonic acid (Emmerling et al., 1993). In rat brain tissue slices, the regulation of APP
processing appears to be a function of neuronal activity: APPs
secretion is stimulated by electrical depolarization in a
frequency-dependent, tetrodotoxin-sensitive fashion (Nitsch et al., 1993), and by muscarinic receptor agonists (Farber et al., 1995). The rate of A
formation appears to be inversely
coupled to the rate of APPs secretion: in several cell culture systems,
stimulated APPs secretion was accompanied by reductions in secreted
A
(Buxbaum et al., 1993; Gabuzda et al., 1993;
Hung et al., 1993; Wolf et al., 1995), suggesting
that stimulated secretory processing of APP into secreted APPs is
associated with reduced formation of potentially amyloidogenic
derivatives. The physiological relevance of regulated APPs secretion is
unknown, but may be related to the functions of APPs as a paracrine
neurotrophic factor or a signaling molecule that is secreted in an
activity-dependent manner in response to neuronal activation by
neurotransmitters.
In Alzheimer's disease brains, neurotransmission is impaired. In particular, cortical serotoninergic, glutamatergic, and peptidergic systems as well as the subcortical cholinergic projection systems are heavily damaged, indicating deafferentation of multiple cortical and hippocampal target regions. Inasmuch as neuronal activation may be involved in regulating APP processing, impaired neuronal signaling may cause alterations in APP processing pathways (for review, see Nitsch and Growdon(1994)).
In the series of experiments reported here, we examined the effects of serotoninergic receptors on APP processing using 3T3 fibroblast lines that stably overexpress 5-HT2a and 5-HT2c receptor subtypes. Wild-type 3T3 cells lack endogenous expression of serotonin receptors, and they provide the necessary cell biological background to assure appropriate signaling functions of transfected receptor subtypes (Julius et al., 1988; Stam et al., 1992). Furthermore, 3T3 cells process APP via regulated cleavage and secretion (Slack et al., 1993). They thus provide a valid model system to study the effects of specific transfected serotonin receptor subtypes on signaling as well as on APPs secretion.
In 3T3 fibroblasts stably transfected with cDNA expression
constructs encoding either 5-HT2aR or 5-HT2cR, serotonin caused rapid
and dose-dependent increases in PI turnover (Fig. 1). Serotonin
increased PI turnover to 29-fold (range 7.8-53, n = 4) compared to that observed basally in cells
overexpressing 5-HT2aR (Fig. 1A) and to 7.9-fold (range
4.1-9.9, n = 4) basal levels in cells
overexpressing 5-HT2cR (Fig. 1B). The EC of the inositol phosphate (IP) accumulation in response
to serotonin was 30 nM in the 5-HT2aR-expressing cells and 2
nM in the 5-HT2cR-expressing cells. Serotonin also caused APPs
secretion to increase in both cell lines (Fig. 2). The maximal
stimulation of APPs secretion was 4.5-fold (range 2.3-10.8, n = 9) basal levels in cells expressing 5-HT2aR and 3-fold
(range 1.6-3.9) in cells expressing 5-HT2cR. The EC
of 5-HT-mediated APPs secretion was 200 nM in cells
expressing 5-HT2aR (Fig. 2A) and 120 nM in the
5-HT2cR-expressing cells (Fig. 2B). Time course
analyses showed that the 5-HT-mediated stimulation of APPs secretion
was rapid, with significant increases in both cell lines occurring as
soon as 5 min following the application of 5-HT (Fig. 2, C and D). The increase in APPs secretion caused by 5-HT2aR
stimulation was blocked by the serotoninergic antagonists ketanserin,
mianserin, and ritanserin (Fig. 3), and the 5-HT2cR-mediated
increase was blocked by mianserin (Fig. 3), indicating that the
effects on APP processing in both cell lines were specifically mediated
by activation of the overexpressed 5-HT2 receptor subtypes. Direct
stimulation of PKC by the phorbol ester PMA mimicked the 5-HT-induced
increase in APPs secretion (Fig. 4A and 5A).
