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The Journal of Neuroscience, September 1, 1998, 18(17):6871-6881
Nicotinic Receptor-Induced Apoptotic Cell Death of Hippocampal
Progenitor Cells
Francois
Berger1,
Fred
H.
Gage1, and
Sukumar
Vijayaraghavan2
1 Laboratory of Genetics, The Salk Institute, La Jolla,
California 92037, and 2 Department of Physiology and
Biophysics, University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
Nicotine has many effects on CNS functions, presumably through its
action on neuronal nicotinic acetylcholine receptors (AChRs). One
subclass of AChRs that binds the snake venom toxin  -bungarotoxin (-Bgt-AChRs) has been shown to modulate neurotransmission in the
brain. We now show that -Bgt-AChR activation by low doses of
nicotine results in apoptotic cell death of both primary and immortalized hippocampal progenitor cells. In HC2S2-immortalized hippocampal progenitors, nicotine is cytotoxic to undifferentiated cells, whereas it spares the same cells once differentiation has been
induced. The activation of -Bgt-AChRs by nicotine results in the
induction of the tumor suppressor protein p53 and the cdk inhibitor
p21. The cytotoxic effect of nicotine is dependent on extracellular
calcium levels and is probably attributable to the poor ability of
undifferentiated progenitors to buffer calcium loads. The major calcium
buffer in these cells, calbindin D28K, is present only after
differentiation has been induced. Furthermore transfection of
undifferentiated cells with calbindin results in dramatic protection
against the cytotoxic effects of nicotine. These results show that
nicotine abuse could have significant effects on the survival of
progenitor populations in the developing and adult brain and also
suggest an endogenous role for -Bgt-AChRs in neuronal development
and differentiation.
Key words:
nicotine; acetylcholine receptors; -bungarotoxin; apoptosis; hippocampal progenitors; p53; cell cycle
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INTRODUCTION |
Neuronal nicotinic acetylcholine
receptors (AChRs) are widely distributed in the CNS, but very few
instances of synaptic transmission have been reported, suggesting that
these receptors might have a different, nontraditional role to play in
CNS functions (for review, see Sargent, 1993 ; Clarke, 1993; Role and
Berg, 1996). The belief that activation of AChRs can have profound
physiological consequences, however, has received widespread acceptance
because of the behavioral and physiological effects of nicotine in
smokers. In addition to the addictive and neuroprotective properties of nicotine (Arneric et al., 1995; James and Nordberg, 1995), studies have
also indicated significant cognitive, intellectual, and behavioral impairments in the offsprings of mothers who smoke during pregnancy (Sexton et al., 1990; Olds et al., 1994). Nicotine efficiently reaches
the amniotic fluid and fetal blood achieving concentrations similar to,
if not more than, that in the maternal blood (Lambers and Clark, 1996).
Changes in the development of cholinergic markers have been reported in
the brains of rat pups exposed to nicotine in utero (Navarro
et al., 1989). These studies on the teratogenic effects of nicotine add
an additional concern for the consequences of smoking among the general
population. Although the mechanisms have not been established, it is
possible that nicotine, through its actions on AChRs, can affect early
developmental events in the CNS.
A class of AChRs that binds, and is functionally blocked by, the snake
venom toxin -bungarotoxin (-Bgt-AChRs) is widely distributed in
the mammalian CNS. One area with high levels of the receptor is the
hippocampus (Freedman et al., 1993; Barrantes et al., 1995) in which
they have been shown to be functional on the soma of CA1 interneurons
in the stratum radiatum (Frazier et al., 1998) as well as on
presynaptic terminals of CA3 mossy fibers (Gray et al., 1996).
-Bgt-AChRs have also acquired prominence recently because of their
ability to modulate glutamatergic transmission in chick medial habenula
(McGhee et al., 1995) and rat hippocampus (Gray et al., 1996), by
virtue of their location at or near presynaptic terminals in these
neurons. The ability of -Bgt-AChRs to modulate synaptic events is
probably attributable to their high calcium permeability (Seguela et
al., 1993; Castro and Albuquerque, 1995; Rogers and Dani, 1995) and the
efficient way in which they can raise intracellular free calcium levels
([Ca]i; Vijayaraghavan et al., 1992). These
results have focused attention on calcium signaling by -Bgt-AChRs
and its consequences for neuronal function.
Many different lines of evidence suggest that -Bgt-AChRs might have
a role to play in neuronal development (for review, see Role and Berg,
1996). Expression patterns during development in the rat
thalamocortical terminals suggest that -Bgt-AChRs may affect events
during a critical period of cortical synaptogenesis (Bina et al., 1995;
Broide et al., 1996). The AChR 7 subunit, the main component of
these receptors, appears early in the development of chick ciliary
ganglion neurons (Corriveau and Berg, 1993) and also in presumptive
myoblasts and related cell types (Corriveau et al., 1995). The
significance of this regulation in the expression of -Bgt-AChRs
during neuronal development remains to be elucidated, but their high
calcium permeability supports a role for these receptors in early gene
expression (Greenberg et al., 1986). In addition, recent studies
implicate -Bgt-AChRs in cell proliferation in a neuroendocrine cell
line (Quik et al., 1994). Exposure to -Bgt saves chick motoneurons
from naturally occurring cell death (Hory-Lee and Frank, 1995). In
Caenorhabditis elegans, a mutation in the channel domain of
an AChR homolog showing sequence homology to the vertebrate 7 gene,
causes degeneration of specific early and late onset neurons (Treinin
and Chalfie, 1995).
One problem with examining signal transduction events during
development and differentiation of CNS neurons has been the paucity of
homogeneous cell populations. At the same time, effects observed in
such selected homogeneous populations of cells have to be validated in
more physiological systems. Our approach, therefore, has been to
identify a specific process triggered by AChR activation in primary
systems and then to perform a detailed analysis of the signal
transduction pathways involved, using a more homogeneous population of
cells.
In recent years, immortalized progenitor cells from fetal and adult CNS
have become systems of choice for the examination of developmental
events (for review, see Cepko, 1989; Gage et al., 1995a). These
cells are derived from primary cell cultures and exhibit their native
characteristics (Eves et al., 1992; Hoshimaru et al., 1996). Despite
numerous studies, there is no evidence for uncontrolled growth (Brustle
and McKay, 1996), and progenitors grown in culture for 2 years show
normal differentiation when grafted to neurogenic sites in the rat
brain (Suhonen et al., 1996). It is because of this fidelity that
progenitor cells grown in vitro are now considered as
possible tools for cell replacement therapy in the nervous system
(Brustle and McKay, 1996).
