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INTRODUCTION |
No decision is more important for an individual cell than that
which determines whether it will survive or die. It is not surprising
that recent research has found this biochemical decision-making process
encompasses a regulatory web with a complexity matching the importance
of the decision (1, 2). Thus, throughout life cells mobilize an active
effort to repress programs that could lead to cell death. In many
instances, continued survival is dependent upon the environment of the
cell, because cell-to-cell contact and signals received from
neighboring cells may provide the necessary impetus to bolster defenses
against death programs. Eventually, through senescence, insults, and/or
loss of adequate inputs, cells are eliminated, often to make room for
replacements. In the central nervous system, however, replacement
neurons are only available marginally if at all (3, 4), putting a
greater burden on the survival mechanisms of mature neurons.
Determining how neurons are able to survive manyfold longer than the
average mammalian cell poses one of the great challenges of research in neurobiology (5, 6). This question is more than of academic interest
because failure to survive, either during the developmental process,
during aging, or upon exposure to potentially lethal insults, is the
basis for innumerable neurodegenerative conditions.
Neurodegenerative diseases often arise as the result of increased
burdens placed on the survival-promoting mechanisms of neurons (7). The
accumulation of mutated proteins or toxic insults can challenge
survival mechanisms, causing neurons to prematurely succumb, which in
some instances of adult-onset neurodegenerative disorders follows
decades of heightened challenge (8). For example, in Alzheimer's
disease (AD),1 it is apparent
that neurons survive many years of heightened stress caused by
mutations and/or elevated stressors before the catastrophic effects are
evident (9). Because the establishment of synaptic contacts is a
critical factor for neuronal survival during development (10, 11),
synaptic activity also may be critical for bolstering cellular defenses
from toxic insults that contribute to neurodegenerative diseases. In
this regard, it is well known that activation of receptors by
neurotrophins, such as nerve growth factor, has a profound influence on
cell survival (12). However, much less is known about the
survival-promoting potential of receptors for classical
neurotransmitters, such as cholinergic muscarinic receptors.
The present investigation was undertaken to determine the extent to
which activation of plasma membrane neurotransmitter receptors are
capable of providing protection from widely encountered toxic conditions. Considering the substantial but still controversial evidence that loss of cholinergic signaling activity may be an early
event in AD (13), activation of cholinergic muscarinic receptors was
used as the model for neurotransmitter input. Three toxic conditions
were examined: DNA damage, oxidative stress, and impaired mitochondrial
function. DNA damage is an important initiator of neuronal apoptosis,
which is evident in conditions including ischemia, oxidative stress,
several neurodegenerative diseases, and after exposure to
chemotherapeutic agents, as recently reviewed (14). Experimentally,
camptothecin, a topoisomerase 1 inhibitor, has been used to generate
DNA damage in neuronal model systems. Camptothecin treatment induces
p53-dependent neuronal death, which follows activation of
the pro-apoptotic protein Bax (15-17). Oxidative stress appears to be
one of the most prevalent causes of neuronal dysfunction and demise in
neurodegenerative disorders and can cause DNA damage. This
overproduction of reactive oxygen species or diminished anti-oxidant
capacity, is evident in Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, aging, stroke, and many other conditions
associated with impaired function and loss of neurons (18-22). In
addition, oxidative stress is an early event in AD pathology, and the
extent and distribution of neurodegeneration in AD parallels indices of
oxidative damage (9, 23-25). Mitochondrial dysfunction also has been
implicated in numerous neurodegenerative conditions (26, 27).
Therefore, the mitochondrial complex I inhibitor rotenone (28) was used
in this study because rotenone administration has been used to model
the neuronal dysfunction of Parkinson's disease (29).
