BACKGROUND
amyotrophic lateral sclerosis  is a degenerative disorder  <cit>  characterized by selective degeneration of motor neurons predominantly in the anterior horn of the spinal cord and brainstem, and pyramidal cells of the motor cortex.
als leads to progressive weakness, atrophy of skeletal muscles, and eventual paralysis and death, usually within 2– <dig> years  <cit> .
up to 10% of als cases are familial ; the remaining 90% have no hereditary component and are known as sporadic als  <cit> .
about 20% of fals cases are caused by autosomal dominant mutations in the gene encoding for cu/zn superoxide dismutase   <cit> .
pathophysiological mechanisms of fals due to sod <dig> mutations include a failure to fold or degrade mutant sod <dig>  production of free radicals, release of free copper, or susceptibility of mutant sod <dig> to disulfide reduction  <cit> .
many studies have implicated oxidative stress and an excitotoxic mechanism in the pathogenesis of als.
plasma homocysteine , which is continuously produced from diet and increases with age, has been considered to play a role in motor neuronal death, thus resulting in als.
hc in vascular systems produces reactive oxygen species , such as superoxide anions, and reduces transitional metals by reaction of the metal with molecular oxygen.
no  might be involved in the mechanism of action of hc.
no is scavenged for transformation into peroxynitrite by reaction with the superoxide anion generated by hc.
this reactive peroxynitrite causes cell death by oxidative damage, disruption of energy metabolism, calcium homeostasis, and mitochondrial function  <cit> .
in addition, motor neurons in sod <dig> models are vulnerable to excitotoxicity by glutamate; glutamate receptor-mediated neurodegeneration is associated with calcium influx and substantial intracellular calcium accumulation  <cit> .
growth hormone , a high molecular weight peptide composed of  <dig> amino acids, is produced by the anterior lobe of the pituitary gland.
gh functions either by direct action on tissues or through the insulin-like growth factor- <dig>  it causes proliferation of many types of cells and controls differentiation in adipose or muscle tissues.
it also controls the metabolism of proteins, carbohydrates, or fatty acids  <cit> .
although the effect of gh on the central nervous system  was first reported over 60 years ago, virtual data have appeared in the past decade  <cit> .
gh secretion was studied in myotonic dystrophy.
this condition was associated with an abnormal pattern of gh secretion over 24 hours and a significant decrease in its circulating levels  <cit> .
one study reported a reduction of gh secretion in patients with als  <cit> .
in the present study, we examined the protective effect of gh on motor neuronal death.
we tested the effect of gh on the viability of motoneuron-neuroblastoma hybrid cells  expressing mutated human sod <dig> against cytotoxic stimuli.
in addition, we attempted to determine whether gh treatment has a neuroprotective effect on transgenic mice with the sod1g93a mutation.
lifespan and body weight measurements, rotarod test, and immunohistochemistry for motor neuronal loss were performed.
RESULTS
the effect of gh on mutant human sod <dig> cell lines
cell lines were cultured with various gh concentrations for 24 h .
the number of living cells increased in the a4v cell line  and g93a cell line  as determined by the mtt assay.
however, there was no statistical difference with the number of viable cells in the wt cell line.
when treated with w <dig> , the increasing viability of a4v with pretreated gh decreased  .
however, it was not significantly different from the wt and g93a cell lines.
when treated with p <dig> , the increasing viability of g93a with pretreated gh decreased  .
however, the difference was not significant from that of the wt and a4v cell lines.
for immunoblotting studies, a4v and g93a cell lines were treated with gh  and active pakt and perk forms were identified.
the change of pakt in a4v cell line  and g93a cell line  at different time points had statistical significance.
similarly, perk levels also changed significantly in a4v  and g93a cell lines  at different time points.
in a4v cells, the ratio of pakt and perk were higher than those in g93a but the difference was not significant  .figure  <dig>
pi3-kinase or map-kinase inhibitor treatment and activation of pakt and perk by gh.