The hippocampal progenitors, HC2S2 cells, are advantageous for
studies on cellular aspects of signaling during differentiation because
they allow comparisons between a developmental and a differentiated setting. HC2S2 cells are committed to a neuronal fate, derived from
adult rat hippocampus, and immortalized by the stable expression of
v-myc driven by a tetracycline-controlled transactivator
(Hoshimaru et al., 1996). These cells, therefore, form ideal systems to
examine temporal events during early differentiation and
development.
Here we show that primary progenitors derived from rat hippocampus show
significant cell death when treated with nicotine at the concentrations
to which both adult smokers, as well as fetuses of pregnant mothers who
smoke, are exposed. This effect is mimicked in HC2S2 cells, in which
identical treatment with nicotine causes a dramatic apoptotic cell
death in undifferentiated cells but spares the same cells when
differentiation has been induced. This effect of nicotine is
attributable to the activation of -Bgt-AChRs and the consequent
expression of p53, a cell cycle-related protein. The susceptibility of
undifferentiated HC2S2 cells to the toxic effects of nicotine probably
results from their poor ability to buffer intracellular calcium caused
by the lack of calbindin, a major mobile calcium buffer in these
cells.
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MATERIALS AND METHODS |
Primary progenitor cells. Hippocampal progenitor
cells were isolated from Fisher rats (passages 5-10) as previously
described (Gage et al., 1995b). Briefly, hippocampi were
isolated, and the cells were dissociated enzymatically and plated onto
uncoated culture wells in DMEM/F-12 high-glucose medium containing 10% fetal bovine serum. After 24 hr, the medium was replaced with serum-free medium containing DMEM/F-12, N2 supplement (Life
Technologies, Gaithersburg, MD), and 20 ng/ml FGF-2 (Boehringer
Mannheim, Indianapolis, IN). Cells were passaged every 3-4 d. After
two or three passages, the cells were plated onto 100 mm dishes coated
with polyornithine (10 µg/ml) and laminin (5 µg/ml). Cell numbers
were determined by staining with trypan blue and iodopropidium.
Detection of apoptosis. Apoptosis was detected by the
terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) method using the Apoptag kit (Oncor). Briefly, Apoptag is an in situ apoptosis detection kit used to detect
the multitude of new 3'-OH DNA ends generated by fragmentation.
Digoxygenin-dUTP is used to label DNA fragments that are then
visualized by a peroxidase-conjugated anti-digoxygenin antibody. Intact
cells that showed the reddish brown reaction product in the nucleus
were taken as Apoptag-positive. Nuclear fragmentation was also
visualized using 10 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Sigma,
St. Louis, MO). DNA laddering was examined by running total DNA from
nicotine-treated cells, control cells, and cells treated with nicotine
in the presence of -Bgt on agarose gels according to the protocol
described by Arends et al. (1990).
HC2S2 cell culture. HC2S2 cells were grown in DMEM/F-12
medium along with N2 supplement (Life Technologies) and 2 ng/ml FGF-2 (Boehringer Mannheim) as described (Hoshimaru et al., 1996).
Cells were plated on dishes coated with polyornithine (10 µg/ml;
Sigma) and laminin (5 µg/ml, Collaborative Biomedical Products).
Differentiation was initiated by the addition of 0.1 µg/ml
doxycycline (Sigma). For imaging experiments, the cells were plated on
coated glass coverslips.
For experiments looking at cell loss, cells were grown in 16 mm wells.
At the end of the treatment period they were trypsinized (0.25 ml
ATV trypsin containing 0.125% trypan blue for 3 min). An
aliquot was then transferred to a hemocytometer for counting. Phase-bright cells that excluded the trypan blue were counted as
survivors.
RT-PCR. Total mRNA was isolated from cells using the RNAzol
method according to the manufacturer's instructions (TelTest). The
samples (50 ng of mRNA) were subjected to reverse transcription for 75 min at 42°C and then denatured at 95°C for 5 min. The reaction mixture contained (in µl): H2O, 7; 10× PCR buffer (500 mM KCl and 100 mM Tris-HCl, pH 8.4), 2; 10 mM dNTP, 2; random hexamers, 1; 25 mM
MgCl2, 3; RNAsin, 0.25; and reverse transcriptase,
0.5. The samples then underwent PCR (23 cycles) consisting of the
following steps: 2 min at 94°C, 2 min at 60°C, 2 min at 72°C,
and, after 23 cycles of amplification, 10 min at 70°C. The reaction
mix for the PCR was (in µl): 10× PCR buffer, 8; 25 mM
MgCl2, 2; [32P]dCTP, 0.2;
Taq polymerase (Perkin-Elmer, Emeryville, CA), 0.5; 5' and
3' primers, 2; and H2O, 65.3. For 7, the 5' sense primer was AGA TAT CAC CAC CAT GAC and the 3' primer was GCC TGC GTG GTG GAC.
For the endogenous control, the RPL27 5' primer was GAA CAT TGA
TGA TGG CAC CTC and the 3' primer was GGG GGA TAT CCA CAG AGT ACC.
After PCR, samples were migrated in a 6% acrylamide gel. Quantitation
was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA), and the linearity of the reaction was controlled for 23 cycles of
amplification.
Fluorescence labeling of cells. For fluorescence labeling of
HC2S2 cells, the cells were grown in four- to eight-well Lab-Tek plates
overnight. For -Bgt labeling, cells were incubated with biotin--Bgt (Molecular Probes, Eugene, OR) at 1:500 dilution for 1 hr. They were then washed in PBS and fixed, followed by a 1 hr
incubation with extravidin-FITC. To determine nonspecific labeling, the
first incubation was done in the presence of 1 µM unlabeled -Bgt on sister cultures.
For immunofluorescence experiments, cells were fixed with 4%
paraformaldehyde for 10 min. After fixation they were washed three
times with PBS and preincubated with PBS containing 4% preimmune donkey serum (PBS-DS) and 0.4% Triton X-100 for 30 min. Primary antibodies (Abs) were then added, and the incubation was continued overnight at 4°C. Antibodies used in this study were polyclonal rabbit (rb) antisera against p21, rb Ab-1 (PharMingen, San Diego, CA),
and rb Ab-C-19 (Santa Cruz Biotechnology, Santa Cruz, CA); 2 rb Abs for
calbindin (PharMingen); and one anti-p53 Ab (Do-1, polyclonal;
antisera; PharMingen). After the overnight incubation, the cells
were washed two times for 10 min each with PBS and one time with
PBS-DS. The secondary Abs (donkey anti-rabbit and donkey anti-mouse;
The Jackson Laboratory, Bar Harbor, ME) were conjugated with either
FITC or Cy3 or with biotin and used at a dilution of 1:500 in PBS-DS.