The results show that muscarinic receptor stimulation provides
remarkably effective protection from each of these three disparate insults. These findings support the notion that synaptic activity, through activation of neurotransmitter receptors, can provide substantial support of cellular survival mechanisms, suggesting that
loss of such synaptic input increases vulnerability to insult-induced programmed cell death.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Treatments--
Human neuroblastoma SH-SY5Y
cells were grown in RPMI medium (Cellgro, Herndon, VA) supplemented
with 10% horse serum (Invitrogen), 5% fetal clone II (Hyclone, Logan,
UT), 2 mM L-glutamine, 100 units/ml penicillin,
and 100 µg/ml streptomycin. Cells were maintained at 37 °C in 95%
air, 5% CO2. Experimental agents used include H2O2, 3-aminobenzamide (3AB), oxotremorine-M,
camptothecin, nicotine, carbachol, mecamylamine, atropine, and rotenone
from Sigma and boc-aspartyl(OMe)-fluoromethyl ketone (BAF) from Alexis
Biochemicals (San Diego, CA).
Enzyme Assays--
Caspase-3 activity was assessed by measuring
cleavage of a fluorogenic substrate, as described previously (30).
Lactate dehydrogenase release was detected using the cytotoxicity
detection kit (Roche Molecular Biochemicals) according to the
manufacturer's protocol.
Immunoblotting--
Cells were harvested, washed twice with
phosphate-buffered saline, and lysed in lysis buffer (20 mM
Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
10 µg/ml aprotinin, 5 µg/ml pepstatin, 1 nM okadaic
acid, and 0.5% Nonidet P-40). The lysates were sonicated for 10 s
on ice and centrifuged at 16,000 × g for 15 min, and supernatants were collected. Protein concentrations were determined using the Bradford method (31) or the bicinchoninic (BCA) method (Pierce). Where indicated, nuclear fractions were prepared as described
previously (32). For preparation of cytosolic and mitochondrial
fractions, harvested cells were disrupted by nitrogen cavitation (33)
followed by sucrose gradient ultracentrifugation (34), and immunoblots
of tubulin and cytochrome oxidase were used as markers of cytosolic and
mitochondrial fractions, respectively. Proteins were resolved in
SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and
incubated with primary antibodies followed by incubation with
horseradish peroxidase-conjugated secondary antibodies. The primary
antibodies used were: poly (ADP-ribose) polymerase (PARP), p21,
cytochrome c (BD Pharmingen), p53, bcl-2, Bax, tubulin
(Upstate Biotechnology, Lake Placid, NY), cytochrome oxidase (Molecular
Probes, Eugene, OR). Immunoblots were developed with enhanced
chemiluminescence (Amersham Biosciences) and were quantified by densitometry.
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RESULTS |
Oxotremorine-M Protects against Camptothecin-Induced Caspase-3
Activation and Cell Death--
We first examined if muscarinic
receptor activation is capable of providing protection from apoptosis
after DNA damage. To do this, we used camptothecin, a topoisomerase 1 inhibitor, to generate DNA damage-induced apoptosis, which we recently
characterized in SH-SY5Y cells (35). As reported, treatment with 1 µM camptothecin caused a large and relatively rapid
increase in the activity of caspase-3 (Fig.
1A). Activation
of muscarinic receptors with the selective agonist oxotremorine-M (300 µM; 30 min of pretreatment) attenuated by ~75%
caspase-3 activation induced by camptothecin (above the basal
level of activity). This protective effect of oxotremorine-M was
completely blocked by the specific muscarinic receptor antagonist 1 µM atropine. Equivalent results were obtained when the
proteolysis of PARP was used as an indicator of the degree of apoptotic
signaling activation. PARP proteolysis increased time-dependently after camptothecin treatment, and
oxotremorine-M pretreatment greatly attenuated this response (Fig.
