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The Journal of Neuroscience, September 15, 1998, 18(18):7361-7371
Cyclic AMP Elevation Is Sufficient to Promote the Survival of
Spinal Motor Neurons In Vitro
Martin G.
Hanson Jr1,
Shiliang
Shen1,
Anthony P.
Wiemelt2,
F. Arthur
McMorris2, and
Ben A.
Barres1
1 Stanford University School of Medicine, Department of
Neurobiology, Stanford, California 94305-5125, and
2 Wistar Institute, Philadelphia, Pennsylvania 19104
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ABSTRACT |
The short-term survival of highly purified embryonic spinal motor
neurons (SMNs) in culture can be promoted by many peptide trophic
factors, including brain-derived neurotrophic factor (BDNF), ciliary
neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor
(HGF). We have asked whether these peptides are sufficient to promote
the long-term survival of purified E15 SMNs. Contrary to previous
reports, we find that when SMNs are cultured in serum-free medium
containing a single peptide trophic factor only approximately one-third
of the cells survive for 3 d in culture. When multiple factors are
combined, additive effects on survival are observed transiently, but by
7 d of culture the majority of SMNs has died. Surprisingly,
when cAMP levels are elevated, the majority of SMNs extend processes
and survive for 1 week in culture in the absence of peptide trophic
factors, even in low-density cultures. A combination of five peptide
trophic factors, together with cAMP elevation, promotes the long-term
survival of most of the SMNs in serum-free culture for 3 weeks. These
findings provide useful culture conditions for studying the properties
of SMNs and have implications for the treatment of motor neuron
diseases.
Key words:
rat; trophic factors; forskolin; IBMX; injury; regeneration; bone morphogenetic proteins
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INTRODUCTION |
Recent studies have suggested that
the signaling mechanisms that promote the survival of CNS and
peripheral nervous system (PNS) neurons may differ (Meyer-Franke et
al., 1995 , 1998). The survival of purified, defined types of PNS
neurons can be promoted in serum-free medium by single peptide trophic
factors (Barbin et al., 1984; Levi-Montalcini, 1987; Barde, 1989,
1990). In addition, PNS neurons survive in the absence of growth
factors when their intracellular levels of cAMP are elevated or when
they are depolarized (Wakada et al., 1983; Rydell and Greene, 1988;
Koike and Tanake, 1991; Franklin and Johnson, 1992). In
contrast, several studies have shown that the survival of CNS neurons
in culture depends on combinatorial signaling by several peptide
trophic factors (Arakawa et al., 1990; Mitsumoto et al., 1994;
Meyer-Franke et al., 1995; Wong et al., 1997). Furthermore, whereas PNS
neurons are intrinsically responsive to peptide trophic factors, the
responsiveness of glutamatergic CNS neurons such as retinal ganglion
cells and cerebral cortical neurons depends on signals from neighboring cells (Meyer-Franke et al., 1995, 1998; McAllister et al., 1996). For
instance, Meyer-Franke et al. (1995, 1998) showed that purified retinal
ganglion cells in culture do not respond to a variety of peptide
trophic factors unless their levels of cAMP are elevated, which can be
induced by depolarization. Thus cAMP elevation induces the retinal
ganglion cells to become growth factor-responsive, whereas it induces
the PNS neurons to become growth factor-independent. Together, these
studies indicate that the signaling mechanisms that promote the
survival of CNS neurons are more complex than those that promote the
survival of PNS neurons.
In this paper we have asked to what extent the survival requirements of
spinal motor neurons (SMNs) are like other CNS neurons, including
retinal ganglion cells and cerebral cortical neurons. SMNs, unlike
these CNS neurons, are cholinergic and project out of the CNS into the
PNS; moreover, unlike most CNS neurons they share the property of PNS
neurons of being able to survive and regenerate after axotomy. Previous
studies have demonstrated that the survival of SMNs depends on Schwann
cell and muscle-derived trophic signals and that their survival in
culture can be promoted by peptide trophic factors normally produced by
muscle, including hepatocyte growth factor (HGF), cardiotrophin-1
(CT-1), and brain-derived neurotrophic factor (BDNF) as well as by
peptide trophic factors normally made by Schwann cells, including
insulin-like growth factor 1 (IGF-1), ciliary neurotrophic factor
(CNTF), neurotrophin-3 (NT-3), and glial-derived neurotrophic factor
(GDNF) (Arakawa et al., 1990; Martinou et al., 1992; Sendtner et al.,
1992a,b, 1996; Yan et al., 1992; Henderson et al., 1993, 1994; Hughes
et al., 1993; Koliatsos et al., 1993; Masu et al., 1993; Neff et al.,
1993; Jung et al., 1994; Zurn and Werren, 1994; L. Li et al., 1995; M. Li et al., 1995; Oppenheim et al., 1995; Ebens et al., 1996; McKay et
al., 1996; Oppenheim, 1996; Pennica et al., 1996; Wong et al., 1997;
Arce et al., 1998). Single peptide trophic factors have been reported
to be sufficient to promote the short-term survival of the majority of
SMNs in culture (Henderson et al., 1993, 1994; Pennica et al., 1996).
These studies, however, were performed in serum-containing medium,
which contains many undefined trophic signals, and peptide trophic
factors have been reported to act combinatorially to promote the
long-term survival of SMNs (Arakawa et al., 1990; Mitsumoto et al.,
1994; Wong et al., 1997).