When biotin-conjugated Abs were used, cells were then incubated with
extravidin-FITC conjugate diluted at 1:500 in PBS. In all cases, the
last wash included DAPI to stain the nuclei (see above). Slides were
then mounted in 100 mM Tris-HCl, pH 8.5, containing 25%
glycerol, 10% polyvinyl alcohol (Air Products), and 2.5%
1,4-diazobicyclo-(2.2.2)-octane (Sigma).
Specificity was verified in each case by using controls in which the
primary Ab was omitted and also, when possible, by using a different
irrelevant primary Ab. Fluorescence imaging was performed using
four-color confocal scanning laser microscopy [Zeiss Axiovert and
Bio-Rad (Hercules, CA) MRC1000].
Western blotting. Cell extracts were run on SDS-PAGE.
Immunoblotting was done using polyvinylidene difluoride membranes
(Millipore, Bedford, MA). Ab-1 (PharMingen) was used for the detection
of p21, and a mixture of polyclonal antibody (pAb) 240 and pAb 421 (a
generous gift from J. Baudier, Institut National de la Santé et
de la Recherche Médicale) was used for p53 immunodetection. Actin
monoclonal antibody (mAb; Boehringer Mannheim) was used to evaluate
relative amounts of sample loaded.
Calbindin transfection. Rat calbindin D28 cDNA (Hunziker and
Schrickel, 1988), a generous gift from Dr. Hilmar Bading (Medical Research Council, Cambridge, UK) was cloned in
pBK-cytomegalovirus (Stratagene) at SalII-Knp1
restriction sites. The Lipofectin method (DOTAP; Boehringer Mannheim)
was used for transient transfections. Seventy microliters of the stock
DOTAP were diluted in 100 µl of PBS and mixed with 10 µg of
calbindin in 100 µl of PBS. The mixture was incubated for 10 min at
room temperature and then added onto undifferentiated HC2S2 cells in a
10 cm dish. Nicotine was added 1 d after transfection, and
calbindin immunochemistry was performed 12 hr later, as described
above. Numbers of fragmented nuclei per 100 cells were determined by
DAPI staining in calbindin-positive and calbindin-negative cells in the
same dish.
For the immunochemistry, cells were fixed in 4% paraformaldehyde in
PBS for 10 min, rinsed three times with PBS (5 min each wash), and
preincubated in PBS containing 0.4% Triton X-100 and 4% PBS-DS for 30 min. The cells were then incubated with primary mouse anti-calbindin
mAb (Sigma) at 1:1000 dilution overnight at 4°C. The next day the
primary Ab was washed away with two washes of PBS and one with PBS-DS
(each wash 10 min). Cells were then incubated with
peroxidase-conjugated secondary Ab (donkey anti-mouse; The Jackson
Laboratory) in PBS-DS and then washed three times in PBS. In the last
wash, 10 ng/ml DAPI was included to visualize the nuclei. Peroxidase
staining was visualized using the diaminobenzidine kit (Vector
Laboratories, Burlingame, CA).
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RESULTS |
Nicotine causes DNA fragmentation in primary hippocampal
progenitor cells
We first tested the effects of nicotine, at doses to which
smokers and their offspring are exposed, on the survival of primary hippocampal progenitor cells. Primary progenitors were isolated from
adult Fisher rats and grown in culture for 5-10 passages (see
Materials and Methods; Gage et al., 1995b). Cells were then exposed to 0.5 µM nicotine overnight. Cell numbers were
determined by trypan blue exclusion. The total number of cells in
control dishes were not significantly different from those in
nicotine-treated dishes. Because these cells are a heterogeneous,
dividing population, and cell counting would clearly underestimate the
effects of nicotine, the numbers of apoptotic cells were directly
examined. Apoptosis was detected by the TUNEL staining method. Cells
that retained their morphology but showed staining of their nuclei were
taken as Apoptag-positive. Significantly more stained nuclei were
detected in nicotine-treated dishes. Averaging nine fields from three
separate experiments showed that the number of apoptotic cells in
nicotine-treated dishes was ~30-fold higher than in untreated
controls (3.6 ± 0.04 vs 0.1 ± 0.02%; mean ± SEM;
p < 0.005, Mann-Whitney U test; Fig. 1). Pretreatment of the cells with 100 nM -Bgt followed by its continuous presence during
nicotine treatment reduced the number to 0.6 ± 0.1%, a reduction
in cell death by 83%. The toxin, by itself, did not significantly
affect cell numbers at this dose.
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Figure 1.
Nicotine-induced cell death in primary hippocampal
progenitors. Primary hippocampal progenitor cells were treated
overnight with either control solution (Control)
or 0.5 µM nicotine (Nicotine). Cell death
was visualized using the Apoptag Kit (Oncor). Peroxidase-positive
nuclei in cells that still retained their morphology were counted.
Treatment with nicotine resulted in an increase in the number of
apoptotic cells. Cell counts revealed a 30-fold increase in the number
of Apoptag-positive cells after nicotine treatment
(p < 0.005; Mann-Whitney U
test). This effect of nicotine was blocked by preincubation with 100 nM -Bgt for 30 min, followed by its continued presence
during nicotine treatment (Nic.+ Bgt).
Five hundred cells per field, three fields per dish were counted. The
values expressed in the y-axis are Apoptag-positive
cells as a percentage of total cells counted from three separate
experiments (mean ± SEM).
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Nicotine induces cell death in undifferentiated HC2S2 cells
Primary progenitor cells are extremely heterogeneous, and putative
neuronal progenitors make up only a small fraction of the total, thus
making it very difficult to examine molecular transduction events
in these cells. Therefore, we used HC2S2-immortalized hippocampal progenitors (Hoshimaru et al., 1996) to examine these events. In
addition, the use of HC2S2 cells also enabled us to examine nicotinic
effect in the context of neuronal development and differentiation.
Treatment of undifferentiated HC2S2 cells with a relevant concentration
of nicotine (0.5 µM) for 36 hr caused a >50% reduction in live cell numbers (Fig.
2A, a vs
c, B; Table 1)
compared with untreated controls, as evidenced by trypan blue exclusion
and cell counting. This time point allowed us to consistently and reliably estimate cell loss. Remaining cells in the dish did not show
any morphological changes and were indistinguishable from controls.