1B). Muscarinic receptor stimulation did not block the
initial response to camptothecin, as evidenced by the lack of effect of
oxotremorine-M treatment on the accumulation of p53 that followed
camptothecin treatment (Fig. 1C). Additionally, the
transcriptional activity of p53 was not blocked by oxotremorine-M
pretreatment, as increases in the level of p21waf1/cip1,
which is regulated by p53, followed the accumulation of p53 equivalently in the absence or presence of oxotremorine-M (Fig. 1C). This indicates that muscarinic receptor stimulation
provided protection at a site downstream from the initial toxic insult, in this case DNA damage and the subsequent accumulation and activation of p53. Examination of lactate dehydrogenase release into the medium as
a measure of cell death revealed that oxotremorine-M greatly attenuated
cell death (Fig. 1D). Oxotremorine-M pretreatment provided a
degree of protection that was nearly as great as that afforded by the
general caspase inhibitor BAF (Fig. 1D), an inhibitor previously reported to block the early, caspase-3-dependent
phase of cell death after camptothecin treatment of primary cortical neurons (16). Furthermore, protection by oxotremorine-M was blocked by
atropine. Thus, caspase-3 activation and cell death induced by the
DNA-damaging agent camptothecin was substantially attenuated by
stimulation of muscarinic receptors.

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Fig. 1.
Oxotremorine-M protects cells from
camptothecin-induced caspase-3 activation and cell death.
Caspase-3 activity (A) and PARP proteolysis (B)
were measured in cells treated with 1 µM camptothecin
(CT), 300 µM oxotremorine-M (OxoM)
30 min before camptothecin, and 1 µM atropine (Atr) 15 min before oxotremorine-M followed by camptothecin. C, p53
and p21 levels were measured in cells treated with camptothecin alone
or with pretreatment with 300 µM oxotremorine-M for 30 min. D, lactate dehydrogenase (LDH) release into
the media was measured 4-10 h after treatment with camptothecin with
or without pretreatments with 100 µM BAF or 300 µM oxotremorine-M and in cells treated with 1 µM atropine before oxotremorine-M treatment. Quantitative
values are means ± S.E.; n = 4-5; *,
p < 0.05.
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Muscarinic Receptor Activation Protects against
H2O2-induced Caspase-3 Activation and Cell
Death--
To test if muscarinic receptor stimulation provided
protection from oxidative stress, it was first necessary to
characterize the H2O2 treatment conditions
needed to activate caspase-3 and induce cell death in SH-SY5Y cells.
The primary mechanism of apoptotic signaling after oxidative stress
ultimately involves activation of the effector enzyme, caspase-3 (1).
Subsequently, activated caspase-3 cleaves susceptible substrates, such
as PARP (36). Caspase-3 activity increased after 2.5 h of
treatment with 100 µM H2O2 and
began to plateau between 4.5 and 5.5 h after treatment (Fig.
2A). The activation of PARP
(37), which may follow oxidative stress and DNA damage, can lead to
depletion of cellular NAD+ and ATP, an effect that can be
avoided by the use of PARP inhibitors, such as 3AB (38, 39). Therefore,
in addition to samples treated with 100 µM
H2O2 alone, in-parallel
H2O2-treated samples PARP was inhibited by
pretreatment with 100 µM 3AB. Similar rates and magnitudes of caspase-3 activation were obtained after treatment with
100 µM H2O2 with or without
inhibition of PARP (Fig. 2A). The effects of
H2O2-induced caspase-3 activation were
reflected in measurements of the proteolysis of PARP, which is mediated by caspase-3 and results in the production of a stable 85-kDa product
cleaved from the 116-kDa intact PARP. Treatment with 50-200 µM H2O2 caused a
concentration-dependent increase in PARP proteolysis (Fig.
2B) and in caspase-3 activity (see below) that was
similar in the presence and absence of 3AB. Cell death, assessed by
measuring the release of lactate dehydrogenase into the media, was
prominent between 8 and 24 h after treatment with 100 µM H2O2 in the absence or
presence of 3AB (Fig. 2C). After 24 h, 60% of cells
treated with H2O2 had died, and only slightly
less (50%) had died after treatment with H2O2
plus 3AB.

