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The Journal of Neuroscience, May 15, 1998, 18(10):3708-3714
Nitric Oxide-Dependent Production of cGMP Supports the Survival
of Rat Embryonic Motor Neurons Cultured with Brain-Derived Neurotrophic
Factor
Alvaro G.
Estévez1, 6, 8,
Nathan
Spear1, 6,
J. Anthony
Thompson2, 4, 6,
Trudy L.
Cornwell5,
Rafael
Radi7,
Luis
Barbeito8, 9, and
Joseph S.
Beckman1, 2, 3, 6
Departments of 1 Anesthesiology,
2 Biochemistry and Molecular Genetics,
3 Neuroscience, 4 Surgery, and
5 Pathology, Division of Molecular and Cellular Pathology,
and 6 The University of Alabama at Birmingham Center for
Free Radical Biology, The University of Alabama at Birmingham,
Birmingham, Alabama 35233, and 7 Departamento de
Bioquímica, Facultad de Medicina, 8 Sección
Neurociencias, Facultad de Ciencias, Universidad de la República
11200 Montevideo, Uruguay, and 9 División
Neurobiología Celular y Molecular, Instituto Clemente Estable,
11600 Montevideo, Uruguay
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ABSTRACT |
Trophic factor deprivation induces neuronal nitric oxide synthase
(NOS) and apoptosis of rat embryonic motor neurons in culture. We
report here that motor neurons constitutively express endothelial NOS
that helps support the survival of motor neurons cultured with
brain-derived neurotrophic factor (BDNF) by activating the nitric
oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated
motor neurons to nitro-L-arginine methyl ester
(L-NAME) decreased cell survival 40-50% 24 hr after
plating. Both low steady-state concentrations of exogenous nitric oxide
(<0.1 µM) and cGMP analogs protected BDNF-treated motor
neurons from death induced by L-NAME. Equivalent
concentrations of cAMP analogs did not affect cell survival. Inhibition
of nitric oxide-sensitive guanylate cyclase with 2 µM
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the
survival of BDNF-treated motor neurons by 35%. cGMP analogs also
protected from ODQ-induced motor neuron death, whereas exogenous nitric
oxide did not. In all cases, cell death was prevented with caspase
inhibitors. Our results suggest that nitric oxide-stimulated cGMP
synthesis helps to prevent apoptosis in BDNF-treated motor neurons.
Key words:
motor neurons; BDNF; endothelial nitric oxide synthase; nitric oxide; apoptosis; guanylate cyclase soluble; cGMP
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INTRODUCTION |
Nitric oxide (NO) production has
been implicated in the induction of neuron death by glutamate (Dawson
et al., 1991 , 1993; Strijbos et al., 1996) and in the pathogenesis of a
variety of neurodegenerative diseases (Beckman, 1991; Varner and
Beckman, 1994), including Alzheimer's disease (Good et al., 1996;
Smith et al., 1997), multiple sclerosis (Sherman et al., 1993), and amyotrophic lateral sclerosis (Beckman et al., 1993; Abe et al., 1995,
1997; Chou et al., 1996a,b). Ventral root avulsion in the rat results
in the induction of nitric oxide synthase (NOS) and motor neuron loss 6 weeks after treatment (Wu, 1993). Inhibition of NOS activity or
prevention of NOS expression by brain-derived neurotrophic factor
(BDNF) improves motor neuron survival after ventral root avulsion (Wu
and Li, 1993; Novikov et al., 1995, 1997).
Motor neuron survival in culture is highly dependent on trophic factor
supply (Henderson et al., 1993, 1994; Hughes et al., 1993; Pennica et
al., 1996). Trophic factor deprivation of cultured motor neurons
stimulates de novo synthesis of neuronal NOS, increases nitrotyrosine immunoreactivity (Estévez et al., 1998), and
induces apoptosis after 18-24 hr (Milligan et al., 1995; Pennica et
al., 1996; Estévez et al., 1998). Inhibition of nitric oxide
synthesis supports the survival of trophic factor-deprived motor
neurons for up to 3 d. Production of nitric oxide by
(Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino] diazen-1-ium-1,2-diolate (DETA-NONOate) reverses protective
effects of NOS inhibitors against apoptosis induced by trophic factor deprivation. In contrast, extracellular NO production does not affect
the number of motor neurons surviving either in the presence or absence
of trophic factors (Estévez et al., 1998), showing that nitric
oxide itself is not sufficient to initiate apoptosis.
Nitric oxide also plays an important role in cell signaling in the
nervous system and is implicated in synaptic plasticity (Garthwaite,
1991; Dinerman et al., 1994; O'Dell et al., 1994; Zhuo et al., 1994;
Boulton et al., 1995; Garthwaite and Boulton, 1995; Kantor et al.,
1996). Nitric oxide is known to contribute to spinal motor neuron
maturation stimulated by supraspinal inputs (Kalb and Agostini,
1993).