Preincubating the cells with 50-100 nM -Bgt for 45 min,
followed by the continued presence of the toxin, completely protected
the cells from nicotine-mediated toxicity (Fig. 2A, b, B). The toxin by itself had no effect on cell
numbers. Similar treatment with 20-50 nM
methyllycaconitine (MLA), an antagonist known to target -Bgt-AChRs
(Ward et al., 1990), also protected the cells from the cytotoxic
effects of nicotine. Culture wells treated with 0.5 µM
nicotine in the presence of 20 nM MLA had 97 ± 6% of
the cells in control wells treated with MLA alone (mean ± SEM,
two experiments). Unlike with -Bgt, the total number of cells in
MLA-treated dishes was slightly lower (80 ± 3% of untreated controls). Whether this result is attributable to some nonspecific toxicity of the insecticide, a possible partial agonist function of
MLA, or hydrolysis products of the norditerpenoid alkaloid that might
have agonistic properties (Hardick et al., 1995) is not known. Nicotine
had significant effects on cell survival at concentration ranges from 5 nM to 5 µM (Fig. 2C), spanning the plasma levels of nicotine in smokers. Whether the small but significant decrease in the efficacy at 5 µM nicotine is attributable
to possible desensitization of AChRs remains to be seen.
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Figure 2.
Effect of nicotine on the survival of HC2S2
progenitor cells. HC2S2 cells plated in 24 mm culture wells were
treated with nicotine for a 36 hr period after which cell survival was
assessed by trypan blue exclusion and counting. Values represent the
mean ± SEM from two to four experiments, each done in triplicate.
A, Bright-field images of HC2S2 cells that were
untreated (a, Con), treated with 0.5 µM
nicotine in the presence of 100 nM -Bgt (b,
Nic + Bgt), or treated with 0.5 µM
nicotine alone (c, Nic). Nicotine caused a dramatic
reduction in the number of trypan blue excluding cells; that reduction
was completely reversed in the presence of -Bgt. Scale bar, 50 µm.
B, Quantitation of the cytotoxic effects of nicotine by
cell counts. Nicotine-treated wells showed a 50% decrease in live cell
numbers that was reversible by pretreating the cells with 100 nM -Bgt followed by the continued presence of the toxin.
The toxin by itself did not significantly affect cell numbers. Cell
numbers in untreated control = 411 ± 59 × 103 cells/24 mm well (mean ± SEM from 4 experiments). Values are expressed as percent live cells compared with
untreated controls (% Con). C,
Dose-response for the effect of nicotine on the survival of
undifferentiated HC2S2 cells. Nicotine significantly decreased the
survival of undifferentiated progenitors at all the concentrations
tested between 5 nM and 5 µM. The lesser
efficacy of the 5 µM concentration could be attributable
to a more rapid desensitization of the receptors. Values are mean ± SEM from two experiments done in triplicate.
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Total cell counts were done for cells treated with nicotine for various
time points. Live cells, dead adherent cells, and floating cells were
counted. Cell numbers were compared with total cells in untreated
sister cultures (Table 1). The total number of cells did not change
between the controls and the nicotine-treated dishes, but the number of
dead cells increased progressively with time from 13% at 12 hr after
nicotine treatment to 56% of the total cell number by 36 hr (Table 1).
These results rule out a significant contribution to nicotine-induced
loss of live cell numbers by general cell cycle arrest and suggest that
cell death is the major contributor to this effect. The surprising
result was that the total number of cells did not change significantly. If the percentage of dividing cells at 24 hr is only 71.5% that of the
untreated control (Table 1), then even if no additional cell death
occurs in the next 12 hr period (the percentage of dead cells is
actually more than double at 36 hr; Table 1), we should still expect at
least a 30% decrease in total cell number. This implies that either
the rate of cell division is much faster than the rate of cell death or
there are compensatory mechanisms triggered directly or indirectly by
nicotine treatment. We come back to this point below.
HC2S2 cells that are under the control of a tet
transactivator can be induced to differentiate by the addition of 0.1 µg/ml doxycycline for 5-10 d, by which time they become terminally
differentiated (Hoshimaru et al., 1996). Identical treatment of
differentiated cells with 0.5 µM (Fig.
3A, Con vs
Nic, B) or with 5 µM nicotine (data
not shown) had no effect on cell survival. These results show that low
concentrations of nicotine, such as those found in the serum of
smokers, can induce cell death in undifferentiated, rapidly dividing
progenitor cells but spare the same cells when they have
differentiated.
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Figure 3.
Nicotine is not cytotoxic to differentiated HC2S2
cells. Differentiated HC2S2 cells were treated with 0.5 mM
nicotine as described in Figure 2. Values represent the mean ± SEM from two experiments, each done in triplicate. A,
Both control (Con) and nicotine-treated
(Nic) cells showed robust neurons with well-defined
processes. Scale bar, 50 µm. B, Cell counts from two
independent determinations done in triplicate revealed no significant
difference in cell numbers between the two conditions.
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Presence of -Bgt-AChRs on HC2S2 cells
The ability of -Bgt and MLA to reverse the effect of nicotine
suggests that it is mediated by the activation of -Bgt-AChRs. Surface-binding experiments (Halvorsen and Berg, 1989) using 10 nM 125I--Bgt indicate that HC2S2 cells
express low levels of toxin binding on their surface. Results show that
undifferentiated cells had 3.9 ± 0.81 fmol/106
cells. Surprisingly, differentiated HC2S2 cells had 12.5 ± 3.37 fmol/106 cells (mean ± SEM from two or three
experiments, each done in triplicate), suggesting that the lack of
nicotine-mediated toxicity in these cells was not attributable to lack
of surface receptors. Assuming only 50% of the cells express surface
toxin binding at a given time (see below), there would be ~4800
-Bgt-binding sites on the surface of an undifferentiated cell.
Fluorescence experiments using biotinylated -Bgt followed by
extravidin-FITC showed specific detectable fluorescence on
approximately half of the cells in both the undifferentiated and
differentiated conditions (Fig. 4A), indicating that
there might be variations in the levels of surface expression of the
receptor between cells.
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Figure 4.
Presence of -Bgt-AChRs on HC2S2 cells.
-Bgt-AChRs on HC2S2 cells were detected by surface radiolabeled
toxin binding (see Results), fluorescence labeling, and RT-PCR
experiments. A, Fluorescence labeling of HC2S2 cells.
Cells were labeled with biotinylated -Bgt followed by Cy3-conjugated
extravidin. Background label was assessed by labeling in the presence
of 1 µM -Bgt. Both the differentiated
(Diff) and the undifferentiated
(Undiff) cells showed detectable levels of toxin
binding on their surface (position of cells is represented by the DAPI
staining in blue). In the differentiated cells, punctate
labeling was also observed on the processes. Scale bar, 25 µm.