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Fig. 2.
H2O2 treatment
induces apoptosis. A, the time course of caspase-3
activation was measured in cells treated with 100 µM
H2O2 with or without a 30-min pretreatment with
100 µM 3AB. (B) PARP proteolysis was measured in cells
treated with 50 to 200 µM H2O2
for 5 h, with or without a 30 min pretreatment with 100 µM 3AB, by immunoblot analysis. The immunoblot shows the
85-kDa product of PARP cleaved by caspase-3. C, the time
course of lactate dehydrogenase release was measured in cells treated
with 100 µM H2O2 with or without
a 30-min pretreatment with 100 µM 3AB, in cells treated
with only 3AB, and in control untreated cells. Quantitative data are
means ± S.E.; n = 3-4.
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Using these established conditions, we examined if muscarinic receptor
stimulation with oxotremorine-M modulated cellular responses to
H2O2. Pretreatment with 300 µM
oxotremorine-M (30 min) greatly reduced caspase-3 activity induced by
100, 150, and 200 µM H2O2 in the
presence or absence of 3AB (Fig.
3A). Remarkably, nearly
complete blockade of caspase-3 activation was attained with
oxotremorine-M in cells incubated with 100 µM
H2O2, and even with the highest concentration
of H2O2 tested, 200 µM, caspase-3 activity was reduced more than 50% by treatment with oxotremorine-M. Measurements of PARP proteolysis showed that it mirrored changes in
caspase-3 activity, as oxotremorine-M treatment also greatly attenuated
the H2O2 concentration-dependent
induction of PARP proteolysis (Fig. 3B). Thus, stimulation
of muscarinic receptors provided substantial protection from the
activation of caspase-3 caused by oxidative stress induced by
H2O2.

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Fig. 3.
Oxotremorine-M treatment protects cells from
H2O2-induced apoptosis. SH-SY5Y cells were
pretreated with 100 µM 3-AB for 30 min, 100 µM BAF for 60 min, or 300 µM oxotremorine-M
(OxoM) (with or without a 15-min pretreatment with 1 µM
atropine (Atr)) for 30 min where indicated followed by
treatment with H2O2. Caspase-3 activity
(A) and PARP cleavage (B) were measured 6 h
after treatment with 50-200 µM
H2O2. C, proteolyzed PARP was
measured 4, 5, and 6 h after treatment with 100 µM
H2O2 in cells pretreated with 3AB and, where
indicated, with oxotremorine-M or BAF. D, lactate
dehydrogenase (LDH) release into the media was measured 11 and 24 h after treatment with 100 µM
H2O2 and the indicated agents. Nuclear p53
levels (E) and p21 levels (F) were measured 2, 3, and 4 h after treatment with 100 µM
H2O2 without or with a 30-min pretreatment with
300 µM oxotremorine-M. Quantitative values are the
means ± S.E.; n = 3-4; *, p < 0.05.
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To evaluate the significance of the protective effect of muscarinic
receptor activation, the protection provided by oxotremorine-M was
compared with that of a general caspase inhibitor, BAF. These experiments demonstrated that the substantial protection provided by
treatment with oxotremorine-M against 100 µM
H2O2-induced PARP proteolysis was similar to
that afforded by 100 µM BAF (Fig. 3C). Furthermore, the effects of oxotremorine-M and BAF were compared on
H2O2-induced cell death, as assessed by
measurements of lactate dehydrogenase release into the media at early
(11 h) and late (24 h) times of cell death that were determined
previously (Fig. 2C). Treatment with the general caspase
inhibitor, 100 µM BAF, blocked cell death by 80% 11 h after treatment, indicating that early cell death induced by 100 µM H2O2 plus 3AB was
predominantly caspase-dependent (Fig. 3D).