increased viability of a4v was reduced by w <dig>  .
increased viability of g93a was reduced by treatment with pd <dig>  a map-kinase inhibitor  .
gh was applied at  <dig>  i.u/ml and active pakt and perk forms were identified  <dig>   <dig>  and 20 min later.
the change in levels of pakt and perk in a4v and g93a cell lines at different time points were analyzed .
results are expressed as mean values and sd .
the protective effect of gh on cytotoxicity
in previous studies, treatment with a <dig>  calcium influx, gsno, no-donor, and homocysteine had a selective cytotoxic effect in the mutated sod- <dig> cell lines, g93a, and a4v.
viabilities of g93a and a4v decreased compared to that of wt  <cit> .
in this study, these effects were reconfirmed.
lack of the protective effect of gh on cytotoxicity of a23187
when treated with a <dig> , cell viabilities decreased but did not reach significance.
viabilities in a4v and g93a decreased by 10% and 20%, respectively, which was higher than that in wt.
pretreatment with  <dig>  i.u./ml and  <dig>  i.u./ml gh had no protective effect on wt, a4v, and g93a cell lines.
the protective effect of gh on the cytotoxicity of gsno
gsno  treatment caused a non-significant reduction in viabilities.
the reduction was 20% in a4v and 10% in g93a, and was higher than in wt.
pretreatment with  <dig>  i.u./ml and  <dig>  i.u./ml gh caused viabilities in wt and a4v to increase, but not significantly .
nuclear fragmentation was observed after gsno  treatment which decreased by pretreatment with  <dig>  i.u./ml gh, but there were no statistically significant difference .
while there were no changes in bax expression, parp expression  increased after treatment with gsno 200 μm, but protective effect of gh was not observed in a4v and g93a cell lines .figure  <dig>
cytoprotective effect of gh.
the protective effect of gh with gsno pretreatment was observed in mutant cells .
in homocysteine-treated cells, reduced viabilities in a4v were partly reversed by gh at  <dig>  i.u/ml  .
gsno treatment increased nuclear fragmentation; gh treatment reduced this phenomenon.
homocysteine also reduced cell viability and the number of fragmented nuclei; this was reversed by pretreatment with gh  .
gsno treatment increased  <dig> kd and  <dig> kd parp fragments in a4v and g93a cells, which were reduced by gh treatment .
results are expressed as mean values and sd .
the protective effect of gh on the cytotoxic effect with homocysteine
when treated with homocysteine , cell viabilities decreased but was not significantly different compared to wt.
viabilities in a4v and g93a decreased more than that in wt .
the protective effect of gh was observed in a4v, especially at  <dig>  i.u./ml  .
homocysteine also caused nuclear fragmentation which decreased by pretreatment with  <dig>  i.u./ml gh  ; however, there were no changes in bax and parp expression.
gh improves motor performance
to investigate improvements in balance and coordination, we used the rotarod test.
gh treatment significantly improved motor performance at 16 weeks, compared to saline-treated sod1g93a mice  .figure  <dig>
in vivo
study.
in the rotarod test, gh treatment significantly improved motor performance at 16 weeks, compared to saline-treated sod1g93a mice  .
gh treatment significantly improved weight loss at  <dig>  and 19 weeks  .
gh treatment resulted in a significant improvement in lifespan, compared with saline-treated sod1g93a transgenic mice  .
y error bars represent standard error of the mean  .
gh improves weight loss
in order to examine weight loss, we monitored mouse body weights weekly.
mice in both groups were weighed from 11 weeks of age.
gh treatment significantly improved weight loss at 18 weeks  and 19 weeks  .
gh improves lifespan
to determine the protective effects of gh on lifespan, gh was injected weekly from postnatal 11 weeks till death  in gh-treated sod1g93a transgenic mice.
after treatment, lifespan was compared with saline-treated sod1g93a transgenic mice.