It has been proposed that nitric oxide toxicity involves its reaction
with superoxide to form the strong oxidant peroxynitrite (Beckman,
1991; Beckman et al., 1993: Dawson et al., 1993; Lipton et al., 1993;
Troy et al., 1996; Estévez et al., 1998), although its protective
and regulatory effects may be attributable to either stimulation of
cGMP synthesis by the soluble guanylate cyclase (Garthwaite and
Boulton, 1995; Farinelli et al., 1996) or S-nitrosylation of the NMDA
receptor and probably other cellular targets (Lipton et al., 1993;
Stamler et al., 1997). Activation of the soluble guanylate cyclase is a
major signaling pathway activated by nitric oxide (Ignarro, 1989;
Garthwaite and Boulton, 1995). Early studies showed that cultured
spinal cord neurons produce cGMP (Weill, 1982). Furthermore, cGMP
analogs prevented spinal cord motor neuron apoptosis during the period
of programmed cell death (Weill and Greene, 1984), suggesting a role
for cGMP in motor neuron survival and differentiation. We report that
nitric oxide-stimulated cGMP synthesis helps prevent apoptosis of motor
neuron cultures in the presence of BDNF, suggesting that low levels of
nitric oxide can have a role in the survival of motor neurons.
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MATERIALS AND METHODS |
Materials. Monoclonal antibodies to p75 low-affinity
neurotrophin receptor and to Islet-1/2 were obtained from the culture medium of the MC192 (Chandler et al., 1984) and 4D5 (Ericson et al.,
1992; Tsuchida et al., 1994) hybridoma cells obtained from C. E. Henderson (Institut National de la Santé et de la Recherche Médicale Unité 382, Developmental Biology Institute of
Marseille, Marseille, France) and the Developmental Studies Hybridoma
Bank (Iowa City, IA), respectively. Polyclonal antibodies to neuronal and endothelial NOS were from Transduction Laboratories (Lexington, KY)
and a generous gift from B. Mayer (Karl-Franzes-Universität Graz,
Graz, Austria). Monoclonal antibodies to endothelial NOS were kindly
provided by T. Michel (Harvard Medical School, Boston, MA).
Affinity-purified anti-mouse IgG was from Cappel (Durham, NC). Cy3- and
FITC-conjugated goat anti-mouse and anti-rabbit secondary antibodies
were obtained from Jackson ImmunoResearch (West Grove, PA). Recombinant
mouse BDNF was a gift of R. W. Scott and J. D. Hirsch
(Cephalon, Inc., West Chester, PA). Culture media, serum, insulin, and
antibiotics were from Life Technologies (Grand Island, NY). The NO
donor DETA-NONOate, the guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and 8-bromo and
8-(4-chlorophenylthio) analogs of cGMP and cAMP were from Alexis
Biochemicals (San Diego, CA). The caspase inhibitor Ac-YVAD-CHO was
from Calbiochem (San Diego, CA), and caspase inhibitors z-VAD-fmk and
Ac-DEVD-CHO were from Alexis Biochemicals (San Diego, CA). Other
reagents used were from Sigma (St. Louis, MO).
Motor neuron purification and culture. Purified motor
neurons were prepared from rat embryonic day 15 spinal cord by
combination of metrizamide gradient centrifugation and immunopanning
with the monoclonal antibody IgG192 against the p75 low-affinity
neurotrophin receptor, as described previously (Henderson et al., 1995;
Estévez et al., 1998). Motor neurons were cultured in L15 media
supplemented with 0.63 mg/ml sodium bicarbonate, 5 µg/ml insulin, 0.1 mM putrescine, 0.1 mg/ml conalbumin, 30 nM
sodium selenite, 20 nM progesterone, 20 mM
glucose, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2% horse
serum. Before plating, culture dishes and slides were precoated with
polyornithine-laminin. Cultures maintained for 24 hr in the presence
of BDNF were mainly composed of large neurons with long-branched
neurites. More than 94% of the cells showed immunofluorescence for the
motor neuron markers p75 neurotrophin receptor (Yan and Johnson, 1988;
Henderson et al., 1993; Estévez et al., 1998) and Islet-1/2
(Henderson et al., 1993; Tsuchida et al., 1994; Estévez et al.,
1998).
Determination of mRNA expression. Total RNA from 50,000 motor neurons plated on 60 mm dishes was isolated using Trizol (Life Technologies) according to manufacturer's instructions,
reverse-transcribed with a reverse transcription-PCR (RT-PCR) kit
(Stratagene, La Jolla, CA), and amplified with the GeneAmp PCR reagent
kit (Perkin-Elmer, Norwalk, CT) (1 cycle at 91°C for 5 min, 54°C
for 5 min, followed by 30 cycles at 91°C for 1 min; 1 cycle at 54°C
for 1 min; 1 cycle at 72°C for 2 min; and a final cycle of 72°C for
10 min). Sense and antisense primers were for endothelial NOS
5'-TACGGAGCAGCAAATCCAC and 5'-CAGGCTGCAGTCCTTTGATC-3' as described by
Shaul et al. (1995). These endothelial NOS primers did not yield a
detectable product using the neuronal NOS cDNA as a template.
Furthermore, the results of a search for the primer sequences in the
National Institutes of Health BLAST indicate that the only homologous
sequence contained in the library corresponds to the rat endothelial
NOS. The products of the PCR were separated by electrophoresis in a 2%
agarose gel and visualized in a UV transilluminator after staining with
ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to normalize the levels of RNA (Estévez et al.,
1998).