In both cell lines, however, the kinase inhibitors staurosporine and
chelerythrine chloride failed to block the response to 5-HT (Fig. 4A and 5A). Moreover, down-regulation of
PKC in these cells by chronic (24 h) pretreatment with PMA also failed
to block the ability of 5-HT to stimulate APPs secretion (Fig. 4A and 5A). The PMA response, however,
was completely blocked by prior chronic pretreatment with PMA (Fig. 4A and 5A). In 5-HT2aR-expressing cells,
the 5-HT-mediated increase in APPs secretion was effectively blocked by
the PLA
-inhibitors manoalide (ML),
dimethyleicosadienoic acid (DEDA), and oleyloxyethyl
phosphorylcholine (OPC) (Fig. 4B), indicating
that PLA
may be also involved in the cellular signaling
cascade that couples 5-HT2aR activation to APPs secretion. This
hypothesis is supported by the finding that activation of PLA
by melittin mimicked the 5-HT-mediated increase in APPs secretion (Fig. 4B), while the calcium-releasing agent
thapsigargin failed to change APPs secretion from these cells (Fig. 4B). Down-regulation of PKC by prior chronic
preincubation with PMA (Fig. 4C) failed to block either
the inhibition of APPs secretion by PLA
inhibitors or its
stimulation by melittin, indicating that the signaling mediated by
PLA
is independent of cellular PKC activity. In the
5-HT2cR-expressing cells, the PLA
inhibitor
dimethyleicosadienoic acid only partly blocked 5-HT2cR-induced APPs
secretion, and melittin was less effective in stimulating this
secretion than in the 5-HT2aR-expressing cells (Fig. 5B). When PKC was down-regulated by prior chronic
preincubation with PMA, all PLA
inhibitors effectively
blocked the 5-HT2cR-mediated increase in APPs secretion (Fig. 5C), suggesting that PKC and PLA
are
both involved in coupling of 5-HT2cR activation to APPs secretion. The
serotoninergic agonist dexnorfenfluramine (DNF) stimulated PI
breakdown in 3T3 cells expressing 5-HT2cR, indicating that it is a
potent agonist of these receptors (Fig. 6A). DNF
dose-dependently stimulated APPs secretion in the 5-HT2cR expressing
cells (Fig. 6B), and this effect was inhibited by the serotonin
receptor antagonists ketanserin, mianserin, and ritanserin (Fig. 6C). In related experiments with cells that
stably overexpress 5-HT2aR, serotonin also increased PI turnover as
well as the secretion of APPs, indicating that DNF is a nonselective
agonist of both 5-HT2aR and 5-HT2cR (data not shown). In contrast to
the responses of stably transfected cells lines, the untransfected
parent cell line failed to exhibit changes in APPs secretion when
exposed to serotonin (100 pM to 100 µM).
Figure 1:
Serotonin
stimulates phosphatidylinositol turnover in 3T3 fibroblasts stably
expressing 5-HT2a or 5-HT2c receptors. Cells were metabolically labeled
with [H]inositol overnight, stimulated with
increasing concentrations of 5-HT in the presence of 10 µM lithium, and radiolabeled inositol phosphates derived from PI
breakdown were measured by scintillation counting. A, 3T3
cells stably expressing 5-HT2aR. B, 3T3 cells stably
expressing 5-HT2cR. Data are from representative experiments and are
means ± S.D. of triplicate culture dishes. Similar dose-response
curves were obtained in four independent experiments for each cell
line. IP, radiolabeled inositol
phosphates.
Figure 2: APPs secretion is increased by 5-HT receptor stimulation in 3T3 cells overexpressing 5-HT2aR or 5-HT2cR. Cells were stimulated with 5-HT and APPs secreted into the culture medium was measured by Western blotting and densitometry. A, dose response of 5-HT2aR-mediated APPs secretion. Filled symbols, stably transfected cells; open symbols, untransfected parent cell line. B, dose response of 5-HT2cR-induced APPs secretion. Filled symbols, stably transfected cells; open symbols, untransfected parent cell line. C, time course of 5-HT2aR-mediated APPs secretion. Filled symbols, 10 µM 5-HT; open symbols, vehicle control. D, time course of 5-HT2cR-mediated APPs secretion. Filled symbols, 10 µM 5-HT; open symbols, vehicle control. Data are means ± S.E. of three independent experiments.