gh-injection resulted in a significant improvement in lifespan  .
mean mouse survival increased from 132 ± 9 days to 143 ± 8 days.
immunoreactivity analysis of the effect of gh on motor neuron loss
in gh-treated sod1g93a transgenic mice, there was a reduction in the number of cresyl violet-positive neurons, compared to non-transgenic mice, but a significant increase was noted compared to that in saline-treated sod1g93a transgenic mice  .
to determine whether gh exerted a neuroprotective effect in sod1g93a transgenic mice, we examined gfap expression in astrocytes by immunohistochemistry.
lumbar spinal cord sections of saline-treated sod1g93a transgenic mice showed strong gfap immunoreactivity.
in contrast, the gfap signal was attenuated in the lumbar spinal cord of gh-treated sod1g93a mice and non-transgenic mice .
next, we performed immunostaining for bcl- <dig> in order to determine if gh inhibits apoptotic neuronal death in sod1g93a transgenic mice,  and found stronger immunoreactivity compared to saline-treated sod1g93a mice.figure  <dig>
histopathological analysis by cresyl violet, gfap, and bcl- <dig> staining.
cresyl violet staining revealed few numbers of neurons in saline-treated sod1g93a mice, and higher numbers in non-transgenic and gh-treated sod1g93a mice .
gfap immunoreactivity was observed at very low levels in non-transgenic mice, and at significantly higher levels in saline-treated sod1g93a mice .
increasing gfap immunoreactivities in saline-treated sod1g93a mice were attenuated by gh treatment.
bcl- <dig> staining  is stronger in gh-treated sod1g93a mice than in saline-treated sod1g93a mice.
the number of motor neurons is significantly higher in the non-transgenic and gh-treated sod1g93a mice  .
results are expressed as mean values and sd .
discussion
a few studies on halting disease progression in sod1-dependent als have been carried out.
the effect of gh on the cns appears to involve brain growth and development, and action as a neuroprotective factor  <cit> .
we tested the neuroprotective effects of gh on human sod-1-expressing cultured motor neurons and sod1g93a transgenic mice.
the results showed that gh protects against motorneuronal death caused by gsno and homocysteine, but not by a <dig>  gh activated mapk and pi3k.
prolongation of survival was observed in gh-treated mice, along with improved motor performance and weight loss.
gh decreased cresyl violet positive motoneuronal loss with stronger bcl- <dig> and lesser gfap immunoreactivity.
our results suggest that gh has a protective effect on mutant sod- <dig> expressing motor neuronal death.
zhang et al.
<cit>  demonstrated that folic acid protects motor neurons against increased homocysteine, inflammation, and apoptosis in sod1g94a transgenic mice.
folic acid, which was applied to sod1g94a transgenic mice, is regarded as an important factor in homocysteine metabolism.
the study showed that folic acid treatment significantly delayed disease onset and prolonged lifespan, accompanied by significantly attenuated plasma homocysteine levels, suppressed activation of microglia and astrocytes, and inhibited expression of inducible nitric oxide synthase  and tumor necrosis factor-alpha in the spinal cord.
pyruvate has been known as an anti-oxidant and an energy source.
when pyruvate was applied in sod1g94a transgenic mice, lifespan increased, disease progression slowed, and motor performance improved.
however, disease onset was not observed to be delayed in this study  <cit> .
gh levels decrease after peaking during adolescence; one third of people over 65 years of age are deficient in gh.
therefore, gh replacement therapy is used as one of the clinical practices for reducing the aging phenomenon  <cit> .
despite the direct application of gh in slowing aging, the mechanism underlying cytoprotection afforded by gh remains unknown.
only a few studies have shown that gh administration prevents organ and tissue deterioration due to aging.
as a possible mechanism of its action, gh has been suggested to act on antioxidant enzymes in the modulation of regulatory pathways in reduction of ros generation.