B, RT-PCR showing the presence of 7 message in HC2S2
cells. Using primers flanking a 460 bp region of the putative
cytoplasmic domain of the rat 7 gene, RT-PCR was performed on RNA
isolated from differentiated HC2S2 cells (lane D), adult
rat hippocampus (lane H), undifferentiated HC2S2
cells (lane U), and rat fibroblasts (lane
F). The level of 7 message was normalized to that of
the control ribosomal protein L 27A message in the same PCR mix. The
7 message is expressed in differentiated HC2S2 cells in levels
comparable to those in the adult hippocampus. Lesser, although
significant, expression was seen in the undifferentiated cells.
Fibroblasts do not show any detectable expression of the 7
message.
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The 7 gene product is the only identified protein that forms a part
of rat brain -Bgt-AChRs, and all the -Bgt binding sites in the
rat hippocampus contain the 7 gene product. RT-PCR experiments showed the presence of 7 message in both undifferentiated and differentiated HC2S2 cells in levels comparable to those in the adult
rat hippocampus (Fig. 4B). Quantitation using a
PhosphorImager demonstrated that differentiated HC2S2 cells have
~40% greater amounts of the message.
Nicotine-induced apoptosis and the cell cycle
We next examined the mechanism underlying nicotine-induced cell
death. For this, we chose a time point (12 hr) when there was
significant cell death, but 98% of the cells were still adherent. The
first set of experiments was designed to determine whether nicotine-induced cell death was apoptotic in nature. Evidence for this
comes from DNA laddering experiments. In apoptosis there is a
characteristic pattern of DNA degradation in multiples of ~200 bp
(Arends et al., 1990). Undifferentiated HC2S2 cells were treated with
0.5 µM nicotine for 12 hr. DNA was then extracted and run
on an agarose gel. As shown in Figure
5A, nicotine-treated cells
showed a characteristic laddering of DNA. The smallest band observed
(Fig. 5A, arrowhead) was ~200 bp in length.
Increasing nicotine concentration to 50 µM did not appear
to cause any more DNA fragmentation. On the other hand, 100 nM -Bgt significantly protected the cells from
nicotine-mediated endonuclease action (Fig. 5A).
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Figure 5.
Nicotine induces apoptosis in
undifferentiated HC2S2 cells. The effect of treatment with 0.5 µM nicotine for 12 hr on the survival of undifferentiated
HC2S2 cells was examined by the following methods. A,
DNA laddering. DNA was extracted from cells 12 hr after no treatment
(Con) or after treatment with 0.5 µM
nicotine (Nic 0.5), 50 µM
nicotine (Nic 50), or 0.5 µM nicotine in
the presence of 100 nM -Bgt (Nic + Bgt). Nicotine-induced DNA fragmentation characteristic
of apoptosis was seen in undifferentiated HC2S2 cells and was prevented
by pretreatment with -Bgt. The arrow shows the
position of the 247 bp DNA marker. B, Fluorescence and
immunocytochemistry. Nicotine-induced cell death in undifferentiated
HC2S2 cells is apoptotic as seen by nuclear fragmentation
(arrow) visualized by staining undifferentiated cells
with DAPI 12 hr after exposure to 0.5 µM nicotine
(bottom panel). Immunofluorescence on the same
field of cells indicates the induction of p53 (red) and
p21 (green) or p53 and p21
(yellow) expression (top
panel). In the presence of bungarotoxin, neither
nuclear fragmentation nor the induction of p53/p21 is seen
(insets). Scale bar, 25 µm. C, Western
blotting. Western blots of untreated cells (lane 1,
C), cells treated with 0.5 µM nicotine in
the presence of 100 nM -Bgt (lane 2,
N + Bgt) or with nicotine alone at 0.5 µM (lane 3, N)
revealed the induction of both p53 and p21 proteins by nicotine. No
induction was observed in control cells or in cells treated with
nicotine in the presence of -Bgt. In differentiated HC2S2 cells
(lanes 4, 5) no p53 signal was seen in
either control (lane 4, C) or in cells
treated with 0.5 µM nicotine (lane 5,
N). Differentiated cells showed endogenous levels
of p21 under both conditions and in a manner independent of the
expression of p53. All lanes were probed with an actin antibody to
estimate the approximate amounts loaded.
|
|
Further confirmation of the apoptotic nature of nicotine-mediated cell
death comes from the following experiments. Cells were exposed to 0.5 µM nicotine for a 12 hr period, at the end of which they
were fixed and stained with 10 ng/ml DAPI. Visualization of
DAPI-stained cells indicated the presence of fragmented nuclei, characteristic of apoptosis (Fig. 5B, bottom
panel). Such fragmentation was not detectable in
cells treated with nicotine in the presence of -Bgt (Fig.
5B, bottom panel, inset; Table
2). The number of fragmented nuclei on
nicotine treatment (10 ± 1% total; Table 2) correlated well with
the number of adherent cells that were Apoptag-positive at this time
point (10.2 ± 1.6; Table 2). Another characteristic of apoptosis
is the requirement for the induction of specific cell death-related
proteins. Induction of expression of the tumor suppressor protein p53
followed by p21, a cdk inhibitor, has been shown to be one pathway for
programmed cell death (for review, see Meikrantz and Schlegel, 1995).
In the same population of cells stained for DAPI, we examined the
induction of p53 and p21 expression by 0.5 µM nicotine.
Robust expression of both p53 and p21 was observed using
immunofluorescence measurements (Fig. 5B, top
panel; Table 2). Once again, treatment with -Bgt
abolished this induction (Fig. 5B, a,
inset). Fifty-three percent of nicotine-treated cells were
p53-positive, whereas 61% were p21-positive (Table 2). No p53-positive
cells were detected in cells treated with nicotine in the presence of
-Bgt, whereas ~6% of the cells showed detectable immunoreactivity
to p21 (Table 2). These experiments clearly demonstrate the ability of
nicotine to induce the expression of two cell cycle-related proteins
shown to play a role in the apoptotic pathway. The discrepancy between
our DAPI and cell count data versus the expression of the two cell
cycle proteins can be explained by the fact that induction of these
genes precedes overt DNA fragmentation and cell death. The explanation
might be that there is a time lag between the induction of programmed cell death and overt signs of cell death (namely, complete nuclear fragmentation and trypan blue inclusion). A recent study demonstrated the existence of at least 14 gene products that are induced by p53
expression and are probably involved in the apoptotic process, and that
maximal cell death might occur as late as 36 hr after induction of p53
(Polyak et al., 1997).