Figure 3: APPs secretion induced by stimulating 5-HT receptors is blocked by 5-HT receptor antagonists. Cells were incubated with the antagonists 10 min prior to treatment with 10 µM 5-HT, and the antagonists and APPs in the culture media were quantitated by Western blotting and densitometry. 5-HT, 10 µM serotonin; KS, 20 µM ketanserin; MS, 50 µM mianserin; RS, 20 µM ritanserin. Data are means ± S.E. of three independent experiments.
Figure 4:
PLA couples activation of
5-HT2aR receptors to increased APPs secretion. A, no
inhibition of 5-HT-mediated APPs secretion by the protein kinase
inhibitors staurosporine (1 µM; Stau) and
chelerythrine chloride (1 µM, CC). Direct
activation of PKC by phorbol myristate acetate (1 µM; PMA) increased APPs secretion. After down-regulation of PKC by
24-h pretreatment with PMA, the response to PMA was indifferent from
the vehicle control condition (CTR). In contrast, 5-HT
stimulated APPs secretion after PMA pretreatment. B, PLA
inhibitors manoalide (3.2 µM; ML),
dimethyleicosadienoic acid (20 µM; DEDA), and
oleyloxyethyl phosphorylcholine (0.1 µM; OPC)
blocked 5-HT-mediated increase in APPs secretion. No alteration of
basal APPs secretion by these PLA
inhibitors. The PLA
activator melittin (2.5 µg/ml) increased APPs secretion. No
effect of the intracellular calcium-releasing agent thapsigargin (TG) on APPs secretion. C, inhibition and stimulation
of PLA
after PKC down-regulation. 5-HT-mediated APPs
secretion is effectively blocked by the PLA
inhibitors, and
melittin (2.5 µg/ml) retained its ability to increase APPs
secretion. Data are means ± S.E. of three
experiments.
Figure 5:
Combined action of PKC and PLA in coupling 5-HT2cR receptor activation to increased APPs
secretion. A, no inhibition of 5-HT-mediated APPs secretion by
the protein kinase inhibitors staurosporine (1 µM; Stau) and chelerythrine chloride (1 µM; CC). Direct activation of PKC by phorbol myristate acetate (PMA) increased APPs secretion. After down-regulation of PKC
by 24-h pretreatment with PMA, the response to PMA was indifferent from
the vehicle control condition (CTR). In contrast, 5-HT
stimulated APPs secretion after PMA pretreatment. B, PLA
inhibitors manoalide (ML), dimethyleicosadienoic acid (DEDA), and oleyloxyethyl phosphorylcholine (OPC)
failed to block 5-HT-mediated increase in APPs secretion. No alteration
of basal APPs secretion by the PLA
activator melittin. No
effect of the intracellular calcium releasing agent thapsigargin (TG) on APPs secretion. C, inhibition and stimulation
of PLA
after PKC down-regulation. 5-HT-mediated APPs
secretion is effectively blocked by all three PLA
inhibitors, and melittin retained its ability to increase APPs
secretion. Data are means ± S.E. of three
experiments.
Figure 6: Dexnorfenfluramine increases APPs secretion. A, stimulation of 5-HT2aR-coupled PI turnover by increasing concentrations of dexnorfenfluramine (DNF). IP, radiolabeled inositol phosphates. B, DNF increased APPs secretion in 3T3 cells expressing 5-HT2aR. C, DNF-induced stimulation of APPs secretion was blocked by the 5-HT receptor antagonists ketanserin (20 µM; KS), mianserin (50 µM, MS), and ritanserin (20 µM; RS). Data are means ± S.E. from triplicate culture dishes of representative experiments.
Increased APPs secretion was detected by the nonselective monoclonal
antibody 22C11 (Fig. 7), as well as by the antiserum R1736 (Fig. 8), an APP-specific antiserum directed against the
N-terminal 16 residues of the A domain. This observation, in
combination with the finding that the antiserum anti-C8 raised against
the C terminus did not detect any protein in the culture medium (data
not shown), suggests that secreted APPs was derived by conventional
-secretase processing. The anti-serum D2-1, raised against
full-length APLP2 expressed in a baculovirus system (Slunt et al., 1994), detected a secreted APLP2 derivative of the expected
molecular mass in the conditioned culture media from cells
overexpressing 5-HT2cR (Fig. 8). Both serotonin and
dexnorfenfluramine substantially increased this APLP2s secretion.