moreover, gh has been shown to prevent age-induced reduction in expression of some components, including cytochrome b and c of the mitochondrial respiratory chain  <cit> .
therefore, we hypothesized that gh has a cytoprotective effect associated with mitochondria in the sod- <dig> mutant.
studies on the cns have shown that gh has a neuroprotective effect against ischemic brain injury, can attenuate trauma-induced depression of spinal cord evoked potentials, and can ameliorate motor dysfunction resulting from spinal cord injury  <cit> .
cns weight increases in overexpression of gh and so does the size of lumbar spinal motor neurons.
however, there is no relative evidence to demonstrate a lack of alteration in motoneuron numbers in the cns of gh receptor-deficient mice.
some studies have shown that gh, insulin-like growth factor- <dig>  and insulin concentrations in cerebrospinal fluid  are significantly lower in patients with als in comparison with the control group  <cit> .
however, it is unknown whether gh affects the survival of motor neurons in als.
morselli et al.
<cit>  suggested that two-thirds of patients with als present with growth hormone  deficiency.
based on this data, several clinical trials were performed with gh on patients with als  <cit> .
however these studies failed to show any significant impact on motor symptoms or patient survival, except in a few patients who seemed to be good gh-responders.
therefore, more studies are needed to investigate the effect of gh on motor neurons.
in our study, gh was protective against gsno and homocysteine in mutant sod-1-expressing cells  <cit> .
however, this effect was not observed in cells treated with a <dig>  suggesting that gh is not related to calcium-influx-mediated cell death.
additionally, the protective effect of gh appears to be related to nuclear fragmentation and not the bax protein, an indicator of mitochondria-related cell death.
parp cleavage and caspase  <dig> represent the signals of apoptosis, which are related to mitochondria-related cell death.
reactive astrocytes in the cns of sod1g93a transgenic mice have been shown to express cleaved parp  <cit> .
with gsno treatment, gh decreases parp cleavage, suggesting that gh may affect caspase  <dig>  however, with homocysteine treatment, gh does not affect parp cleavage, suggesting that it is not related to caspase  <dig>  therefore, the protective effect of gh in our study may be attributed to a non-mitochondrial mediated apoptosis, i.e., another non-developmental pathway.
in case of figure 1a, we showed the proliferating effect of gh on a4v that is blocked by w <dig>  however, in figure 2a, we attempted the no cytotoxicity and checked whether this cytotoxicity can be protected by gh.
we assumed that figure 1a could show the significant difference the viability of a4v cells because w <dig> is directly related with the gh receptor cascade.
figure 2a showed the tendency of increasing viability of a4v cells.
the main signaling pathway of gh is through the gh receptor related to the tyrosine kinase janus tyrosine kinase- <dig> .
jak <dig> is activated when a gh molecule binds to a dimer of the gh receptor and jak <dig>  which promotes the phosphorylation of both jak <dig> and the gh receptor.
subsequently, stat  <dig>   <dig>  and  <dig> dimerize and translocate to the nucleus in order to activate transcription of the target gene.
jak <dig> also phosphorylates and potentiates the mitogen-activated protein kinase  and phosphatidyl inositol- <dig> kinase  cascades to proliferate survival related cells  <cit> .
the proliferative effect of gh was observed at concentrations between  <dig> – <dig>  i.u., which was negated by the inhibitors of pi <dig> kinase or map kinase, suggesting that this proliferation is mediated by survival signaling.
the effect of pi3k or mapk appeared to differ among the cell lines.
the pi <dig> kinase inhibitor mainly affected a4v, but not wt and g93a.
however, the map kinase inhibitor mainly affected g93a, and not wt and a4v.
pakt levels suggested that pi3k signaling  <cit>  is activated in a4v and g93a cell lines.
increased perk levels also suggested that the mapk signaling pathway is active in a4v and g93a cell lines.
it would be ideal to use mice of the same sex to avoid possible gender differences.
unintentionally, random assignment results in different sex ratio.