However, after 24 h, pretreatment with BAF only reduced cell death
by 30%, indicating that later cell death is mediated by a
caspase-independent mechanism. A similar transition, with protection by
BAF at shorter times after oxidative stress followed by loss of
protection at 24 h, has been reported previously (40). In the same
experiments in which BAF was tested, parallel samples were used to
determine whether cell death was modulated by activation of muscarinic
receptors. Once again, 300 µM oxotremorine-M (30 min
preincubation) afforded the same extent of protection as did inhibition
of caspases with BAF (Fig. 3D), reducing cell death by 80%
11 h after treatment with H2O2. The protective effect of oxotremorine-M was completely blocked by pretreatment with the muscarinic antagonist atropine (1 µM), confirming that oxotremorine-M action was mediated
by a muscarinic receptor-dependent mechanism. There was a
lower protective effect of oxotremorine-M 24 h after treatment
with H2O2, which matched the diminished effect of BAF. Thus, treatment with H2O2 caused
activation of caspase-3 and an early caspase-dependent cell
death, and these effects were greatly attenuated by the caspase
inhibitor BAF and to a similar extent by muscarinic receptor activation.
The complete signaling pathway mediating
H2O2-induced caspase-3 activation is not known,
but an early event in the response to H2O2 is
stabilization, accumulation, and transcriptional activation of the
tumor suppressor p53. Treatment with 100 µM
H2O2 caused a large increase in the level of
p53 that was evident 2 h after treatment, before activation of
caspase-3 (Fig. 3E). There was a corresponding increase in
the level of the p53-regulated protein p21, indicating the p53 was
transcriptionally active (Fig. 3F). Pretreatment with 300 µM oxotremorine-M (30 min) did not reduce the
H2O2-induced increases in p53 or p21 (Fig. 3,
E and F), indicating that oxotremorine-M did not
exert an antioxidant effect blocking initial responses to
H2O2 but, rather, attenuated signaling leading to caspase-3 activation and cell death.
Stimulation of Muscarinic Receptors Protects against
Rotenone-induced Caspase Activation but Not from Staurosporine--
To
examine if the protection afforded by muscarinic receptor stimulation
also was evident in another condition that causes apoptosis,
experiments were carried out using rotenone to inhibit the
mitochondrial complex 1, which causes oxidative stress (41). In
accordance with our recent report that characterized rotenone-induced caspase-3-mediated apoptosis in SH-SY5Y cells (42), there was a nearly
6-fold increase in caspase-3 activity after treatment with 5 µM rotenone (Fig.
4A). Pretreatment with 300 µM oxotremorine-M almost completely prevented
rotenone-induced activation of caspase-3, whereas activation of
nicotinic cholinergic receptors with nicotine provided no protection
from rotenone. Treatment with carbachol, a nonspecific cholinergic
receptor agonist, prevented caspase-3 activation by rotenone to a
similar extent as oxotremorine-M, and the protective effect of
carbachol was prevented by the muscarinic receptor antagonist atropine
but was unaffected by the nicotinic receptor antagonist mecamylamine.
Thus, activation of muscarinic receptors greatly attenuated caspase-3
activation induced by rotenone as well as by camptothecin and
H2O2.

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Fig. 4.
Muscarinic receptor stimulation attenuates
caspase-3 activation induced by rotenone but not staurosporine.
A, caspase-3 activity was measured in cells 16 h after
treatment with 5 µM rotenone (Rot). Where
indicated, cells were pretreated for 30 min with 300 µM
oxotremorine-M (Oxo), 300 µM carbachol
(Cb), or 10 µM nicotine (Nic).
Cells were treated with the antagonists 10 µM atropine
(Atr) or 10 µM mecamylamine (Mec)
15 min before treatment with agonists. Values are presented as the
percent of caspase-3 activity in untreated control (Ctl)
cells and are the means ± S.E.; n = 3-4; *,
p < 0.05 compared with values from samples treated
with rotenone alone. B, caspase-3 activity was measured in
cells treated with 0.5 µM staurosporine alone or after a
30-min pretreatment with 300 µM oxotremorine-M. Values
are presented as the percent of caspase-3 activity in untreated control
cells (means ± S.E.; n = 3).