Figure 7: Western blot of secreted APPs with the monoclonal antibody 22C11. A, 3T3 cells stably transfected with cDNA encoding 5-HT2aR. B, 3T3 cells stably transfected with cDNA encoding 5-HT2cR. Cell were incubated for 60 min with 10 µM serotonin (5-HT), 1 µM phorbol myristate acetate (PMA), 1 µM staurosporine (Stau), 1 µM chelerythrine chloride (Chel). Lanes 6-8, cells were preincubated with 1 µM PMA for 24 h prior to the experiments. Media were analyzed for APPs using the monoclonal antibody 22C11 (lanes 1-5).
Figure 8:
Western blots of secreted APPs and APLP2s
with the specific antisera R1736 and D2-1. APPs, secreted APPs detected
by the APP-specific antiserum R1736 directed against the 16 N-terminal
residues of the A domain (absent in APLP2). R1736 detects
increased signals in 5-HT-treated 3T3 cells (lane 3, 0.01
µM 5-HT; lane 4, 20 µM 5-HT stably
transfected with 5-HT2cR as compared with vehicle-treated transfected
cells (lane 1). APLPs, secreted APLP2s detected by the
APLP2-specific antiserum D2-1 raised against baculovirus-expressed
full-length APLP2. This antiserum does not cross-react with APPs.
Compared with the vehicle-treated control condition (lane 5),
D2-1 detected increased signals in 5-HT-treated (10 µM; lanes 6 and 7) and DNF-treated (1 µM; lane 8) 3T3 cells transfected with
5-HT2cR.
The results of this study show that the rates at which APPs is secreted by cultured 3T3 cells stably overexpressing 5-HT2aR and 5-HT2cR can be accelerated by serotonin or by the serotoninergic agonist DNF. This finding, coupled with previous observations that stimulating muscarinic acetylcholine receptor subtypes m1 and m3 (Buxbaum et al., 1992; Nitsch et al., 1992; Wolf et al., 1995) or metabotropic glutamate receptors (Lee et al., 1995) can increase APPs secretion, suggests that the regulation of APP processing may be a function of various cell surface receptor types, linked to a number of common second messenger systems.
Stimulation of 3T3 cells overexpressing 5-HT2aR or 5-HT2cR caused rapid and dose-dependent increases in phosphatidylinositol breakdown, as indicated by the accumulation of labeled inositol phosphates in the presence of lithium after metabolic labeling of phosphatidylinositol. These results confirm that both receptor subtypes are coupled to PI turnover signaling and assure that the stably transfected cell lines express functionally intact surface receptor proteins. Two additional lines of evidence demonstrated that stimulated APPs secretion was mediated by the activation of serotoninergic cell surface receptors: 5-HT failed to change APPs secretion from the untransfected parent 3T3 fibroblast line, and the increase in APPs secretion from the stably transfected cells was blocked by 5-HT receptor antagonists.
In
agreement with previous reports (Caporaso et al., 1992; Slack et al., 1993), direct activation of PKC with the phorbol ester
PMA increased APPs secretion in the cell lines used in this study. In
contrast to our previous observations with 293 cells overexpressing m1
and m3 acetylcholine receptors (Nitsch et al., 1992), however,
inhibition of PKC by the kinase inhibitors staurosporine or
chelerythrine chloride failed to block 5-HT-mediated increases in APPs
secretion from 3T3 fibroblasts. Moreover, down-regulation of PKC by
prior chronic pretreatment with PMA also failed to block the
5-HT-induced increase in APPs secretion (Fig. 4A).