however, no behavioral or phenotypic gender difference related to g93a mutation was found in g53a c57bl/6 j transgenic mice  <cit> .
a significant reduction in astrogliosis, as assessed by glial fibrillary acidic protein  staining was observed, which suggests delayed astrocyte activation in gh-treated sod1g93a transgenic mice.
reduced expression of bcl- <dig> has been observed in the spinal cord of transgenic mice expressing sod <dig> with the g93a mutation  <cit>  and in human patients having als without sod <dig> mutations  <cit> .
bcl- <dig> is an important inhibitor of most types of apoptotic cell death, and may reduce motor neuron losses by obstructing the activation of apoptosis induced by cu/zn-sod mutation  <cit> .
in addition, it has been demonstrated that bcl- <dig> expression is reduced in symptomatic sod1g93a transgenic mice  <cit> .
CONCLUSIONS
our results demonstrate that gh has a protective effect in mutant sod-1-expressing motor neuronal death.
we confirmed these results with in vitro and in vivo studies in mutant sod <dig> cells and mice, respectively.
our results further indicate that the protection afforded by gh to mutant sod- <dig> expressing motor neurons could be through a novel non-mitochondrial mediated pathway that is different from the established pathway.
however, more trials are needed in order to further clarify the neuroprotective mechanism of gh action.

 amyotrophic lateral sclerosis is a degenerative disorder characterized by selective degeneration of motor neurons predominantly in the anterior horn of the spinal cord and brainstem, and pyramidal cells of the motor cortex. als leads to progressive weakness, atrophy of skeletal muscles, and eventual paralysis and death, usually within 2– years. up to 10% of als cases are familial; the remaining 90% have no hereditary component and are known as sporadic als. about 20% of fals cases are caused by autosomal dominant mutations in the gene encoding for cu/zn superoxide dismutase. pathophysiological mechanisms of fals due to sod mutations include a failure to fold or degrade mutant sod production of free radicals, release of free copper, or susceptibility of mutant sod to disulfide reduction. many studies have implicated oxidative stress and an excitotoxic mechanism in the pathogenesis of als. plasma homocysteine, which is continuously produced from diet and increases with age, has been considered to play a role in motor neuronal death, thus resulting in als. hc in vascular systems produces reactive oxygen species, such as superoxide anions, and reduces transitional metals by reaction of the metal with molecular oxygen. no might be involved in the mechanism of action of hc. no is scavenged for transformation into peroxynitrite by reaction with the superoxide anion generated by hc. this reactive peroxynitrite causes cell death by oxidative damage, disruption of energy metabolism, calcium homeostasis, and mitochondrial function. in addition, motor neurons in sod models are vulnerable to excitotoxicity by glutamate; glutamate receptor-mediated neurodegeneration is associated with calcium influx and substantial intracellular calcium accumulation. growth hormone, a high molecular weight peptide composed of amino acids, is produced by the anterior lobe of the pituitary gland. gh functions either by direct action on tissues or through the insulin-like growth factor- it causes proliferation of many types of cells and controls differentiation in adipose or muscle tissues. it also controls the metabolism of proteins, carbohydrates, or fatty acids. although the effect of gh on the central nervous system was first reported over 60 years ago, virtual data have appeared in the past decade. gh secretion was studied in myotonic dystrophy. this condition was associated with an abnormal pattern of gh secretion over 24 hours and a significant decrease in its circulating levels. one study reported a reduction of gh secretion in patients with als. in the present study, we examined the protective effect of gh on motor neuronal death. we tested the effect of gh on the viability of motoneuron-neuroblastoma hybrid cells expressing mutated human sod against cytotoxic stimuli. in addition, we attempted to determine whether gh treatment has a neuroprotective effect on transgenic mice with the sod1g93a mutation. lifespan and body weight measurements, rotarod test, and immunohistochemistry for motor neuronal loss were performed.  