Immunofluorescence. Cultures were fixed for 15 min with a
combination of 4% paraformaldehyde and 0.1% glutaraldehyde in PBS on
ice. Then the cells were rinsed three times with PBS, permeabilized with 0.1% Triton X-100 for 15 min, blocked for 1 hr with 10% goat serum and 2% bovine serum albumin in PBS, incubated with the primary antibody overnight at 4°C, rinsed three times with PBS, incubated with FITC- or Cy3-conjugated secondary antibody for 30 min at room
temperature, rinsed again three times with PBS, fixed with 4%
paraformaldehyde, and rinsed with PBS. After a brief rinse in
double-distilled water, the cells were mounted in a SlowFade-Light antifade kit (Molecular Probes, Eugene, OR). Primary antibodies were
used at a dilution of 1:100 for supernatant from 4D5 hybridoma against
Islet-1/2 and 5 µg/ml for the IgG 192 to p75 neurotrophin receptor.
Rabbit polyclonal antibody against neuronal NOS from Transduction
Laboratories was used at a 1:100 dilution. Antibodies to endothelial
NOS included a monoclonal provided by T. Michel used at a 1:200
dilution and two different polyclonal antibodies obtained from B. Mayer
and Transduction Laboratories used at 1:400 and 1:100 dilutions,
respectively. Similar results were obtained with all three antibodies
to endothelial NOS, but the best results were obtained using the
polyclonal antibody obtained from B. Mayer. Controls used included
omission of the primary antibody or nonpermeabilizing cells when using
antibodies directed against the intracellular antigens endothelial NOS
and Islet-1/2.
Determination of motor neuron survival. Motor neuron
survival was quantified 24 hr and 3 d after plating by counting
all healthy neurons with neurites longer than four soma diameters under
phase contrast in a 1 cm2 field in the center of the
dish, as described previously (Henderson et al., 1993; Pennica et al.,
1996; Estévez et al., 1998). Motor neurons considered viable by
this method also stained with the vital dye fluorescein diacetate. Most
of the degenerating neurons considered nonviable by this method showed
morphological characteristics of apoptosis, including disintegration of
the neurites and soma shrinkage (see Fig. 2). Nuclear condensation and
fragmentation were visualized by terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling as described
previously (Estévez et al., 1998). To compare the results of
different experiments, the number of cells attached with neurites 4-5
hr after plating in the presence of BDNF was taken as 100% survival
and was ~50-60% of the cells initially plated (Henderson et al.,
1993; Estévez et al., 1998).
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RESULTS |
Motor neurons in culture express endothelial NOS
BDNF-treated motor neurons expressed endothelial NOS mRNA (Fig.
1) and were immunoreactive for
endothelial NOS protein 24 hr after plating (Fig.
2). Both mRNA (Fig. 1) and
immunoreactivity (Fig. 2) for endothelial NOS were also present after
trophic factor deprivation, which causes motor neurons to undergo
apoptosis (Milligan et al., 1995; Estévez et al., 1998). In
contrast, mRNA and protein immunoreactivity for neuronal NOS were found
only after trophic factor deprivation and were not detectable in
BDNF-treated neurons, as reported previously (Estévez et al.,
1998). Endothelial NOS immunoreactivity was localized predominately in
cytoplasmic regions of the soma of BDNF-treated motor neurons. Less
immunoreactivity was found in the neurites. In contrast, endothelial
NOS immunoreactivity in trophic factor-deprived cells was disordered
because of the loss of nuclear and cellular structure during apoptosis.
These results showed that motor neurons constitutively express both the
messenger and the protein for endothelial NOS.
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Figure 1.
Rat embryonic motor neurons in culture express
endothelial NOS mRNA. Total RNA from 50,000 motor neurons was extracted
24 hr after plating. Half of the RNA was incubated with reverse
transcriptase (+RT) before PCR amplification,
whereas the other half was amplified without RT
( RT). The amplification product for the GAPDH
was used to standardize the quantities of sample used in the gels. A
cDNA (100 pg) from an endothelial NOS vector was used as the standard,
and the specificity of the primers was assessed using a similar
quantity of neuronal NOS cDNA (data not shown).
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Figure 2.
Endothelial NOS immunoreactivity in cultured motor
neurons. Fluorescence micrographs of motor neurons cultured for 24 hr
with 100 pg/ml BDNF (A) or without trophic
factors (B) and stained using antibodies to
endothelial NOS. Staining was performed as described in Materials and
Methods. Similar results were obtained using two other antibodies.
Omission of the primary antibody and nonpermeabilized cells revealed no
detectable stain. Scale bar, 25 µm.
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Inhibition of nitric oxide synthesis initiates BDNF-treated motor
neuron death
Inhibition of nitric oxide production by L-NAME
supported trophic factor-deprived motor neuron survival (Fig.
3A) but stimulated the death
of >40% of the BDNF-treated neurons 24 hr after plating (Fig.
3B). Low steady-state concentrations of NO (<100
nM) produced by 20 µM DETA-NONOate were
measured in the culture medium, as described previously (Beckman and
Conger, 1995; Estevez et al., 1998). Production of low steady-state
concentrations of exogenous nitric oxide by 20 µM
DETA-NONOate prevented BDNF-treated motor neuron death induced by
L-NAME for 24 hr (Fig. 3) and for up to 3 d (data not
shown) but did not affect motor neuron survival in either the presence
or absence of BDNF (Fig. 3). The inactive stereoisomer
nitro-D-arginine methyl ester (D-NAME) did not
modify neurotrophin-treated motor neuron survival (Fig. 3B).