Here we show that single peptide trophic factors are sufficient to
promote only the short-term survival of a small percentage of SMNs in
serum-free medium and that, whereas combinations of two to six peptide
trophic factors act additively to promote long-term survival, the so
far identified trophic factors for SMNs are insufficient to promote the
long-term survival of the majority of SMNs. To our surprise, we found
that, as for PNS neurons, cAMP elevation is sufficient to promote the
short-term survival and growth of the majority of SMNs in the absence
of peptide trophic factors. The long-term survival of the majority of
SMNs is promoted by collaborative signaling by several peptides,
together with cAMP elevation.
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MATERIALS AND METHODS |
Detailed step-by-step protocols for all procedures are available
on request (barres{at}leland.stanford.edu).
Reagents
Recombinant human trophic factors were obtained from Regeneron
(BDNF, CNTF; Tarrytown, NY), Genentech (HGF; South San Francisco, CA),
Creative Biomolecules (BMP-7/OP-1; South San Francisco, CA), Genetics
Institute (BMP-2, BMP-4; Cambridge, MA). All other recombinant peptides
were obtained from Peptrotech (Rocky Hill, NJ).
Preparation of spinal cord suspensions
Embryonic day 15 (E15) spinal cords were obtained from Sprague
Dawley rats (Simonsen Labs, CA) and dissociated enzymatically to make a
suspension of single cells, essentially as described by Camu and
Henderson (1992). In brief, the ventral half of the spinal cord tissue
was incubated at 37°C for 15 min in a trypsin solution (0.05%; Life
Technologies, Gaithersburg, MD) in calcium-free and
magnesium-free PBS (Life Technologies). Then the tissue
was washed with 10% fetal calf serum and triturated in a Leibowitz-15 (L-15) solution containing bovine serum albumin (4% BSA; Sigma, St.
Louis, MO) and DNase (0.004%; Sigma).
Purification of spinal motor neurons
Spinal motor neurons from E15 rats were purified to >85%
purity as determined by islet-1 immunostaining essentially as described by Camu and Henderson (1992). The SMNs were purified from the spinal
cord cell suspensions by using a density gradient, followed by
sequential immunopanning to yield 20,000 SMNs per E15 spinal cord
(Ericson et al., 1992).
Gradient. The spinal cord cell suspension was centrifuged
through a 4% BSA cushion at 125 × g for 10 min. The
pellet was resuspended and centrifuged through 6.8% metrizamide (w/v;
Sigma) at 500 × g for 15 min to remove cells of higher
density from the spinal cell suspension. The lower-density cells were
resuspended in L-15 and centrifuged through a 4% BSA cushion at
125 × g for 10 min.
Preparation of panning dishes. A Petri dish (100 × 15 mm; Fisher Scientific, Pittsburgh, PA) was incubated with 12 ml of Tris buffer solution, pH 9.5, with 40 µg/ml affinity-purified goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) for 12 hr
at 4°C. The dish was washed three times with 8 ml of PBS and then was
incubated with 10 ml of anti-p75 monoclonal IgG supernatant (MAB 192, Eric Shooter) for 12 hr at 4°C. The supernatant was removed
and the plate was washed three times with PBS. A Petri dish coated with
an anti-galactocerebroside (GC) monoclonal supernatant was prepared
similarly.
Panning procedure. The spinal cell suspension was
resuspended in 10 ml of L-15 and incubated on the Petri dish coated
with anti-GC antibodies to remove microglia and oligodendrocytes. The nonadherent cells were placed in a 37°C incubator for 1 hr to allow
new undigested p75 to appear on the cell surface; this step is crucial,
because the extracellular domain of p75 contains ~10 trypsin cleavage
sites; thus p75 is digested in large part during the preparation of the
spinal cord cell suspension. To select for motor neurons, we
filtered the spinal cell suspension through Nitex mesh (15 µM, Tetko, Elmsford, NY) to remove any cell aggregates and placed the suspension on the Petri dish coated with anti-p75 antibodies for 1 hr at room temperature. The nonadherent cells were
removed by washing the dish five times with PBS.
Removal of adherent cells from the panning plate. A trypsin
solution (4 ml; 0.125%) was prepared by diluting a trypsin 20× stock
(Sigma) into Earle's balanced salt solution. Cells on the panning dish
were incubated with this solution for 2 min in a 10% CO2
incubator at 37°C. The cells were dislodged by gently pipetting
trypsin solution around the plate. Ten milliliters of a 25% FCS
solution were added to inactivate the trypsin, and the cells were
centrifuged and collected as above.
Culture of purified motor neurons
Approximately 2500 purified motor neurons were cultured in
96-well plates that had been coated with merosin (2 µg/ml; Life Technologies) in 100 µl of serum-free medium containing L-15 and sodium bicarbonate. The serum-free additives that were used included BSA, selenium, putrescine, thyroxine, and transferrin [modified from
Bottenstein and Sato (1979), as previously described in Lillien and
Raff (1990)] (B-S medium) pyruvate (1 mM), glutamine (1 mM), penicillin/streptomycin, and trophic factors as
indicated. In some cases, when indicated, the serum-free additive B27
(Life Technologies; Brewer et al., 1993) also was added at a dilution of 1:50. The percentage of surviving cells was assessed after 3 d
by MTT assay (see below). All values were normalized to the percentage
of surviving cells at 1 hr after plating. All peptide trophic factors
were used at a plateau concentration of 10 ng/ml, as established by
dose-response curves, except for NT-3, NT-4/5, transforming growth
factor  (TGF), TGF  2, and TGF3, which were used at 50 ng/ml. Insulin was used at 5 µg/ml.