To test this possibility, we withdrew nicotine at 12 hr, thoroughly
washed the cells, and counted cells for cell death after a 12 or 24 hr
drug-free period. Cell death increased after nicotine withdrawal, and
the numbers of apoptotic cells were 22 and 28% less than those in the
continued presence of nicotine at the 24 and 36 hr time points,
respectively. This finding suggests that the initial 12 hr period of
nicotine exposure is enough to trigger most of cell death-initiating
events that last over the next 24 hr. The percentage of p53-positive
cells at 24 hr remained more or less the same after nicotine withdrawal
(at hour 12) and increased by 50% in the chronic presence of nicotine
(Table 3). This must mean that a larger
number of cells express p53 at 24 and 36 hr than at 12 hr. In the case
of nicotine withdrawal experiments, it implies that more cells are
being recruited into the p53 pathway in the absence of the agonist, or
the p53 expressing cells continue to divide. One way to explain this
finding and the cell number data (Table 1) is that nicotine might
induce a long-lasting cell proliferation as seen in small lung
carcinoma cells (Codignola et al., 1994), and a considerable proportion
of these cells undergo apoptosis. The upper threshold for this
proliferation might be set by other factors (e.g., FGF) in the medium.
Our data suggest that although apoptosis is the main mechanism
underlying nicotine-induced cell death, the actions of nicotine must be
complex involving downstream factors other than the ones measured in
this study (see Discussion).
Western blots of extracts from both undifferentiated and differentiated
HC2S2 cells were performed to determine whether a similar induction of
p53 occurs in differentiated cells. Although expression of p53 in
response to nicotine was seen in undifferentiated HC2S2 cells, no such
expression of the protein was detected in differentiated neurons (Fig.
5C). The cdk inhibitor p21, which is induced early in the
differentiation of HC2S2 cells (F. Berger and F. H. Gage, unpublished
results) was present in differentiated cells in the absence and
presence of nicotine. In undifferentiated HC2S2 cells, p21 expression
was only observed in conjunction with p53 on treatment with nicotine
(Fig. 5C). These results suggest that the induction of p53
in response to nicotine exposure is only seen in undifferentiated HC2S2
cells and that the presence of p21 in differentiated cells appears
independent of both nicotine exposure and p53 induction.
p53-independent induction of p21 is known and is thought to have a role
in neuronal survival (Poluha et al., 1996).
Role of calcium in nicotine-mediated apoptosis
An obvious mechanism by which -Bgt-AChRs could mediate the
effects of nicotine is by increasing [Ca]i (MacNicol and
Schulman, 1992; Vijayaraghavan et al., 1992, 1995). We examined the
role of calcium in nicotine-mediated apoptosis of undifferentiated HC2S2 cells.
Calcium-imaging studies using cells loaded with fura-2 AM and fluo-3 AM
were done on undifferentiated progenitor cells. As expected, no calcium
transients were observed on treatment with 0.5 µM
nicotine (Vijayaraghavan et al., 1992; also see Discussion). The effect
of nicotine on cell death was, however, dependent on extracellular
calcium. A 12-fold reduction of calcium in the media (from 1.2 mM to 100 µM) protected undifferentiated
HC2S2 cells from nicotine-induced cell death, although at this calcium
concentration cell numbers were lower than in controls with normal
extracellular calcium (Fig. 6).
Furthermore, incubation of HC2S2 cells with 10 µM
KN-62, an inhibitor of calcium/calmodulin-dependent kinase (Cam
kinase), showed significant protection against the toxic effects of
nicotine (Fig. 6), lending additional evidence for the calcium
dependence of this effect.
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Figure 6.
Nicotine-induced apoptosis is calcium-dependent.
The effect of 0.5 µM nicotine on the survival of
undifferentiated HC2S2 cells was examined at two different calcium
concentrations (1.2 mM vs 100 µM). Lowering
external calcium 10-fold to 100 µM reversed the ability
of nicotine to mediate cell death in these cells. Cells were tested for
nicotine-induced cytotoxicity in the presence or absence of 10 µM KN-62 (Calbiochem, La Jolla, CA), an inhibitor of CAM
kinase II. In the presence of KN-62, the ability of nicotine to kill
undifferentiated HC2S2 cells was reduced. The inhibitor by itself does
not significantly affect cell survival. Results show that the effect of
nicotine is calcium-dependent.
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Involvement of calbindin D28K in nicotine-mediated apoptosis
One reason for the calcium-dependent susceptibility of
undifferentiated HC2S2 cells could be the lack of adequate endogenous calcium-buffering mechanisms. A major calcium buffer in hippocampal neurons is calbindin D28K. Studies show that hippocampal cells that
express calbindin are much more resistant to calcium cytotoxicity than
those that do not (Mattson et al., 1991), suggesting that expression of this protein is important in protecting neurons from
external calcium insults.
Examining HC2S2 cells for calbindin expression using immunofluorescence
showed very little to no expression of the protein in undifferentiated
HC2S2 cells (Fig. 7A).
Expression of calbindin seems to be initiated only after the induction
of differentiation in these cells (Fig. 7B), suggesting that
the lack of this buffer might make undifferentiated HC2S2 cells more
susceptible to calcium-mediated cytotoxicity. To directly determine
whether calbindin expression is responsible for the susceptibility of
undifferentiated HC2S2 cells to nicotine, we examined nicotine-induced
apoptosis in cells transiently transfected with calbindin D28.
Undifferentiated cells were transiently transfected with rat calbindin
cDNA using DOTAP. The cells were then exposed to nicotine for 12 hr,
after which they were tested for both calbindin expression, using an
anti-calbindin mAb (Sigma) followed by a peroxidase-conjugated
secondary Ab, and for fragmented nuclei using DAPI staining. The degree
of DNA damage in calbindin-positive cells was 38-fold less than that seen in HC2S2 cells that did not express the buffer (Fig.
7C). These results suggest that lack of adequate
calcium-buffering mechanisms might underlie the susceptibility of
undifferentiated HC2S2 cells to nicotine-mediated toxicity and further
confirm the role of changes in [Ca]i in mediating the
effects of -Bgt-AChRs. However, ~50% of calbindin-positive cells
showed changes in morphology that resemble changes in early
differentiation (Fig. 7B). This difference in morphology was
a consequence of calbindin expression and was unaltered by nicotine
treatment or by mock transfection. Although there was no difference in
the extent of protection against the cytotoxicity of nicotine among the
two morphological populations (data not shown), this finding raises the
possibility that protection by calbindin might be attributable to
induction of early differentiation events, and not attributable to a
direct buffering of -Bgt-AChR-mediated calcium increases.