These results suggested that L-NAME induced motor neuron
death by inhibiting nitric oxide production.
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Figure 3.
Effect of NOS inhibitors on BDNF-dependent motor
neuron survival. Motor neuron survival was determined 24 hr after
plating by counting neurons with neurites longer than four soma
diameters, as described in Materials and Methods. Motor neurons were
cultured with (A) or without
(B) 100 pg/ml BDNF plus 1 mM
L-NAME (LNAME), 1 mM
D-NAME (DNAME), 20 µM
DETA-NONOate (NO), or the indicated combinations. The
steady-state concentration of NO produced by 20 µM
DETA-NONOate under our experimental conditions was 100 nM,
as described previously (Beckman and Conger, 1995; Estévez et
al., 1998). Different from BDNF, *p < 0.001. Values are the mean ± SD of at least three experiments performed
in duplicate.
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Most of the cells considered nonviable showed apoptotic morphology
(Fig. 2B). To further determine whether
L-NAME inhibited neurite growth or induced apoptosis,
L-NAME toxicity on BDNF-treated motor neuron cultures was
challenged using the following: Ac-YVAD-CHO, a selective caspase I
inhibitor that prevents cultured motor neuron apoptosis (Milligan et
al., 1995; Estévez et al., 1998); zVAD, a general caspase
inhibitor; and Ac-DEVD-CHO, a selective caspase III inhibitor (for
review, see Nicholson and Thornberry, 1997). Motor neuron death induced
by NOS inhibition was prevented by all three caspase inhibitors for 24 hr (Fig. 4), suggesting that L-NAME induced apoptosis in BDNF-treated motor neurons
rather than inhibited differentiation. Similar results were obtained after 3 d, but the fresh caspase inhibitors had to be added every 24 hr (data not shown). The protection by caspase inhibitors suggests that the change in morphology was attributable to motor neuron death by
apoptosis rather than inhibition of differentiation.
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Figure 4.
Inhibition of caspase activity prevents
L-NAME-induction of BDNF-treated motor neuron death. Motor
neuron survival was determined 24 hr after plating, as indicated in
Figure 3. Cultures were exposed to 100 pg/ml BDNF alone or with the
indicated combinations of 1 mM L-NAME
(LNAME), 10 µM Ac-YVAD-CHO
(LNAME+YVAD), 50 µM
zVAD-fmk (LNAME+zVAD), and 30 µM Ac-DEVD-CHO
(LNAME+DEVD). Values are the mean ± SD of two experiments performed in duplicate.
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cGMP supports motor neuron survival
The cGMP analogs 8-Br-cGMP and 8-CPT-cGMP abolished the toxic
effects of L-NAME on BDNF-treated motor neurons (Table
1). The protective effect of 8-Br-cGMP
was dependent on the concentration, with an apparent EC50
of 30 µM, and was maximal at ~100 µM
(Fig. 5). No protection was provided by
100 µM 8-CPT-cAMP (Table 1) or 10-1000 µM
8-Br-cAMP (Fig. 5), showing that cGMP analogs were not protective by
cross-activation of a cAMP-dependent pathway (Jiang et al., 1992;
Cornwell et al., 1994).
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Figure 5.
Concentration-dependent prevention of
L-NAME toxicity by 8-Br-cGMP but not by cAMP. Motor neuron
survival was determined 24 hr after plating, as described in Figure 3.
 , Trophic factor deprivation;  , BDNF (100 pg/ml) plus
L-NAME (1 mM);  , BDNF plus
L-NAME plus 8-Br-cGMP;  , BDNF plus L-NAME
plus 8-Br-cAMP. Values are the mean ± SD of at least two
experiments performed in duplicate.
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To further test the role of cGMP production on motor neuron survival,
BDNF-treated cultures were exposed to the selective soluble guanylate
cyclase inhibitor ODQ (Garthwaite et al., 1996). ODQ (2 µM) did not affect the survival of motor neurons deprived of trophic factors but stimulated ~40% of BDNF-treated motor neurons to die after 24 hr (Fig. 6). After 72 hr,
ODQ stimulated the death of 55 ± 7% of the motor neurons
cultured with BDNF. Analogs of cGMP abolished the toxic effects of ODQ
after 24 hr (Fig. 6) and for up to 3 d (data not shown), whereas
extracellular generation of nitric oxide from DETA-NONOate was not
protective (data not shown). These results suggest that ODQ increased
apoptosis was attributable to selective inhibition of cGMP synthesis by
guanylate cyclase.
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Figure 6.
Effect of the inhibition of soluble guanylate
cyclase on BDNF-treated motor neuron survival. Motor neuron survival
was determined 24 hr after plating, as described in Figure 3. Motor
neurons were cultured with 100 pg/ml BDNF (bars
3-6), 2 µM ODQ (bars 2,
4-6), 1 mM 8Br-cGMP
(B-cG; bar 5), and 100 µM
CTP-cGMP (C-cG; bar 6) in the
indicated combinations. Values are the mean ± SD of at least
three experiments performed in duplicate.