MTT survival assay
The MTT survival assay was performed as described by Mosmann
(1983). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma] was dissolved in PBS at 5 mg/ml and sterilized by
passage through a Millipore filter (0.22 µm; Millipore, Bedford, MA).
This stock solution was added to the culture well (1:9) and incubated
at 37°C for 1 hr. Viable cells with active mitochondria cleaved the
tetrazolium ring into a visible dark blue formazan reaction product.
The viable and dead cells in each well were counted by bright-field
microscopy. In all cases the percentage of survival determined by the
MTT assay was nearly identical to the values determined by morphology
alone. All values are given as the mean ± SEM of at least three
cultures. All experiments were repeated at least three times. The
results of representative experiments are shown.
Immunofluorescence staining
After fixation with 4% paraformaldehyde for 10 min at room
temperature, the cells were incubated for 30 min in a 50% goat serum
solution containing 1% BSA and 100 mM L-lysine
to block nonspecific binding and containing Triton X-100 0.4% to
permeabilize the membrane. To stain for purity of spinal motor neurons,
we incubated cells in monoclonal islet-1/2 antibody (4D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA),
respectively, followed by fluorescein-coupled goat anti-mouse IgG (10 µg/ml; Jackson Laboratories, Bar Harbor, ME). The coverslips
were mounted in Citifluor on glass slides, sealed with nail varnish,
and examined with a Nikon fluorescence microscope.
Cyclic AMP immunostaining was performed with a polyclonal antiserum and
with a procedure developed by Wiemelt et al. (1997). In brief, an
antibody to cAMP was prepared by immunizing a rabbit with a protein
carrier coupled to cAMP with acrolein. For immunostaining, acutely
isolated purified SMNs were incubated for 60 min in Neurobasal with
0.2% BSA containing either nothing, forskolin (10 µM),
IBMX (0.1 mM), forskolin and IBMX together, or trophic
factors as indicated. The cells were fixed with 5.5% acrolein (v/v) in
a sodium acetate-buffered solution (0.1 M, pH 4.75) for 1 hr at room temperature. The cells were washed in a quenching solution
containing glycine (1 mg/ml) for 30 min, followed by sodium borohydride
(1%) for 30 min. Then the cultures were stained by incubating them in
the anti-cAMP antiserum diluted 1:50 in a Tris-HCl solution (50 mM; pH 7.5) containing Triton X-100 (0.5%) and goat serum
(50%) overnight. The cultures were rinsed three times in the Tris
solution and incubated in a biotin-conjugated goat anti-rabbit IgG
(Amersham, Arlington Heights, IL) at 10 µg/ml for 1 hr. The cultures
were rinsed three times in the Tris solution and incubated in a
FITC-conjugated streptavidin solution (10 µg/ml) for 1 hr. Finally,
the cultures were rinsed in the Tris solution three times and then
mounted as described above. The cAMP antiserum is highly specific for cAMP (Wiemelt et al., 1997).
Cellular cAMP assay
The cAMP radioimmunoassay was performed by using the BIOTRAK
cAMP [125I] assay system (Amersham RPA 509). In
all, 5000 SMNs were plated per well in a 24-well plate and incubated
for 24 hr in serum-free medium containing HGF, BDNF, CNTF, and GDNF.
Then the cells were washed three times for 20 min intervals with 0.2%
BSA. The cells were incubated for 1, 6, and 24 hr at 37°C with 0.2%
BSA containing either nothing, forskolin (10 µM), IBMX
(0.1 mM), forskolin and IBMX together, trophic factors, or
forskolin and trophic factors as indicated. After incubation, the cells
were permeabilized with ice-cold ethanol. The extracts were lyophilized
in a speed vacuum (SC110A, Savant Industries, Farmingdale, NY) and
stored at  20°C until analysis. The acetylation protocol was used to
increase the signal size of the amount of cAMP. The extracts were
solubilized in assay buffer and then diluted in a mixture of one part
acetic anhydride to two parts triethylamine. 3',5'-Cyclic phosphoric acid 2'-0-succinyl-3-[125I]-iodotyrosine methyl
ester, which binds to cyclic AMP, was added, followed by the anti-cAMP
antiserum. After incubation for 15-18 hr at 4°C, Amerlex-M secondary
antibody was added to the sample, and the precipitate was obtained by
centrifugation. Each sample was counted for 60 sec by an autosampling
gamma scintillation counter (Beckman, Fullerton, CA), and the cAMP
values were obtained with a standard curve.
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RESULTS |
Evaluation of yield and purity of the spinal motor neurons
The procedure for purifying SMNs developed by Camu and Henderson
(1992, 1994) involves two steps: first, a density gradient is used to
select low-density cells from an E15 spinal cord cell suspension;
second, immunopanning is used to select the low-density cells that
express the p75 nerve growth factor receptor. We modified this
procedure by adding a 1 hr recovery step to allow for the reappearance
of p75 on cell surfaces, because p75 has 10 trypsin cleavage sites and
is digested in large part during the enzymatic procedure used to
prepare the spinal cord cell suspensions (see Materials and Methods).