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Figure 7.
The apoptotic effects of nicotine are dependent on
the expression of calbindin D28K. A, Expression of
calbindin-D28K. Undifferentiated and differentiated HC2S2 cells were
incubated overnight with Abs against calbindin D28K followed by an
FITC-conjugated secondary. Little to no expression of calbindin was
seen in undifferentiated HC2S2 cells, whereas robust signals were
obtained from differentiated cells. These results suggest that
calbindin is expressed only after the induction of differentiation in
these cells. Scale bar, 25 µm. B, Rescue of
undifferentiated HC2S2 cells by transient transfection of calbindin.
Undifferentiated HC2S2 cells were transiently transfected with
calbindin D28K. Cells were then treated with 0.5 µM
nicotine and stained for nuclear fragmentation with DAPI (top
panel) and with an anti-calbindin mAb (bottom
panel). Calbindin-positive cells (top and
bottom panels, long arrows) showed very
little nuclear fragmentation, whereas cells that do not express the
gene had large numbers of fragmented nuclei (top panel,
short arrows). Scale bar, 25 µm. C,
Quantitation of calbindin-mediated rescue. The numbers of fragmented
nuclei were counted in undifferentiated cells treated with 0.5 µM nicotine that were calbindin-positive
(D28 +ve) or did not express the gene
(D28  ve). Cells that did not express
calbindin showed a 38-fold greater number of fragmented nuclei than
calbindin-positive cells (p < 0.003, Mann-Whitney U test). Results are mean ± SEM from
three independent determinations.
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|
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DISCUSSION |
The results presented here demonstrate that nicotine can induce
apoptotic cell death in undifferentiated hippocampal progenitor cells
but spares the same cells when they are differentiated. This effect of
nicotine is antagonized by -Bgt and results in the induction of the
tumor suppressor protein p53. The apoptotic effect is dependent on the
ability of the cells to tolerate changes in [Ca]i.
Sensitivity to calcium insults, in turn, is probably dependent on the
levels of calbindin expression in these cells.
A mechanistic explanation for the various actions of nicotine on the
CNS has only recently begun to emerge. Major players for mediating some
of these effects have turned out to be the -Bgt-AChRs. Recent
studies have shown that these receptors can play an important role as
modulators of synaptic strength in the CNS (McGhee et al., 1995; Gray
et al., 1996; Alkondon et al., 1996) and could also have roles in the
pathophysiology of diseases such as Alzheimer's disease (Gray et al.,
1996) and schizophrenia (Freedman et al., 1993, 1997).
The role that -Bgt-AChRs play during neuronal development has
previously been inferred from their expression profiles in both CNS and
peripheral neurons, as well as from studies on their effects on neurite
outgrowth and survival (see introductory remarks). These findings
indicate that the receptors might be expressed early and could play a
role in early development and differentiation events (Role and Berg,
1996). Our study on cell death in primary hippocampal progenitor cells
shows a 30-fold increase in the number of apoptotic cells after
nicotine treatment. The small numbers of apoptotic cells are expected
because the action of nicotine depends on the expression of
-Bgt-AChRs. If the receptors are expressed only in a subpopulation
of neuronally committed progenitors, then one would expect only a tiny
percentage of the total progenitor population to be affected. This fact
makes the examination of transduction mechanisms quite difficult and
motivated us to examine more homogeneous populations.
In our study on HC2S2 cells, the expression of -Bgt-AChRs in
undifferentiated neuronal progenitors precedes most neuronal markers
examined (Hoshimaru et al., 1996). The level of expression of surface
-Bgt binding in undifferentiated HC2S2 cells is very low
(~2400-4800 binding sites per cell). This level is approximately three orders of magnitude less than that on chick ciliary ganglion neurons (Halvorsen and Berg, 1989) and ~50-fold lower than that obtained from primary rat hippocampal neurons in culture (S. Vijayaraghavan, unpublished data). The presence of these sites
in other neuronally committed hippocampal progenitors (Komourian and
Quik, 1996) suggests that low levels of toxin binding might be
prevalent among immature hippocampal neurogenic cells. Nevertheless,
this low number is clearly sufficient to trigger nicotine-mediated
effects.
The cause of nicotine-induced loss of live cell numbers is mainly
attributable to cell death. It does not appear, from our data, that
general arrest of cell division or induction of differentiation plays a
significant role in these effects. The apoptotic nature of
nicotine-induced cell death has been confirmed in multiple ways in this
study. Cell counts, DNA laddering, TUNEL reactivity, DAPI staining, and
the induction of p53 and p21 all indicate that cells undergo apoptotic
cell death when treated with nicotine and also implicate at least one
of the two cell cycle-dependent proteins in the mediation of this
effect. Nicotine seems to have the ability to trigger the expression of
p53 only in undifferentiated cells. One possibility is that, in
undifferentiated HC2S2 cells, influx of calcium on -Bgt-AChR
activation triggers the expression of p53. Excitotoxicity in neurons
has been shown to involve the induction of p53 (Jordan et al., 1997;
Sakhi et al., 1997). Differentiated HC2S2 cells might be better
equipped to handle calcium loads and thus might be in a position to
resist the nicotine-dependent induction of p53 expression. The role of
p53 in calcium-mediated toxicity is evident from studies on p53 null
mutants. Knock-out mice lacking p53 are resistant to excitotoxic
insults (Xiang et al., 1996).
Quantitation of nicotine-mediated changes in the various parameters
measured (Tables 1-3) suggests that there might actually be a
long-lasting proliferative effect of nicotine on HC2S2 cells, despite
the rapid expression of p53 and p21. This finding implies that p53 can
overcome the inhibitory effects of p21 on cell cycle as has been shown
in other systems (Kagawa et al., 1997). The simplest explanation would
be that nicotine induces proliferation of undifferentiated HC2S2 cells,
as seen in lung small cell carcinoma cells (Codignola et al., 1994), a
large proportion of which undergo p53-mediated apoptotic cell death.
This explanation would seem more consistent with current thinking in
the field that changes in the dynamic balance between cell division and
apoptosis triggered by the same stimulus would ultimately decide the
fate of that cell (Xia et al., 1995).