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DISCUSSION |
In the present study, we found that nitric oxide produced by the
endothelial isozyme of nitric oxide synthase contributed to the
survival of motor neurons cultured with BDNF by a cGMP-dependent mechanism. Inhibition of nitric oxide synthesis with L-NAME
or cGMP formation with the guanylate cyclase inhibitor ODQ reduced motor neuron survival by 40%. The cell death appeared to result from
apoptosis based on morphological criteria and protection with caspase
inhibitors. Cell death attributable to NOS inhibition was prevented
either by generating low steady-state concentrations of exogenous
nitric oxide or by the addition of cGMP analogs. In contrast, cell
death induced by the guanylate cyclase inhibitor ODQ was prevented by
cGMP analogs but not by exogenous nitric oxide. These results suggest
that nitric oxide-dependent activation of soluble guanylate cyclase was
necessary for BDNF to support the survival of motor neurons in
culture.
Treatment of chick embryos with cGMP analogs prevents programmed cell
death of motor neurons (Weill and Greene, 1984), suggesting that cGMP
production may be involved in embryonic development. Induction of
neuronal NOS is frequently associated with critical developmental
periods in the brain. However, Kalb and Agostini (1993) report that
differentiation of motor neurons in vivo appears to be
influenced by nitric oxide production during a period in which motor
neurons are not immunoreactive for neuronal NOS, suggesting that either
nitric oxide is produced by neighboring cells or by a different NOS
isoform. Our results indicate that embryonic motor neurons in culture
constitutively express endothelial NOS. We have reported previously
that BDNF-treated motor neurons do not express detectable amounts of
neuronal NOS, but it is rapidly induced in BDNF-deprived cultures
(Estévez et al., 1998). No detectable immunoreactivity for the
inducible macrophage-like nitric oxide synthase could be found in
either BDNF-treated or -deprived motor neuron cultures (A. G. Estevez
and J. S. Beckman, unpublished observations). Consequently, endothelial
NOS was the most likely candidate for stimulating cGMP formation that
contributes to BDNF-stimulated survival of motor neurons in
culture.
Immunoreactivity for endothelial NOS has been found in adult human
motor neurons from amyotrophic lateral sclerosis patients (Chou et al.,
1996b; Abe et al., 1997). Endothelial NOS is also reported in rat and
mouse hippocampal pyramidal neurons in which it may play a role in the
regulation of long-term potentiation (Dinerman et al., 1994; O'Dell et
al., 1994; Kantor et al., 1996). Posttranslational modifications of
endothelial NOS result in a distinct intracellular localization
compared with neuronal NOS. The N terminus of endothelial NOS can be
both palmitoylated and myristylated, the hydrophobic tails of which
anchor the endothelial NOS to the plasma and Golgi membranes (Busconi
and Michel, 1993; Robinson et al., 1995, 1996; Sessa et al., 1995). The
activity of endothelial NOS is further modulated in endothelium by
binding to caveolin proteins that keep endothelial NOS in an inactive state (Feron et al., 1996; Michel et al., 1997). In contrast, neuronal
NOS is a soluble enzyme but contains an additional 20 kDa N-terminal
protein domain known as the PDZ domain (PSD-95/disks large/ZO-1
homology domain) that targets neuronal NOS to membrane-associated proteins (Brenman et al., 1997). These interactions can localize neuronal NOS to NMDA receptor in neurons and to cluster near motor neuron endplates in skeletal muscle in which the influx of calcium after channel opening will rapidly activate neuronal NOS. The distinct
intracellular localization of endothelial NOS compared with neuronal
NOS may result in different physiological regulations and functions
in vivo. Trophic factors such as neurotrophins are known to
elevate intracellular calcium levels in neurons (Hegarty et al., 1997;
Holm et al., 1997; Jiang et al., 1997) that may be responsible for the
activation of endothelial NOS and subsequent stimulation of cGMP
production.
The apparent requirement for nitric oxide-stimulated cGMP production
for the survival of motor neurons cultured with BDNF was surprising,
because we have shown previously that the production of nitric oxide by
neuronal NOS can induce motor neuron death in trophic factor-deprived
motor neurons (Estévez et al., 1998). The toxicity of nitric
oxide in trophic factor-deprived motor neurons appears to result from
the simultaneous production of superoxide and nitric oxide that leads
to the formation of peroxynitrite (Estévez et al., 1998). Both
NOS inhibitors and superoxide scavengers largely prevent motor neuron
death attributable to trophic factor deprivation. In addition, motor
neurons become immunoreactive for nitrotyrosine, a modification induced
by peroxynitrite (Beckman et al., 1994; Beckman, 1996). The protection
by NOS inhibitors in BDNF-deprived motor neurons was overcome by
generating nitric oxide with 20 µM DETA-NONOate. However,
the same concentrations of NO generated under identical conditions from
DETA-NONOate were protective in the present study to motor neurons
treated with NOS inhibitors. Consequently, nitric oxide either can
support the survival of motor neurons by stimulating cGMP synthesis or can induce apoptosis by reacting with superoxide to form peroxynitrite (Estévez et al., 1998).
In parallel with our observations on motor neurons, nitric oxide has
been shown to support survival of PC12 cells deprived of trophic
factors through a cGMP-dependent mechanism (Farinelli et al., 1996).