In addition, we added an initial panning step with an irrelevant
antibody to eliminate microglia from the cell suspension. We assessed
the purity and yield of our SMN preparations with the 4D5
anti-islet-1/2 monoclonal antibody, which specifically labels spinal
motor neurons (Ericson et al., 1992; Tsuchida et al., 1994; Yamamoto et
al., 1997). These modifications allowed us to obtain a fourfold
increase in our yield of SMNs as well as an increase in their purity.
Although only 2% of the cells in the E15 spinal cord cell suspension
were islet-1/2+, the purity of the SMN preparation
was 86% (Table 1; Fig.
1). The remaining 14% of the cells were
neurons, because they did not take up BrdU or stain with glial or
fibroblast markers and they did stain with an anti-neuron-specific
enolase antiserum.
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Figure 1.
Spinal motor neurons after 24 hr in culture.
A, Phase-contrast micrograph. B,
Immunofluorescence micrograph of the same field as in A,
stained with the 4D5 anti-islet-1/2 monoclonal antibody. The SMNs were
cultured in BDNF, CNTF, GDNF, HGF, forskolin, and IBMX. Scale bar, 50 µm.
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Effects of single peptide tropic factors on short-term survival of
SMNs in vitro
To determine the ability of single peptide trophic factors to
promote SMN survival, we cultured the purified E15 SMNs in serum-free medium on a merosin substrate (see Materials and Methods). After 3 d in culture we assessed their survival by using an MTT assay, as
described previously (see Materials and Methods). In the absence of
added growth factors, the majority of the cells died within 3 d
with the characteristic morphology of apoptosis. The nucleus and
cytoplasm of the apoptotic cells were shrunken, and the cells did not
metabolize MTT. Interestingly, as previously reported (Henderson et
al., 1993), ~10% of the cells did not die in the absence of growth
factors and appeared healthy, with a large soma and long processes
(Table 2). These cells were not
contaminating cells but were probably motor neurons because they were
islet-1/2+ (data not shown).
We tested whether plateau concentrations of single peptide growth
factors previously shown to promote the survival of SMNs in
vitro and to be produced normally by Schwann cells or muscle cells
would promote their survival. Over the same 3 d culture period, as
previously reported, we found that a large variety of peptides was
sufficient to significantly promote the survival of the SMNs, although
typically single peptides could promote only the survival of
~20-35% of the cells for a few days or only 10-25% of the cells
if the 10% that would survive without signals were not counted (Table
2). These peptides included CNTF, leukemia inhibitory factor (LIF),
BDNF, NT-3, NT-4/5, nerve growth factor (NGF), IGF-1, fibroblast growth
factor (FGF), GDNF, and HGF. In addition, we observed that several
recently described peptides called bone morphogenetic proteins 2, 4, and 7 (BMPs), which are members of the TGF superfamily and are
produced by a variety of cell types including Schwann cells (Bitgood
and McMahon, 1995; Hogan, 1996; Lein et al., 1996), also promoted the
survival of the SMNs (Table 2). In all cases, the viability of SMNs
cultured in single peptide trophic factors in serum-free medium dropped off considerably after 3 d; by 1 week almost all of the cells were
dead. The omission of merosin as the substrate resulted in significantly less survival (data not shown).
Effects of multiple peptide tropic factors on short-term survival
of spinal motor neurons
Because single factors did not promote 100% survival of spinal
motor neurons at 3 d (Table 2), we investigated whether
combinations of peptide trophic factors used at plateau concentrations
would improve short-term survival. Because factors fell into several classes according to the receptors and signal transduction pathways that they activate, we first tested combinations of factors within the
same class and found that additive effects on survival did not occur,
as expected. For example, over a 3 d culture period neither the
cytokines CNTF and LIF were additive, nor were the neurotrophins NT-3,
BDNF, NT-4/5, and NGF (data not shown).
In contrast, combinations of factors from different classes nearly
always had an additive effect on the survival of the SMNs (Table
3). More cells survived in a combination
of three factors than in two factors, and short-term survival
progressively increased as a function of the number of trophic factors
combined. As the percentage of surviving cells approached 100%, the
additive effects of four or more factors became less noticeable. When
we combined four or more factors from different classes, regardless of
which four factors, we were able to promote the survival of the
majority of SMNs for 3 d in culture (Table 3). Surviving neurons
quickly extended dendrites and axons (Fig.
2). The rate and amount of process
outgrowth correlated closely with the degree of survival.
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Figure 2.
Hoffman micrograph of purified spinal motor
neurons in culture. SMNs were cultured for 24 hr in BDNF, CNTF, GDNF,
HGF, forskolin, and IBMX. Scale bar, 75 µm.