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It is important to note that not all apoptotic-inducing insults are
attenuated by stimulation of muscarinic receptors. Staurosporine is one
of the most widely used agents to activate caspase-3-mediated apoptosis, and we previously characterized the staurosporine
concentration- and time-dependent activation of apoptosis
in SH-SY5Y cells (30). Treatment with staurosporine caused a
time-dependent increase in caspase-3 activity, and there
was no attenuation in cells pretreated with oxotremorine-M (Fig.
4B). Thus, muscarinic receptor activation does not cause a
global inhibition of the activation of caspase-3 caused by apoptotic
stimuli, but it provides protection from a discreet group of insults.
Oxotremorine-M Treatment Prevents Camptothecin-induced
Mitochondrial Cytochrome c Release, bcl-2 Depletion, and Bax
Accumulation--
We focused on the actions of oxotremorine-M against
the deleterious effects of camptothecin to examine the mechanism of
protection. To examine if the protective effect of stimulated
muscarinic receptors was upstream or downstream of the mitochondrial
release of cytochrome c, we monitored the mitochondrial and
cytosolic levels of cytochrome c. Treatment with
camptothecin (1 µM; 4 h) caused a massive release of
cytochrome c into the cytosol, resulting in a nearly 4-fold increase in the cytosolic cytochrome c level and a 60%
decline in the mitochondrial cytochrome c content (Fig.
5). Pretreatment with oxotremorine-M (300 µM; 30 min) completely blocked this displacement of
cytochrome c from the mitochondria into the cytosol. Thus, the protective effect of muscarinic receptor stimulation targeted a
step in the apoptotic signaling pathway preceding cytochrome c release from the mitochondria.

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Fig. 5.
Muscarinic receptor stimulation blocked
cytochrome c release from the mitochondria caused by
camptothecin treatment. Cells were treated with 1 µM
camptothecin (CT) or with 300 µM
oxotremorine-M (Oxo) 30 min before camptothecin, and
cytosolic and mitochondrial fractions were immunoblotted for cytochrome
c (Cyt c). Quantitative values are the means ± S.E.; n = 3.
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Members of the bcl-2 family of proteins are key regulators of the
mitochondrial-dependent apoptotic pathway. For example, the
pro-apoptotic Bax can translocate to the mitochondria to transmit apoptotic signals, an event that can reduce the mitochondrial level of
anti-apoptotic bcl-2. Therefore, we tested if treatment with
oxotremorine-M influenced changes in the mitochondrial levels of Bax
and bcl-2 after exposure to camptothecin. Treatment with camptothecin
increased the level of Bax in the mitochondria by ~4-fold (Fig.
6A). This
translocation of Bax to the mitochondria induced by camptothecin was
nearly completely blocked by pretreatment with oxotremorine-M. The
mitochondrial bcl-2 levels were regulated opposite to those of Bax. As
shown in Fig. 6B, camptothecin treatment caused a reduction
in the mitochondrial level of bcl-2, and pretreatment with
oxotremorine-M prevented this loss of mitochondrial bcl-2. Thus, the
protective action of stimulated muscarinic receptors after camptothecin
treatment is associated with blockade of the activation of
pro-apoptotic Bax, which was associated with retention of mitochondrial
bcl-2 and cytochrome c.

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Fig. 6.
Mitochondrial Bax and bcl-2 levels.
Cells were treated with 1 µM camptothecin (CT)
or with 300 µM oxotremorine-M (Oxo) 30 min
before camptothecin, and mitochondrial fractions were immuno-blotted for Bax (A) or bcl-2 (B).