the effect of gh on mutant human sod cell lines cell lines were cultured with various gh concentrations for 24 h. the number of living cells increased in the a4v cell line and g93a cell line as determined by the mtt assay. however, there was no statistical difference with the number of viable cells in the wt cell line. when treated with w, the increasing viability of a4v with pretreated gh decreased. however, it was not significantly different from the wt and g93a cell lines. when treated with p, the increasing viability of g93a with pretreated gh decreased. however, the difference was not significant from that of the wt and a4v cell lines. for immunoblotting studies, a4v and g93a cell lines were treated with gh and active pakt and perk forms were identified. the change of pakt in a4v cell line and g93a cell line at different time points had statistical significance. similarly, perk levels also changed significantly in a4v and g93a cell lines at different time points. in a4v cells, the ratio of pakt and perk were higher than those in g93a but the difference was not significant.figure pi3-kinase or map-kinase inhibitor treatment and activation of pakt and perk by gh. increased viability of a4v was reduced by w. increased viability of g93a was reduced by treatment with pd a map-kinase inhibitor. gh was applied at i.u/ml and active pakt and perk forms were identified and 20 min later. the change in levels of pakt and perk in a4v and g93a cell lines at different time points were analyzed. results are expressed as mean values and sd. the protective effect of gh on cytotoxicity in previous studies, treatment with a calcium influx, gsno, no-donor, and homocysteine had a selective cytotoxic effect in the mutated sod- cell lines, g93a, and a4v. viabilities of g93a and a4v decreased compared to that of wt. in this study, these effects were reconfirmed. lack of the protective effect of gh on cytotoxicity of a23187 when treated with a, cell viabilities decreased but did not reach significance. viabilities in a4v and g93a decreased by 10% and 20%, respectively, which was higher than that in wt. pretreatment with i.u./ml and i.u./ml gh had no protective effect on wt, a4v, and g93a cell lines. the protective effect of gh on the cytotoxicity of gsno gsno treatment caused a non-significant reduction in viabilities. the reduction was 20% in a4v and 10% in g93a, and was higher than in wt. pretreatment with i.u./ml and i.u./ml gh caused viabilities in wt and a4v to increase, but not significantly. nuclear fragmentation was observed after gsno treatment which decreased by pretreatment with i.u./ml gh, but there were no statistically significant difference. while there were no changes in bax expression, parp expression increased after treatment with gsno 200 μm, but protective effect of gh was not observed in a4v and g93a cell lines.figure cytoprotective effect of gh. the protective effect of gh with gsno pretreatment was observed in mutant cells. in homocysteine-treated cells, reduced viabilities in a4v were partly reversed by gh at i.u/ml. gsno treatment increased nuclear fragmentation; gh treatment reduced this phenomenon. homocysteine also reduced cell viability and the number of fragmented nuclei; this was reversed by pretreatment with gh. gsno treatment increased kd and kd parp fragments in a4v and g93a cells, which were reduced by gh treatment. results are expressed as mean values and sd. the protective effect of gh on the cytotoxic effect with homocysteine when treated with homocysteine, cell viabilities decreased but was not significantly different compared to wt. viabilities in a4v and g93a decreased more than that in wt. the protective effect of gh was observed in a4v, especially at i.u./ml. homocysteine also caused nuclear fragmentation which decreased by pretreatment with i.u./ml gh; however, there were no changes in bax and parp expression. gh improves motor performance to investigate improvements in balance and coordination, we used the rotarod test. gh treatment significantly improved motor performance at 16 weeks, compared to saline-treated sod1g93a mice.figure in vivo study. in the rotarod test, gh treatment significantly improved motor performance at 16 weeks, compared to saline-treated sod1g93a mice. gh treatment significantly improved weight loss at and 19 weeks. gh treatment resulted in a significant improvement in lifespan, compared with saline-treated sod1g93a transgenic mice. y error bars represent standard error of the mean. gh