These data imply that activation of PKC can be sufficient to increase
APPs secretion, but that this activation is not necessary for the
5-HT2R-mediated increase. The concentrations of 5-HT necessary to
elicit maximal acceleration of PI breakdown, as well as the EC of this effect, were 1 to 2 orders of magnitude lower than these
for stimulated APPs secretion. This is compatible with the possibility
that APP processing is also coupled to other cellular signaling
pathways besides PI turnover and PKC activation. To test whether
PLA
is involved in 5-HT-mediated APPs secretion, we
examined the ability of drugs that inhibit this enzyme, and we also
used melittin, a PLA
-activating peptide. Stimulation of
5-HT2aR-expressing cells with 5-HT in the presence of the PLA
inhibitors manoalide, dimethyleicosadienoic acid, or
oleyloxyethyl phosphorylcholine failed to affect basal APPs secretion,
but all three drugs inhibited 5-HT-induced secretion. These results
demonstrate that 5-HT2aR activation can accelerate APPs secretion by
coupling to PLA
. A role of PLA
in the
regulation of APPs processing was also suggested by the finding that
melittin accelerated APPs secretion. Moreover, this effect was not
blocked by down-regulation of PKC by chronic exposure to PMA, and
manoalide, dimethyleicosadienoic acid, or oleyloxyethyl
phosphorylcholine blocked 5-HT-induced increases in APPs secretion
after down-regulation of PKC. Hence, 5-HT2aR-coupled and
PLA
-mediated acceleration of APPs secretion can occur
independently of PKC. These data are consistent with the previous
observation that PLA
can partially mediate the increase in
APPs secretion induced by muscarinic stimulation (Emmerling et
al., 1993).
Cellular signaling mechanisms that couple 5-HT2cR
to APP processing were more complex than in the cells overexpressing
5-HT2aR. As with 5-HT2aR activation, cells expressing 5-HT2cR responded
with increased APPs secretion to 5-HT after down-regulation of PKC, and
the kinase inhibitors staurosporine and chelerythrine chloride failed
to inhibit this increase. In contrast to the findings in 5-HT2aR cells,
however, the PLA inhibitors failed to block consistently
the 5-HT-mediated increase in APPs secretion in 5-HT2cR cells. The
combination of PKC down-regulation and PLA
inhibition did
effectively inhibit the ability of 5-HT to stimulate APPs secretion. It
is therefore possible that the coupling of 5-HT2cR to APPs secretion
requires both PKC and PLA
activities. Treatment of the
5-HT2R-expressing cells with thapsigargin, which discharges
Ca
from internal stores by inhibiting the
Ca
-ATPase of the endoplasmic reticulum membrane
failed to affect APPs secretion in the 3T3 cells used. Perhaps the
previously reported effects of Ca
on APPs secretion
(Buxbaum et al., 1994) are cell type-specific.
APP is a
member of the multigene family of APP-like proteins (APLP). APLP2 is
homologous to APP in the extreme C-terminal region, and has various
homologous regions in the ectodomain (Slunt et al., 1994; von
der Kammer et al., 1994; Wasco et al., 1992, 1993).
However the A domain is not preserved; thus no amyloidogenic
derivatives can be generated from APLP2. Because of its similarities to
APP, APLP2 is detected by nonselective antibodies (Slunt et al., 1994), including the monoclonal antibody 22C11 that was raised
initially against an APP fusion protein (Weidemann et al., 1989). To differentiate between APPs and APLPs, we used the
antiserum R1736 (Haass et al., 1992) directed against the 16
N-terminal residues of the A
domain. R1736 detected the same
receptor-mediated increase in secreted APPs as 22C11 (Fig. 8).
The APLP2-specific antiserum D2-1 (Slunt et al., 1994)
detected secreted APLP2 derivatives (APLP2s) with the expected
molecular masses in the cell culture supernatants obtained from 3T3
cells overexpressing 5-HT2aR or 5-HT2cR. Stimulation of these cell
lines with 5-HT substantially increased APLP2 s secretion (Fig. 8). These data suggest that APP and APLP2 are processed by
similar receptor-regulated proteolytic pathways, and they imply the
possibility that APLP2 may compete with APP for enzymes involved in
signal transduction and proteolytic processing. Levels of APLP2 may
thus influence the metabolism of APP. The cellular mechanism of
receptor-regulated APPs and APLP2s secretion is unclear. As in
previously reported experiments, 5-HT2R-mediated increases in APPs
secretion occurred in the absence protein synthesis confirming that
pre-existing protein is processed in response to receptor activation.
It is possible that secretory vesicle formation, trafficking, or
proteolytic cleavage events are accelerated by surface receptor-coupled
signaling. The first possibility is underscored by the recent finding
that PKC stimulates vesicle budding from the trans-Golgi network and
thus accelerates the formation of APP-containing secretory vesicles in
a reconstituted cell-free system (Xu et al., 1995).