Quantitative values are the means ± S.E.; n = 3. C, mitochondrial Bax levels were measured after treatment
with 100 µM H2O2 (5 h) or 5 µM rotenone (16 h) with or without a 30-min pretreatment
with 300 µM oxotremorine-M.
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Because the mitochondrial accumulation of Bax is likely most proximal
to muscarinic receptors of these events, we examined mitochondrial Bax
levels after treatment with H2O2 and rotenone. A similar oxotremorine-M-induced diminution of Bax translocation to the
mitochondria was detected after treatment with
H2O2 or with rotenone (Fig. 6C),
suggesting that a common survival-promoting mechanism, blockade of Bax
activation, is induced by stimulation of muscarinic receptors after
each of these insults.
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DISCUSSION |
This investigation found that activation of muscarinic receptors
provided substantial protection from the induction of apoptotic signaling caused by three common insults, DNA damage, oxidative stress,
and impaired mitochondrial function. Although cells eventually succumbed because of the extremity of the toxic treatments employed, remarkable protection from the activation of caspase-3 induced by each
of these conditions was evident upon stimulation of muscarinic receptors, a protection that was associated with reduced translocation of the pro-apoptotic protein Bax to the mitochondria. To study this
effect in cells with a relatively uniform level of expression of
muscarinic receptors, human neuroblastoma SH-SY5Y cells were used
because they endogenously express muscarinic receptors predominantly of
the M3 subtype (43), as opposed to mixed populations of cells and
receptors present in most other neuronal preparations. These properties
of physiologically relevant levels of receptors and the uniformity of
receptor expression were deemed sufficiently advantageous to balance
the limitations imposed by the utilization of proliferating cells. In
fact, many studies show that exposure to differentiating agents
enhances defenses against many insults, such as oxidative stress
(44-46), suggesting that this model system imposed a greater challenge
for rescuing cells from these insults. However, with this
identification of the protective capacity of activated muscarinic
receptors, further efforts should be directed toward examining these
interactions in mature neurons and determining whether or not
stimulation of other G-protein-coupled receptors also provides protection.
Stimulation of muscarinic receptors provided protection from three
relatively common insults. Oxidative stress contributes to neuronal
dysfunction and loss in many neurodegenerative conditions (21, 22). For
example, much evidence links oxidative stress to the neuropathology of
AD (9, 18, 19, 20, 25), where there is a concomitant loss of
cholinergic function (13), including loss of muscarinic
receptor-coupled signaling activities (47). Based on this co-existence
of oxidative stress and loss of muscarinic receptor function, it was of
interest to investigate if loss of muscarinic receptor-coupled
signaling could contribute to the deleterious effects of oxidative
stress by testing if active muscarinic receptors provide protection
from caspase activation. The results demonstrated that activation of
muscarinic receptors with the selective agonist oxotremorine-M provided
significant protection from the apoptosis-signaling cascade induced by
H2O2. Muscarinic receptor activation also
provided substantial protection from caspase-3 activation ensuing from
inhibition of mitochondrial function or DNA damage. These findings
suggest that loss of muscarinic receptor activity in neurodegenerative
conditions, such as AD, likely increases the vulnerability of cells to
the detrimental effects of these insults.
The protection provided by muscarinic receptor activation was of a
similar magnitude to that of a general caspase inhibitor. For both of
these, protection was greatest at lower concentrations of
H2O2 and at short times of treatment with
H2O2 or camptothecin. The former suggests that
muscarinic receptor activation may provide considerable protection from
physiologically relevant levels of oxidative stress. The delayed death
observed even in the presence of either oxotremorine-M or the caspase
inhibitor BAF could be due to necrotic mechanisms contributing to the
later cell death, against which these agents were ineffective, or the
agents themselves may have lost the ability to provide protection after
long incubation periods. For example, the agents may have been degraded
or sequestered, or prolonged exposure to oxotremorine-M may have
down-regulated relevant signaling activities, which lessened the
protection provided from cell death. Regardless of the cause of the
temporally limited protection, it is notable that muscarinic receptor
activation was essentially equivalent to inhibition of caspases in
providing protection, indicating that at physiologically relevant
levels of toxic insults, considerable protection is provided by
intracellular signals emanating from muscarinic receptors.