Calcium flux caused by the activation of -Bgt-AChRs seems to play a
major role in the mediation of many of the effects of the receptors. An
indication of the developmental consequence of this receptor activation
comes from studies of an AChR homolog in C. elegans (Treinin
and Chalfie, 1995). In the nematode, a naturally occurring mutation in
the channel domain of this homolog results in cell death, probably
caused by calcium influx through a slowly desensitizing receptor. A
similar calcium-dependent mechanism is indicated for the effects of low
nicotine doses on primary progenitors and HC2S2 cells. The cytotoxic
effect of low levels of nicotine on HC2S2 cells appears to be dependent
on calcium flux from the outside. Reducing extracellular calcium
reduces the susceptibility of the cells to the toxic effects of
nicotine, as does incubating the cells with nicotine in the presence of KN-62, a Cam kinase inhibitor. Induction of Cam kinase on AChR activation has been demonstrated in PC12 cells (MacNicol and Schulman, 1992). Although these studies show the requirement of calcium for the
cytotoxic effects of nicotine, the steps in the pathway in which
calcium is required have yet to be worked out. No calcium transients
were induced by nicotine, which was as expected. Most of the bulk
[Ca]i increases observed with nicotine in neurons come
from the activation of voltage-gated calcium channels (Vijayaraghavan et al., 1992). Very little to no functional expression of calcium channels is seen in undifferentiated HC2S2 cells (Hoshimaru et al.,
1996). Thus, one can infer that, if calcium flux is an initial step in
the pathway, then localized increases in concentrations of the ion
passing through -Bgt-AChR channels must be important. Such localized
differences in ligand-gated ion channel-mediated changes in
[Ca]i unaccompanied by alterations in bulk
[Ca]i underlie changes in gene expression during synaptic
transmission in the hippocampus (Deisseroth et al., 1996).
The effect of localized calcium flux through -Bgt-AChR channels is
proving to be very significant, judging from our results and from the
results of recent studies. In the absence of observable differences in
bulk [Ca]i in the presence or absence of -Bgt, nicotine activates the production of arachidonic acid in chick ciliary
ganglion neurons in culture in a manner sensitive to the toxin
(Vijayaraghavan et al., 1992, 1995). In rat hippocampal neurons,
activation of -Bgt-AChRs causes a calcium-dependent increase in the
efficacy of glutamatergic transmission in the absence of contributions
from voltage-gated calcium channels and at very low concentrations of
nicotine. These results suggest that -Bgt-AChRs are localized in
close proximity to release sites in presynaptic terminals of
hippocampal neurons (Gray et al., 1996). Our data from HC2S2 cells
support a similar spatial relationship between -Bgt-AChRs and their
effector sites.
The acute sensitivity of HC2S2 cells to changes in local calcium levels
suggests a poor ability to maintain calcium homeostasis. This might be
important for these cells developmentally, making them very responsive
to calcium-dependent cues. One reason for the inability of
undifferentiated HC2S2 cells to tolerate calcium insults is the lack of
adequate calbindin expression. The expression of this protein seems to
be differentiation-dependent in these cells. Robust expression of
calbindin is observed in differentiated HC2S2 cells. The idea that lack
of calbindin expression makes HC2S2 cells susceptible to
calcium-mediated toxicity is consistent with studies on cell death in
hippocampal neurons (Mattson et al., 1991; Goodman et al., 1993;
Beck et al., 1994). Strong evidence comes from our finding that
undifferentiated HC2S2 cells transfected with calbindin are resistant
to nicotine-mediated apoptosis. However, although calbindin-protected
cells that were morphologically indistinct from control untransfected
cells, we cannot rule out the possibility that the effects of
expression of this calcium buffer are attributable to its ability to
induce early differentiation. If true, the results would imply that
calbindin expression triggers early differentiation events and also
that protection against the toxic effects of nicotine must be conferred
to the cells very early on in the differentiation process before
morphological changes are observed.
Nicotine appears to have contradictory effects on cell survival in
different systems. The drug protects cultured striatal neurons against
NMDA receptor-mediated neurotoxicity (Marin et al., 1994). In
PC12 cells, nicotine can rescue differentiated cells from NGF
deprivation-induced cell death but cannot prevent cell death under the
same conditions in undifferentiated cells (Yamashita and Nakamura,
1996). In spinal motoneurons, nicotine promotes survival (Messi et al.,
1997). On the other hand, -Bgt, by its action on neuronal AChRs,
also protects motoneurons from naturally occurring cell death (Hory-Lee
and Frank, 1995). Our results strongly suggest that nicotinic effects
in the brain are not simple consequences of expression levels of AChRs.
Phenomena such as nicotine-induced upregulation of receptors might not
be primary determinants of the action of this drug. Rather, the
expression and functional interaction between components of various
signal transduction pathways triggered by nicotine would ultimately
determine the effect for a given cell. The effect of nicotine on cell
survival probably depends on a number of factors such as specific gene expression, cell cycle stage, developmental stage, levels of trophic factors, and calcium-buffering capabilities. These could determine both
the cytotoxic and protective effects of nicotine. At the same time, the
complex requirements would enable the drug to selectively target
defined population of cells, resulting in elimination or protection.
For example, expression of p21 is essential for the survival of
differentiating neurons (Poluha et al., 1996), thus possibly making
nicotine a candidate for neuroprotection under conditions that might
downregulate the expression of the gene (e.g., aging and changes in
growth factor levels).
Finally, our results show that nicotine abuse could have significant
consequences to the developing CNS in fetuses of mothers who smoke and
could alter development of -Bgt-AChR-containing hippocampal
progenitors in the brains of teens and adults using nicotine. Our
results suggest that untimely loss or increase of specific progenitor
populations could underlie some of the cognitive and behavioral effects
of nicotine. Further in vivo experiments, currently under
way, will address this issue in greater detail.
| |
FOOTNOTES |
Received April 28, 1998; revised June 3, 1998; accepted June 18, 1998.
This work was supported by grants from the National Institute on Aging
and the National Institute of Neurological Diseases and Stroke
(F.H.G.), the National Institute on Drug Abuse (S.V.), and a
grant-in-aid from the American Heart Association, California Affiliate
(S.V.). Part of this work was performed at University of California San
Diego, Department of Biology. We thank Profs. Darwin Berg, William
Betz, and Nicholas Spitzer and Dr. Angie Ribera for comments on this
manuscript. We also thank Dr. Hilmar Bading (Medical Research Council,
Cambridge, UK) for the calbindin cDNA and Dr. J. Baudier
(Institut National de la Santé et de la Recherche Médicale)
for p53 antibodies.
Correspondence should be addressed to Sukumar Vijayaraghavan,
Department of Physiology and Biophysics, C-240, University of Colorado
Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262.
Dr. Berger's present address: U.318 Institut National de la
Santé et de la Recherche Médicale-Université Joseph
Fourier De Grenoble, Pavillon B, Centre Hospitalier
Universitaire, BP 217, F 38043, Grenoble Cedex 9, France.
| |
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Top
Abstract
Introduction