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Although combinations of multiple factors could promote survival of
most of the SMNs for 3 d, the percentage of surviving cells fell
rapidly with increasing time in culture (see below), raising the
question of whether there are important peptide trophic factors for
SMNs that have not yet been identified. To test this possibility, we
made extracts of developing embryonic and postnatal rat brain, muscle,
sciatic nerve, and spinal cords. The effects of the extracts separately
and in combination with growth factors on the short-term survival of
SMNs were determined. Although the extracts prepared from spinal cord,
sciatic nerve, and muscles each promoted the survival of approximately
one-third of the SMNs in culture, their effects were not additive with
combinations of three or more peptide factors (data not shown).
Effects of steroid hormones, oxygen tension, and density on
SMN survival
Steroid hormones have been reported previously to enhance the
survival of SMNs and other neurons and, except for progesterone, were
not present in our serum-free medium. We prepared a modified B-S medium
that lacked progesterone and assessed the ability of single steroid
hormones added to the medium to promote SMN survival. Steroid hormones
were insufficient by themselves to promote SMN survival, so we also
added CNTF, BDNF, and insulin to the medium. After 3 d in culture,
the survival of the SMNs was assessed with the MTT assay (Table
4). Whereas progesterone, retinoic acid, and thyroid hormone did not promote survival, a weak but statistically significant enhancement of survival was observed with
dihydrotestosterone, estradiol, and hydrocortisone. Moreover, their
survival-promoting effects were additive (Table 4).
SMNs have been shown previously to be sensitive to oxidative damage,
raising the question of whether the high ambient oxygen tension of
20%, which is approximately four times higher than the oxygen levels
that SMNs normally are exposed to in vivo, could be toxic to
the cultured SMNs. To test this possibility, we compared the survival
of SMNs cultured at 5 and 20% oxygen in a water-jacketed oxygen-controlling incubation chamber (Forma Scientific, Marietta, OH)
for 3 d in serum-free medium containing HGF, BDNF, or CNTF. The
percentages of cells that survived in each condition in the 5 and 20%
oxygen atmospheres were nearly identical (data not shown). Thus the
high oxygen levels typically used in our cultures do not account for
the rapid fall off in survival of the SMNs after 3 d in
culture.
Last, we studied the effects of cell density on survival of the SMNs in
serum-free medium containing a single peptide trophic factor, either
BDNF or HGF. After 3 d in culture, survival at low density was two
to three times lower than at the high density we usually used for our
experiments (Fig. 3).
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Figure 3.
The effect of cell density on spinal motor neuron
survival. Purified spinal motor neurons were cultured in serum-free
medium containing BDNF, HGF, or forskolin and IBMX at the indicated
densities. The percentage of cells surviving was determined by the MTT
assay after 3 d of culture (results are mean ± SEM,
n = 3; a representative experiment is shown; all
experiments were repeated at least three times).
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Effects of cyclic AMP elevation on the survival of spinal
motor neurons
cAMP elevation has been reported previously to enhance the
survival of CNS neurons by enhancing their responsiveness to peptide trophic factors and the survival of PNS neurons by making them independent of growth factors. Thus we next examined the effects of the
pharmacological elevation of cAMP on SMN survival. Forskolin (10 µM), an activator of adenylyl cyclase, is sufficient to
elevate cAMP levels in purified retinal ganglion cells in culture, but it is not sufficient to elevate cAMP levels in sympathetic neuron cultures (Buckmaster and Tolkovsky, 1994; Meyer-Franke et al., 1998),
where the addition of a phosphodiesterase inhibitor, IBMX, is also
necessary. Thus we assessed the effects of pharmacological agents on
cAMP levels by immunostaining with a cAMP-specific polyclonal antiserum
and by radioimmunoassay (see Materials and Methods).
The SMN cultures were treated with serum-free medium containing either
nothing, forskolin (10 µM), IBMX (0.1 mM),
forskolin together with IBMX, or a combination of six peptide trophic
factors for 60 min, and then the cultures were stained with the cAMP
antibody. Only very low levels of basal immunoreactivity were
detectable in the control cultures (Fig.
4A) and in cultured
treated with growth factors alone (Fig. 4B),
forskolin alone (Fig. 4C), or IBMX alone (Fig.
4D). The combination of forskolin and IBMX together, however, produced intense cAMP immunoreactivity throughout the cell
bodies and processes (Fig. 4E,F).
Interestingly, although nearly all cells stained significantly above
control, ~10% of the cells were labeled particularly strongly (data
not shown).
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Figure 4.
cAMP immunoreactivity in spinal motor neurons in
culture. Purified spinal motor neurons in serum-free medium were
treated for 1 hr before immunostaining in nothing
(A), six peptides together (BDNF, CNTF, IGF-1,
HGF, FGF, and GDNF; B), forskolin
(C), IBMX (D), or forskolin
together with IBMX (E, F). Scale
bar, 50 µm.
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These increases in cAMP immunoreactivity corresponded closely with cAMP
contents as measured by a radioimmunoassay (see Materials and Methods).
Although cAMP levels in the SMNs were not increased significantly after
1 hr of treatment with forskolin or IBMX alone, the amount of cAMP was
approximately doubled after 24 hr of culture (Fig.
5). The combination of forskolin plus
IBMX together increased cAMP levels nearly 10-fold when measured both
after 1 and 24 hr of culture (Fig. 5).
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Figure 5.
cAMP content of SMNs in culture. Purified SMNs in
serum-free culture were treated for 1 or 24 hr in the indicated
conditions (see Materials and Methods), and then their cAMP levels were
measured by radioimmunoassay. All values represent mean ± SEM
(n = 4) of a representative experiment; all
experiments were repeated at least three times.