However, nitric oxide can also be toxic to PC12 cells when superoxide
scavenging is impaired with antisense superoxide dismutase (SOD)
oligonucleotides (Troy et al., 1996). The toxicity resulting from SOD
downregulation has also been attributed to the formation of
peroxynitrite in PC12 cells (Troy et al., 1996). Nerve growth factor
(NGF) prevents death, induces differentiation of PC12 cells (Greene and
Tischler, 1976), and stimulates de novo synthesis of all
three isoforms of NOS (Peunova and Enikolopov, 1995). Inhibition of
nitric oxide production prevents differentiation of PC12 cells by NGF
but does not affect cell survival induced by NGF (Peunova and
Enikolopov, 1995; Farinelli et al., 1996). This contrasts with our
results with cultured motor neurons in which inhibition of nitric oxide
synthesis in the presence of trophic factors led to apoptosis. In both
PC12 cells and motor neurons, endogenous production of nitric oxide has
important physiological functions mediated by cGMP that improve
survival and differentiation. On the other hand, NO can become toxic to
motor neurons by forming peroxynitrite when conditions increase
cellular superoxide generation, as occurs during trophic factor
deprivation.
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FOOTNOTES |
Received Dec. 22, 1997; revised Feb. 16, 1998; accepted March 6, 1998.
This work was supported by grants from National Institutes of Health
(NIH) (J.S.B.) and the American Amyotrophic Lateral Sclerosis Association (J.S.B.). N.S. was supported in part by the Basic Mechanisms in Lung Disease Training Fellowship Grant HL07533 from NIH.
We thank C. E. Henderson from the Developmental Biology Institute of Marseille for the MC192 hybridoma and for helpful suggestions and
discussion; R. A. Star from the University of Texas (Dallas, TX)
for the generous gift of endothelial NOS cDNA; R. W. Scott and
J. D. Hirsch from Cephalon, Inc. (West Chester, PA) for BDNF; B. Mayer from Karl-Franzes-Universität Graz (Graz, Austria) for endothelial NOS antibodies; K. Conger, Y. Zhuang, and Y. Z. Ye from University of Alabama at Birmingham for helpful suggestions and
assistance; Q. Li for technical assistance with the RT-PCR; and V. Darley-Usmar for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Alvaro G. Estévez,
Department of Anesthesiology, Research Division, University of Alabama
at Birmingham, 1900 University Boulevard, THT 958, Birmingham, AL
35233.
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REFERENCES |
-
Abe K,
Pan L-H,
Watanabe M,
Kato T,
Itoyama Y
(1995)
Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis.
Neurosci Lett
199:152-154[ISI][Medline].
-
Abe K,
Pan L-H,
Watanabe M,
Konno H,
Kato T,
Itoyama Y
(1997)
Upregulation of protein-tyrosine nitration in the anterior horn of amyotrophic lateral sclerosis.
Neurol Res
19:124-128[ISI][Medline].
-
Beckman JS
(1991)
The double-edged role of nitric oxide in brain function and superoxide-mediated pathology.
J Dev Physiol
15:53-59[ISI][Medline].
-
Beckman JS
(1996)
Oxidative damage and tyrosine nitration from peroxynitrite.
Chem Res Toxicol
9:836-844[ISI][Medline].
-
Beckman JS,
Conger K
(1995)
Direct measurement of nitric oxide in solutions with an ozone based chemiluminescent detector.
Methods
7:35-39.
-
Beckman JS,
Carson M,
Smith CD,
Koppenol WH
(1993)
ALS, SOD and peroxynitrite.
Nature
364:584[Medline].
-
Beckman JS,
Ye YZ,
Anderson P,
Chen J,
Accavetti MA,
Tarpey MM,
White CR
(1994)
Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry.
Biol Chem
375:81-88.
-
Boulton CL,
Southam E,
Garthwaite J
(1995)
Nitric oxide-dependent long-term potentiation is blocked by a specific inhibitor of soluble guanylyl cyclase.
Neuroscience
69:699-703[ISI][Medline].
-
Brenman JE,
Xia H,
Chao DS,
Black SM,
Bredt DS
(1997)
Regulation of neuronal nitric oxide synthase through alternative transcripts.
Dev Neurosci
19:224-231[ISI][Medline].
-
Busconi L,
Michel T
(1993)
Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization.
J Biol Chem
268:8410-8413[Abstract/Free Full Text].
-
Chandler CE,
Parsons LM,
Hosang M,
Shooter EM
(1984)
A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells.
J Biol Chem
259:6882-6889[Abstract/Free Full Text].
-
Chou SM,
Wang HS,
Taniguchi A
(1996a)
Role of SOD-1 and nitric oxide/cyclic GMP cascade on neurofilament aggregation in ALS/MND.
J Neurol Sci [Suppl]
139:16-26.
-
Chou SM,
Wang HS,
Komai K
(1996b)
Colocalization of NOS and SOD1 in neurofilament accumulation within motor neurons of amyotrophic lateral sclerosis: an immunohistochemical study.
J Chem Neuroanat
10:249-258[ISI][Medline].
-
Cornwell TL,
Arnold E,
Boerth NJ,
Lincoln TM
(1994)
Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP.
Am J Physiol
267:C1405-C1413[Abstract/Free Full Text].
-
Dawson VL,
Dawson TM,
London ED,
Bredt DS,
Snyder SH
(1991)
Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.
Proc Natl Acad Sci USA
88:6368-6371[Abstract/Free Full Text].
-
Dawson V,
Dawson T,
Bartley D,
Uhl G,
Snyder S
(1993)
Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures.