improves weight loss in order to examine weight loss, we monitored mouse body weights weekly. mice in both groups were weighed from 11 weeks of age. gh treatment significantly improved weight loss at 18 weeks and 19 weeks. gh improves lifespan to determine the protective effects of gh on lifespan, gh was injected weekly from postnatal 11 weeks till death in gh-treated sod1g93a transgenic mice. after treatment, lifespan was compared with saline-treated sod1g93a transgenic mice. gh-injection resulted in a significant improvement in lifespan. mean mouse survival increased from 132 ± 9 days to 143 ± 8 days. immunoreactivity analysis of the effect of gh on motor neuron loss in gh-treated sod1g93a transgenic mice, there was a reduction in the number of cresyl violet-positive neurons, compared to non-transgenic mice, but a significant increase was noted compared to that in saline-treated sod1g93a transgenic mice. to determine whether gh exerted a neuroprotective effect in sod1g93a transgenic mice, we examined gfap expression in astrocytes by immunohistochemistry. lumbar spinal cord sections of saline-treated sod1g93a transgenic mice showed strong gfap immunoreactivity. in contrast, the gfap signal was attenuated in the lumbar spinal cord of gh-treated sod1g93a mice and non-transgenic mice. next, we performed immunostaining for bcl- in order to determine if gh inhibits apoptotic neuronal death in sod1g93a transgenic mice, and found stronger immunoreactivity compared to saline-treated sod1g93a mice.figure histopathological analysis by cresyl violet, gfap, and bcl- staining. cresyl violet staining revealed few numbers of neurons in saline-treated sod1g93a mice, and higher numbers in non-transgenic and gh-treated sod1g93a mice. gfap immunoreactivity was observed at very low levels in non-transgenic mice, and at significantly higher levels in saline-treated sod1g93a mice. increasing gfap immunoreactivities in saline-treated sod1g93a mice were attenuated by gh treatment. bcl- staining is stronger in gh-treated sod1g93a mice than in saline-treated sod1g93a mice. the number of motor neurons is significantly higher in the non-transgenic and gh-treated sod1g93a mice. results are expressed as mean values and sd. discussion a few studies on halting disease progression in sod1-dependent als have been carried out. the effect of gh on the cns appears to involve brain growth and development, and action as a neuroprotective factor. we tested the neuroprotective effects of gh on human sod-1-expressing cultured motor neurons and sod1g93a transgenic mice. the results showed that gh protects against motorneuronal death caused by gsno and homocysteine, but not by a gh activated mapk and pi3k. prolongation of survival was observed in gh-treated mice, along with improved motor performance and weight loss. gh decreased cresyl violet positive motoneuronal loss with stronger bcl- and lesser gfap immunoreactivity. our results suggest that gh has a protective effect on mutant sod- expressing motor neuronal death. zhang et al. demonstrated that folic acid protects motor neurons against increased homocysteine, inflammation, and apoptosis in sod1g94a transgenic mice. folic acid, which was applied to sod1g94a transgenic mice, is regarded as an important factor in homocysteine metabolism. the study showed that folic acid treatment significantly delayed disease onset and prolonged lifespan, accompanied by significantly attenuated plasma homocysteine levels, suppressed activation of microglia and astrocytes, and inhibited expression of inducible nitric oxide synthase and tumor necrosis factor-alpha in the spinal cord. pyruvate has been known as an anti-oxidant and an energy source. when pyruvate was applied in sod1g94a transgenic mice, lifespan increased, disease progression slowed, and motor performance improved. however, disease onset was not observed to be delayed in this study. gh levels decrease after peaking during adolescence; one third of people over 65 years of age are deficient in gh. therefore, gh replacement therapy is used as one of the clinical practices for reducing the aging phenomenon. despite the direct application of gh in slowing aging, the mechanism underlying cytoprotection afforded by gh remains unknown. only a few studies have shown that gh administration prevents organ and tissue deterioration due to aging. as a possible mechanism of its action, gh has been suggested to act on antioxidant enzymes in the modulation of