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To determine whether cAMP elevation was sufficient to promote survival
in the absence of added growth factors, we cultured the SMNs for 3 d in serum-free medium containing pharmacological agents to elevate
cAMP. IBMX alone and forskolin alone each weakly promoted SMN survival
(Fig. 5). When forskolin and IBMX were combined, however, the majority
of SMNs survived for 3 d (Fig. 5) and rapidly extended dendrites
and axons, although trophic peptides were not added to the culture
medium. The effects of cAMP elevation on survival and process outgrowth
are not likely to be attributed to enhanced production of autocrine
peptides by the SMNs, because even when they were cultured at very low
density, a majority of SMNs still survived and extended processes (see
Fig. 3). The effects of forskolin and IBMX were mimicked by the cell
membrane-permeant cAMP analog, chlorophenylthio-cAMP (CPT-cAMP, 125 µM; Fig. 6), which was also
sufficient to promote the survival of the majority of SMNs. The
enhancement of survival induced by cAMP elevation was blocked by the
specific protein kinase A inhibitors, H89 (10 µM; Chijiwa
et al., 1990) and Rp-cAMP (200 µM; Botelho et al., 1988).
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Figure 6.
The effects of cAMP elevation on survival of
spinal motor neurons in culture. Purified spinal motor neurons were
cultured in serum-free medium containing the indicated peptide trophic
factors or drugs. After 3 d their survival was measured by the MTT
assay. All peptides and drugs were used at plateau concentrations as
follows: BDNF (10 ng/ml), CNTF (10 ng/ml), HGF (10 ng/ml), GDNF (10 ng/ml), forskolin (10 µM), IBMX (0.1 mM),
pertussis toxin (50 ng/ml), CPT-cAMP (125 µM), and
CPT-cGMP (125 µM).
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Activation of several other second messenger pathways did not mimic the
effects of cAMP elevation. CPT-cGMP (125 µM),
which is not degraded by phosphodiesterases, had a weak and transient effect (Fig. 6), as previously noted (Weill and Green, 1984). However,
CPT-cGMP significantly enhanced intracellular cAMP levels in the SMNs,
possibly by slowing its rate of degradation (our unpublished
observations). The phorbol ester tissue plasminogen activator
(TPA; 10 nM), which activates protein kinase C, and depolarization by high extracellular K+ (50 mM) or glutamate receptor agonists did not mimic the
effects of cAMP elevation (data not shown). For instance, only 2.6 ± 0.5 (mean ± SEM; n = 3) of SMNs survived for
3 d in high K+, which did not elevate cAMP
levels. Interestingly, the survival-promoting effects of CPT-cAMP were
mimicked when pertussis toxin (50 ng/ml), an inhibitor of a
Gi-protein that inhibits adenylyl cyclase, was combined
with IBMX (Fig. 6), suggesting that the SMNs in culture may be
secreting a substance that decreases their cAMP levels. Acetylcholine
was an obvious candidate because it has been shown to activate
muscarinic receptors coupled to Giprotein; however, atropine, an antagonist of muscarinic receptors, did not mimic the
effect of pertussis toxin (data not shown).
Effects of growth factors and cAMP elevation on long-term
survival of SMNs
Last, we asked to what extent combinations of peptide trophic
factors and cAMP elevation could promote the long-term survival of SMNs
in culture. Remarkably, cAMP elevation alone was sufficient to promote
the survival of the majority of cells for 1 week in culture; however,
by the end of 2 weeks most of the cells had died (Fig.
7). Similarly, a combination of five
peptide trophic factors (BDNF, CNTF, GDNF, HGF, and insulin) promoted
the survival of the majority of the SMNs for 1 week, but at 2 weeks
only approximately one-half were still alive; by the end of 3 weeks
most had died. When the combination of five peptide trophic factors was
combined with forskolin and IBMX to elevate cAMP levels, however,
nearly two-thirds of the SMNs were still alive at 2 weeks. When the
serum-free additive B27 (Brewer et al., 1993), which contains lipid
precursors, antioxidants, and several steroid hormones, including
hydrocortisone, also was added, the majority of cells survived for 3 weeks (Fig. 7).
View larger version (18K):
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Figure 7.
The effects of combining survival factors on
long-term survival. The survival of purified spinal motor neurons
cultured in the indicated factors was assessed after 3, 7, 14, and
21 d of culture by the MTT assay. cAMP Elevation
was produced by forskolin (10 µM) plus IBMX (0.1 mM). Hormones are a combination of DHT,
estradiol, and hydrocortisone at plateau levels. The medium also
contained insulin (5 µg/ml). Results are mean ± SEM
(n = 3) of a representative experiment; all
experiments were repeated at least three times.
|
|
| |
DISCUSSION |
Single peptide trophic factors are not sufficient to promote the
short- or long-term survival of the majority of SMNs in
vitro
It has been reported repeatedly that single peptide trophic
factors are sufficient to promote the short-term survival of the majority of SMNs (Henderson et al., 1993, 1994; Pennica et al., 1996).