J Neurosci
13:2651-2661[Abstract].
-
Dinerman JL,
Dawson TM,
Schell MJ,
Snowman A,
Snyder SH
(1994)
Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity.
Proc Natl Acad Sci USA
91:4214-4218[Abstract/Free Full Text].
-
Ericson J,
Thor S,
Edlund T,
Jessell T,
Yamada T
(1992)
Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1.
Science
256:1555-1560[Abstract/Free Full Text].
-
Estévez AG,
Spear N,
Manuel SM,
Radi R,
Henderson CE,
Barbeito L,
Beckman JS
(1998)
Nitric oxide and superoxide contribute to motor neuron apoptosis induced by trophic factor deprivation.
J Neurosci
18:923-931[Abstract/Free Full Text].
-
Farinelli SE,
Park DS,
Greene LA
(1996)
Nitric oxide delays the death of trophic factor-deprived PC12 cells and sympathetic neurons by a cGMP-mediated mechanism.
J Neurosci
16:2325-2334[Abstract/Free Full Text].
-
Feron O,
Belhassen L,
Kobzik L,
Smith TW,
Kelly RA,
Michel T
(1996)
Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells.
J Biol Chem
271:22810-22814[Abstract/Free Full Text].
-
Garthwaite J
(1991)
Glutamate, nitric oxide and cell-cell signaling in the nervous system.
Trends Neurosci
14:75-82.
-
Garthwaite J,
Boulton CL
(1995)
Nitric oxide signaling in the central nervous system.
Annu Rev Physiol
57:683-706[ISI][Medline].
-
Garthwaite J,
Sotham E,
Boulton CL,
Nielsen EB,
Schmidt K,
Mayer B
(1996)
Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.
Biochem Pharmacol
48:184-188.
-
Good PF,
Werner P,
Hsu A,
Olanow CW,
Perl DP
(1996)
Evidence for neuronal oxidative damage in Alzheimer's disease.
Am J Pathol
149:21-28[Abstract].
-
Greene LA,
Tischler AS
(1976)
Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.
Proc Natl Acad Sci USA
73:2424-2428[Abstract/Free Full Text].
-
Hegarty JL,
Kay AR,
Green SH
(1997)
Trophic support of cultured spiral ganglion neurons by depolarization exceeds and is additive with that by neurotrophins or cAMP and requires elevation of [Ca2+]i within a set range.
J Neurosci
17:1959-1970[Abstract/Free Full Text].
-
Henderson CE,
Camu W,
Mettling C,
Gouin A,
Poulsen K,
Karihaloo M,
Rullamas J,
Evans T,
McMahon SB,
Armanini MP,
Berkemeier L,
Phillips HS,
Rosenthal A
(1993)
Neurotrophins promote motor neuron survival and are present in embryonic limb bud.
Nature
363:266-270[Medline].
-
Henderson CE,
Phillips HS,
Pollock RA,
Davies AM,
Lemeulle C,
Armanini M,
Simmons L,
Moffet B,
Vandlen RA,
Koliatsos VE,
Rosenthal A
(1994)
GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.
Science
266:1062-1064[Abstract/Free Full Text].
-
Henderson CE,
Bloch-Gallego E,
Camu W
(1995)
Purification and culture of embryonic motorneurons.
In: Neural cell culture: a practical approach (Cohen J,
Wilkin G,
eds), pp 69-81. Oxford: IRL.
-
Holm NR,
Christophersen P,
Olesen AP,
Gammeltoft S
(1997)
Activation of calcium-dependent potassium channels in rat brain neurons by neurotrophin-3 and nerve growth factor.
Proc Natl Acad Sci USA
94:1002-1006[Abstract/Free Full Text].
-
Hughes RA,
Sendtner M,
Thoenen H
(1993)
Members of several gene families influence survival of rat motoneurons in vitro and in vivo.
J Neurosci Res
36:663-671[ISI][Medline].
-
Ignarro LJ
(1989)
Heme-dependent activation of soluble guanylate cyclase by nitric oxide: regulation of enzyme activity by porphyrins and metalloporphyrins.
Semin Hematol
26:63-76[ISI][Medline].
-
Jiang H,
Shabb JB,
Corbin JD
(1992)
Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues.
Biochem Cell Biol
70:1283-1289[ISI][Medline].
-
Jiang H,
Ulme DS,
Dickens G,
Chabuk A,
Lavarreda M,
Lazarovici P,
Guroff G
(1997)
Both p140trk and p75NGFR nerve growth factor receptors mediate nerve growth factor-stimulated calcium uptake.
J Biol Chem
272:6835-6837[Abstract/Free Full Text].
-
Kalb RG,
Agostini J
(1993)
Molecular evidence for nitric oxide-mediated motor neuron development.
Neuroscience
57:1-8[ISI][Medline].
-
Kantor DB,
Lanzrein M,
Stary SJ,
Sandoval GM,
Smith WB,
Sullivan BM,
Davidson N,
Schuman EM
(1996)
A role for endothelial NO synthase in LTP revealed by adenovirus-mediated inhibition and rescue.
Science
274:1744-1748[Abstract/Free Full Text].
-
Lipton SA,
Choi YB,
Pan ZH,
Lei SZ,
Chen HSV,
Sucher NJ,
Loscalzo J,
Singel DJ,
Stamler JS
(1993)
A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.