The signaling mechanisms responsible for the protective effect of
muscarinic receptor activation on the detrimental effects of these
insults appeared to be directed at Bax. The results clearly showed that
oxotremorine-M did not prevent the initial detrimental effects of
H2O2 or camptothecin, as exemplified by the
equal activation of p53 by each of these agents in the presence and
absence of oxotremorine-M. However, oxotremorine-M treatment blocked
the translocation of Bax to mitochondria, blocked loss of mitochondrial bcl-2, and blocked cytochrome c release to the cytosol. The
latter action leads to activation of caspase-3, indicating that the
muscarinic receptor-induced protection from caspase-3 activation likely
results from these actions, originating at the point of Bax activation, which is known to mediate apoptosis after a variety of insults, such as
DNA damage (48). The mechanism by which stimulated muscarinic receptors
block apoptotic activation of Bax remains to be determined. In the
absence of apoptotic conditions, Bax is predominantly cytosolic, at
least partially sequestered by the scaffold protein 14-3-3 (49, 50).
Conformational changes in Bax appear to lead to its translocation to
the mitochondria and oligomerization (51-53) followed by release of
cytochrome c (54), although precise details of the molecular
mechanisms underlying this transition during apoptotic signaling remain
unknown. Our findings suggest that signals from muscarinic receptors
attenuate apoptotic activation of Bax, an action that may be mediated
by signals (e.g. kinase activities, calcium transients)
impinging on Bax, 14-3-3, or other regulatory molecules known to
participate in Bax activation (e.g. Refs. 55 and 56),
retarding its activation and translocation to the mitochondria.
However, the precise intermediate steps accounting for Bax activation
as well as muscarinic receptor-induced neuroprotection await identification.
AD is associated with both increased neuronal oxidative stress and loss
of cholinergic function. The results of this study demonstrate that
these two conditions likely exacerbate neuronal dysfunction since
activation of muscarinic receptor-coupled signaling provides protection
from oxidative stress-induced apoptosis. Thus, as muscarinic
receptor-coupled signaling activity becomes impaired during the
progression of AD, neurons lose this important mechanism of
counteracting the detrimental effects of oxidative stress. Interpretation of the results presented here must take into
consideration the current controversy about the extent to which
apoptosis contributes to Alzheimer's disease as well as other
neurodegenerative conditions (57). Therefore, we suggest that the
protection provided by muscarinic receptors against oxidative stress
and the other insults examined might be of greater importance for
maintaining neural function and plasticity, which likely are impaired
temporally before apoptosis after exposure to these insults, although
indicators of apoptotic signaling were used as outcome measures in this
study. The findings reported here also may have relevance for
developmental regulation of the central nervous system. There is a
wealth of information that cholinergic afferents to the cortex and
hippocampus serve a growth regulatory function in ontogeny (reviewed in
Refs. 58 and 59). The present results suggest that one action of cholinergic activity may be to promote survival of innervated neurons
during development by inhibiting apoptotic mechanisms. In this regard,
it is of interest that cell loss induced by alcohol exposure has been
hypothesized to stem at least in part from inhibitory actions of
alcohol on muscarinic receptor signaling (60). We would speculate that
stimulation of other G-protein-coupled receptors activating similar
signaling pathways also would bolster neuronal survival, providing a
means for innervated cells to survive under conditions that are lethal
to cells not connected to sufficiently active networks.
In conclusion, these results indicate that in many instances continued
neuronal survival is dependent upon the environment of the cell, as
cell-to-cell contact and signals received from neighboring cells may
provide the necessary impetus to bolster defenses against death programs.