Our findings are not in agreement with these latter reports. Although
we were able to confirm that a large variety of single peptides is
sufficient to promote the survival of some SMNs, in no case did we find
single peptides that were sufficient to promote short- or long-term
survival of the majority of SMNs. At most, we observed that a single
peptide could promote the survival of approximately one-third of the
cells for ~3 d. Using similar immunopanning methods and culture
medium, however, we have found repeatedly that we can promote the
survival of the majority of cells in cultures of defined types of PNS
neurons with single peptides (Meyer-Franke et al., 1998). We believe
that there are two explanations for the previously reported ability of
single peptides to promote the survival of most SMNs in culture. First,
horse serum, which contains a large variety of known and unknown
peptide trophic factors and also doubles intracellular levels of cAMP
(our unpublished observations), was added to the culture medium in
these previous reports. Second, the survival of the cells in these
studies was normalized for initial viability by dividing all survival
values by the initial viability at 15-24 hr. Although this is intended to normalize for necrotic injury to cells during the isolation procedure, it also subtracts death caused by apoptosis. The large majority of SMNs die by apoptosis in 24 hr when appropriate trophic signals are not present. We have avoided these issues by omitting the
use of serum in our culture medium and by normalizing all of our
survival values to the 1 hr viability (typically ~91%).
Our data are also not in agreement with the possibility that single
peptide trophic factors might significantly promote the long-term
survival of subsets of motor neurons; if this were the case, then
single peptide trophic factors should have been sufficient to promote
the long-term survival of a subset of motor neurons. We did not observe
this; rather, we observed that single peptides promoted the survival of
a small percentage of cells for 3 d, but by 7 d nearly all of
the SMNs had died.
The large variety of different peptides that can promote the survival
of SMNs is remarkable. Peptides from nearly all major classes of
trophic factors are able to promote the survival of the embryonic SMNs.
In addition to the previously reported ability of FGF, GDNF, CNTF, LIF,
CT-1, TGF-, IGF-1, HGF, and neurotrophins to promote SMN survival,
we found that a variety of bone morphogenetic proteins, including
BMP-2, BMP-4, and BMP-7, also significantly promotes the short-term
survival of a subset of SMNs. Schwann cells have been reported to
release trophic activities that promote motor neuron survival,
including neurotrophins, cytokines, and GDNF; they also produce BMP-7
(Lein et al., 1996), and thus it is likely that BMP-7 contributes to
this Schwann cell-derived trophic activity for SMNs. Interestingly,
whereas BMPs such as BMP-7 have been reported to promote the
differentiation of neural cells (Liem et al., 1995; Lein et al., 1996;
Mabie et al., 1997), to our knowledge this is the first example of a
survival-promoting effect by the BMPs.
Multiple trophic factors collaborate to promote survival of
each SMN
Our experiments confirm the remarkable ability of peptide trophic
factors to collaborate in promoting the survival of embryonic SMNs
(Arakawa et al., 1990; Mitsumoto et al., 1994; Wong et al., 1997;
Yamamoto et al., 1997). It has not been clear, however, to what extent
a combination of the known peptides is sufficient to promote long-term
survival of most of the SMNs. Our results indicate that a combination
of up to seven different peptide trophic factors is sufficient only to
promote the survival of approximately one-half of the SMNs in
serum-free medium. Although it is likely that there are important
trophic activities for SMNs that await identification, our data
indicate that the activation of second messenger pathways not normally
activated by peptide trophic factors also may collaborate in promoting
survival. Thus long-term survival of most SMNs is attained when they
are cultured in a combination of peptides normally expressed by Schwann
cells and muscle cells, together with cAMP elevation.
cAMP elevation is sufficient to promote the survival of the
majority of SMNs
The most important finding in this study is that the elevation of
intracellular levels of cAMP is sufficient to promote the survival and
growth of SMNs cultured in serum-free medium in the absence of peptide
trophic factors. Although cAMP elevation has been found previously to
promote survival and growth factor independence in a variety of PNS
neurons, SMNs are the first class of CNS neurons for which this is the
case. cAMP elevation has been reported to enhance the responsiveness of
retinal ganglion cells to their peptide trophic factors (Meyer-Franke
et al., 1995, 1998), but in contrast to these cells the SMNs clearly
responded well to single peptides without the need to elevate cAMP
levels or other second messengers. It is unlikely, however, that cAMP
promotes survival of the SMNs by potentiating the effects of autocrine peptides released into the culture medium, because cAMP-stimulated survival was not strongly dependent on cell density. In contrast, survival of the SMNs cultured in single peptide trophic factors was
strongly dependent on density; this is not surprising in view of the
collaborative effects of peptides on SMN survival and the fact that
SMNs express a variety of trophic peptides such as NT-3.