The
physiological relevance of regulated APPs secretion is unclear. We
speculate that having APP processing under neurotransmitter control may
allow neuronal activity to control the formation of a secretory
derivative (Farber et al., 1995; Nitsch et al., 1993)
with possible paracrine neurotrophic and neuroprotective activities.
Regulated cleavage of a membrane precursor followed by secretion of the
ectodomain has been described for a variety of transmembrane proteins,
including transforming growth factor- (Bosenberg et al., 1992; Pandiella et al., 1991), and the tumor necrosis
factor receptor (Brakebusch et al., 1992), and it was proposed
that regulated secretion is involved in switching transforming growth
factor-
's activity from that of a juxtacrine to a paracrine
growth factor (Pandiella and Massague, 1991). Growth factor-like
activities of APPs have been observed in several cell culture models.
In particular, a neurotrophin-like stimulation of neurite outgrowth and
branching was observed in PC-12 cells (Milward et al., 1992).
Conversely, antisense constructs directed against APP transcripts
inhibit neurite outgrowth in primary neurons (Allinquant et al., 1995). A biologically active domain that may promote trophic
activities of APP in vivo was mapped to the N terminus of APPs
(Roch et al., 1994). APPs also protects primary neurons from
glutamate-induced excitotoxic damage (Mattson et al., 1993),
presumably by suppressing potentially toxic increases in intracellular
calcium (Barger et al., 1995). Thus, it is possible that
secreted APPs has trophic functions in brain that are unrelated to
APP's role as an amyloid precursor.
Regulated APPs secretion
can be mediated by cholinergic (Buxbaum et al., 1992; Nitsch et al., 1992; Wolf et al., 1995) and glutamatergic
(Lee et al., 1995) agonists, and electrical activity of brain
cells activated APPs secretion from freshly prepared tissue slices.
Hence, receptor-coupled APP processing may normally occur throughout
the brain at muscarinic, glutamatergic, and, now, serotoninergic
synapses. In most (Buxbaum et al., 1993; Gabuzda et al., 1993; Hung et al., 1993; Wolf et al., 1995), but
not all (Dyrks et al., 1994) cell types, receptor-mediated
activation of APPs secretion is associated with decreased generation of
A. We were unable to measure A
secreted from the 3T3
fibroblasts within the short time intervals used for receptor
stimulation in this study. Because cultured fibroblasts secrete very
little amounts of A
, time periods of more than 24 h are necessary
to detect measurable levels with current immunoadsorbent assays. Thus
it remains to be investigated whether serotonin receptor-induced
stimulation of APPs secretion is associated with changes in the rate of
A
secretion.
In Alzheimer's disease brain, amyloid deposits are present throughout the brain cortex, and they are not co-localized with any specific neurotransmitter system. Similarly, many neurotransmitter systems, including the cholinergic, serotoninergic, glutamatergic, and peptidergic systems, are heavily damaged in Alzheimer's disease brains, and this damage is associated with significant losses in cortical synapses (Terry et al., 1991). Inasmuch as these pathological alterations and the resulting deafferentation of target cells are associated with amyloidogenic APP processing, they may be involved in promoting the amyloid formation in Alzheimer's disease brain.
Identifying cell surface receptors,
such as 5-HT2aR and 5-HT2cR, whose stimulation increases APPs
secretion, could constitute a useful novel pharmacological strategy for
manipulating of APP processing in brain, for promoting the potential
functions of APPs as a paracrine neurotrophic/neuroprotective factor,
and for concomitantly reducing the formation of amyloidogenic
derivatives. A possible candidate compound may be dexfenfluramine, a
widely used anti-obesity drug which is metabolized to
dexnorfenfluramine in vivo. Brain levels of dexnorfenfluramine
in subjects taking therapeutic doses of dexfenfluramine (30 mg/day) are
probably in the order of 1-3 µM. ()In our
transfected cell lines, this concentration of dexnorfenfluramine was
sufficient to promote both APPs secretion and PI turnover (Fig. 6). Clinical studies with highly selective receptor
agonists are needed to determine whether this approach can modify the
clinical course of Alzheimer's disease.