Nature
364:626-631[Medline].
-
Michel JB,
Feron O,
Sacks D,
Michel T
(1997)
Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin.
J Biol Chem
272:15583-15586[Abstract/Free Full Text].
-
Milligan CE,
Prevette D,
Yaginuma H,
Homma S,
Cardwell C,
Fritz LC,
Tomaselli KJ,
Oppenheim RW,
Scwartz LM
(1995)
Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.
Neuron
15:385-393[ISI][Medline].
-
Nicholson DW,
Thornberry NA
(1997)
Caspases: killer proteases.
Trends Biochem Sci
22:299-306[ISI][Medline].
-
Novikov L,
Novikova L,
Kellerth J-O
(1995)
Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion.
Neurosci Lett
200:45-48[ISI][Medline].
-
Novikov L,
Novikova L,
Kellerth J-O
(1997)
Brain-derived neurotrophic factor promotes axonal regeneration and long-term survival of adult rat spinal motoneurons in vivo.
Neuroscience
79:765-774[ISI][Medline].
-
O'Dell TJ,
Huang PL,
Dawson TM,
Dinerman JL,
Snyuder SH,
Kandel ER,
Fishman MC
(1994)
Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS.
Science
265:542-546[Abstract/Free Full Text].
-
Pennica D,
Arce V,
Swanson TA,
Vejsada R,
Pollock RA,
Armanini M,
Dudley K,
Phillips HS,
Rosenthal A,
Kato AC,
Henderson CE
(1996)
Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons.
Neuron
17:63-74[ISI][Medline].
-
Peunova N,
Enikolopov G
(1995)
Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells.
Nature
375:68-73[Medline].
-
Robinson LJ,
Busconi L,
Michel T
(1995)
Agonist-modulated palmitoylation of endothelial nitric oxide synthase.
J Biol Chem
270:995-998[Abstract/Free Full Text].
-
Robinson JL,
Ghanouni P,
Michel T
(1996)
Posttranslational modifications of endothelial nitric oxide synthase.
Methods Enzymol
268:436-448[Medline].
-
Sessa WC,
Carcia-Cardeña G,
Liu J,
Keh A,
Pollock JS,
Bradley J,
Thiru S,
Braverman IM,
Desai KM
(1995)
The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide.
J Biol Chem
270:17641-17644[Abstract/Free Full Text].
-
Shaul PW,
North AJ,
Snyder SH,
Star RA
(1995)
Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.
Am J Respir Cell Mol Biol
13:167-174[Abstract].
-
Sherman MP,
Griscavage JM,
Ignarro LJ
(1993)
Nitric oxide-mediated neuronal injury in multiple sclerosis.
Med Hypotheses
39:143-146.
-
Smith MA,
Harris PL,
Sayre LM,
Beckman JS,
Perry G
(1997)
Widespread peroxynitrite-mediated damage in Alzheimer's disease.
J Neurosci
17:2653-2657[Abstract/Free Full Text].
-
Stamler JS,
Toone EJ,
Lipton SA,
Sucher NJ
(1997)
(S)NO signals: translocation, regulation, and a consensus motif.
Neuron
18:691-696[ISI][Medline].
-
Strijbos PJ,
Leach MJ,
Garthwaite J
(1996)
Vicious cycle involving Na+ channels, glutamate release, and NMDA receptor mediates delayed neurodegeneration through nitric oxide formation.
J Neurosci
16:5004-5013[Abstract/Free Full Text].
-
Troy CM,
Derossi D,
Prochiantz A,
Greene LA,
Shelanski ML
(1996)
Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway.
J Neurosci
16:253-261[Abstract/Free Full Text].
-
Tsuchida T,
Ensini M,
Morton SB,
Baldassare M,
Edlund T,
Jessell TM,
Pfaff SL
(1994)
Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.
Cell
79:957-970[ISI][Medline].
-
Varner PD,
Beckman JS
(1994)
Nitric oxide toxicity in neuronal injury and degeneration.
In: Nitric oxide in the nervous system (Vincent SR,
ed), pp 191-206. London: Academic.
-
Weill CL
(1982)
cGMP accumulation by spinal cord neurons during the period of neuromuscular junction formation.
Neurosci Lett
30:263-268[Medline].
-
Weill CL,
Greene DP
(1984)
Prevention of natural motoneurone cell death by dibutyryl cyclic GMP.
Nature
308:452-454[Medline].
-
Wu W
(1993)
Expression of nitric-oxide synthase (NOS) in injured CNS neurons as shown by NADPH diaphorase histochemistry.
Exp Neurol
120:153-159[ISI][Medline].
-
Wu W,
Li L
(1993)
Inhibition of nitric oxide synthase reduces motoneuron death due to spinal root avulsion.
Neurosci Lett
153:121-124[ISI][Medline].
-
Yan Q,
Johnson Jr EM
(1988)
An immunohistochemical study of the nerve growth factor receptor in developing rats.
J Neurosci
8:3481-3498[Abstract].
-
Zhuo M,
Kandel ER,
Hawkins RD
(1994)
Nitric oxide and cGMP can produce either synaptic depression or potentiation depending on the frequency of presynaptic stimulation in the hippocampus.
NeuroReport
5:1033-1036[ISI][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18103708-07$05.00/0
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Top
Abstract
Introduction