How does the elevation of cAMP promote survival of the SMNs? The
ability of cAMP elevation to promote the survival of PNS neurons has
been well described for >30 years, but the mechanism is unknown. In
the SMNs, cAMP elevation mimicked several different actions of peptide
growth factors, because it promoted cell survival as well as survival
and growth of their processes, suggesting that growth factors normally
might promote SMN survival by elevating cAMP. This is not the case,
however, because combinations of trophic peptides did not elevate cAMP
levels in the SMN cultures. cAMP elevation might promote survival by
blocking the apoptosis pathway, for instance by elevating bcl-2 levels
or decreasing bax levels, both of which previously have been shown to
be sufficient to promote SMN survival (Dubois-Dauphin et al., 1994;
Farlie et al., 1995; Sagot et al., 1995). However, sympathetic ganglion
neurons that lack bax survive in culture in the absence of peptide
trophic factors but do not extend processes (Deckwerth et al., 1996). An interesting alternative possibility is that cAMP elevation in SMNs
activates a signal transduction pathway normally activated by multiple
growth factors. Consistent with this possibility, protein kinase A
activation recently has been shown to activate the serine-threonine
kinase B-raf, which in turn activates MAP kinase that promotes the
survival of PC12 cells in culture, thus mimicking the effects of NGF
(Vossler et al., 1997). Consistent with this possibility, we were able
to block the cAMP-promoted survival of the SMNs with several protein
kinase A inhibitors. Moreover, transgenic animals deficient in B-raf
have reduced neuronal survival dramatically (Pritchard and McMahon,
1997; Wojnowski et al., 1997). Because SMNs in culture express B-raf
(P. Stork, M. Hanson, and B. Barres, unpublished observations), this
possibility merits future investigation.
Our data do not rule out the possibility that cAMP also helps to
promote the survival of motor neurons by enhancing their responsiveness
to trophic factors, in addition to its ability to make them growth
factor-independent. Several lines of evidence suggest that cAMP may, in
fact, enhance trophic responsiveness. First, the survival of the SMNs
cultured in single peptide trophic factors was increased substantially
by cAMP elevation, and protein kinase A inhibitors blocked the ability
of peptides to promote SMN survival. Moreover, we recently have found
that cAMP elevation significantly increases TrkB immunoreactivity on
the surfaces of the SMNs in culture (Meyer-Franke et al., 1998). Thus
optimal trophic responsiveness of the SMNs very well may depend on
basal cAMP levels. These levels might be disrupted in disease
processes, and dying SMNS, for instance in amyotrophic lateral
sclerosis, may have impaired responsiveness to peptide trophic factors.
If so, this would help to account for the so far limited success of the
delivery of exogenous peptide trophic factors such as BDNF, CNTF, and
IGF-1 in treating ALS patients.
Does cAMP normally help to promote SMN survival in vivo?
Although cAMP elevation was sufficient to promote survival of most of
the SMNs for at least 1 week in culture, survival decayed with longer
culture periods, as it did with combinations of peptides in the absence
of cAMP elevation. Remarkably, however, we found that we could promote
the long-term survival of most of the cells for 3 weeks when we used
cAMP elevation together with a combination of peptide trophic factors.
These findings suggest the possibility that the regulation of cAMP
levels normally may play an important role in the control of SMN
survival in vivo. In any case, these simple defined culture
conditions now provide a preparation in which the behavior of motor
neurons and their interactions with other cell types may be studied in
long-term culture.
Implications for survival and regeneration of SMNs after injury and
in neurodegenerative disease
In addition to possible therapeutic applications of our findings,
our data are also potentially relevant to understanding why CNS and PNS
neurons have radically different abilities to survive and regenerate
their axons in response to axotomy. Whereas PNS neurons survive and
regenerate, many types of CNS neurons die and do not regenerate,
particularly long projection glutamatergic neurons. It recently has
been hypothesized that this lesser ability of CNS neurons to survive
and regenerate is attributable at least in part to the more complex
signaling mechanisms that regulate their survival and growth (Snider,
1994; Meyer-Franke et al., 1995). Remarkably, SMNs, although they are
CNS neurons, share the ability with PNS neurons to both survive and
regenerate after injury and to survive and grow in response to cAMP
elevation. When the axons of CNS and PNS neurons are severed, they are
cut off from important glial-derived and target-derived trophic peptide signals. The presence of another mechanism that is sufficient to
promote survival and growth in the absence of trophic peptide signaling
obviously could be crucial for SMNs and PNS neurons to survive and
regenerate after injury. It is possible that target-derived signals
normally might act to depress cAMP signals and that this inhibition is
released after axotomy. Consistent with this possibility, calcitonin
gene-related peptide, which increases cAMP levels, is upregulated in
SMNs after axotomy (Zigmond and Sun, 1997), but it is not known whether
cAMP levels increase in axotomized SMNs and PNS neurons. Last, our
findings raise the question of whether the survival of other types of
CNS cholinergic neurons, such as cholinergic neurons in the basal
forebrain, which share the ability of SMNs to survive and regenerate
after axotomy, is promoted by cAMP elevation.
| |
FOOTNOTES |
Received April 15, 1998; revised June 26, 1998; accepted July 1, 1998.
This work was supported by grants from the March of Dimes (B.A.B.), by
National Institutes of Health Grants NS32122 and CA09171 (F.A.M.), and
by the National Multiple Sclerosis Society (F.A.M. and A.P.W.). We
thank Genetics Institute for recombinant BMP-2 and BMP-4, Creative
Biomolecules for recombinant BMP-7/OP-1, Genentech for recombinant HGF,
and Regeneron for recombinant BDNF and CNTF.
Correspondence should be addressed to Dr. B. A. Barres, Stanford
University School of Medicine, Department of Neurobiology, Fairchild
Science Building D235, 299 Campus Drive, Stanford, CA 94305-5125. E-mail: barres{at}stanford.edu
| |
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Top
Abstract
Introduction