regulatory pathways in reduction of ros generation. moreover, gh has been shown to prevent age-induced reduction in expression of some components, including cytochrome b and c of the mitochondrial respiratory chain. therefore, we hypothesized that gh has a cytoprotective effect associated with mitochondria in the sod- mutant. studies on the cns have shown that gh has a neuroprotective effect against ischemic brain injury, can attenuate trauma-induced depression of spinal cord evoked potentials, and can ameliorate motor dysfunction resulting from spinal cord injury. cns weight increases in overexpression of gh and so does the size of lumbar spinal motor neurons. however, there is no relative evidence to demonstrate a lack of alteration in motoneuron numbers in the cns of gh receptor-deficient mice. some studies have shown that gh, insulin-like growth factor- and insulin concentrations in cerebrospinal fluid are significantly lower in patients with als in comparison with the control group. however, it is unknown whether gh affects the survival of motor neurons in als. morselli et al. suggested that two-thirds of patients with als present with growth hormone deficiency. based on this data, several clinical trials were performed with gh on patients with als. however these studies failed to show any significant impact on motor symptoms or patient survival, except in a few patients who seemed to be good gh-responders. therefore, more studies are needed to investigate the effect of gh on motor neurons. in our study, gh was protective against gsno and homocysteine in mutant sod-1-expressing cells. however, this effect was not observed in cells treated with a suggesting that gh is not related to calcium-influx-mediated cell death. additionally, the protective effect of gh appears to be related to nuclear fragmentation and not the bax protein, an indicator of mitochondria-related cell death. parp cleavage and caspase represent the signals of apoptosis, which are related to mitochondria-related cell death. reactive astrocytes in the cns of sod1g93a transgenic mice have been shown to express cleaved parp. with gsno treatment, gh decreases parp cleavage, suggesting that gh may affect caspase however, with homocysteine treatment, gh does not affect parp cleavage, suggesting that it is not related to caspase therefore, the protective effect of gh in our study may be attributed to a non-mitochondrial mediated apoptosis, i.e., another non-developmental pathway. in case of figure 1a, we showed the proliferating effect of gh on a4v that is blocked by w however, in figure 2a, we attempted the no cytotoxicity and checked whether this cytotoxicity can be protected by gh. we assumed that figure 1a could show the significant difference the viability of a4v cells because w is directly related with the gh receptor cascade. figure 2a showed the tendency of increasing viability of a4v cells. the main signaling pathway of gh is through the gh receptor related to the tyrosine kinase janus tyrosine kinase-. jak is activated when a gh molecule binds to a dimer of the gh receptor and jak which promotes the phosphorylation of both jak and the gh receptor. subsequently, stat and dimerize and translocate to the nucleus in order to activate transcription of the target gene. jak also phosphorylates and potentiates the mitogen-activated protein kinase and phosphatidyl inositol- kinase cascades to proliferate survival related cells. the proliferative effect of gh was observed at concentrations between – i.u., which was negated by the inhibitors of pi kinase or map kinase, suggesting that this proliferation is mediated by survival signaling. the effect of pi3k or mapk appeared to differ among the cell lines. the pi kinase inhibitor mainly affected a4v, but not wt and g93a. however, the map kinase inhibitor mainly affected g93a, and not wt and a4v. pakt levels suggested that pi3k signaling is activated in a4v and g93a cell lines. increased perk levels also suggested that the mapk signaling pathway is active in a4v and g93a cell lines. it would be ideal to use mice of the same sex to avoid possible gender differences. unintentionally, random assignment results in different sex ratio. however, no behavioral or phenotypic gender difference related to g93a mutation was found in g53a c57bl/6 j transgenic mice. a significant reduction in astrogliosis, as assessed by glial fibrillary acidic protein staining was observed, which suggests delayed astrocyte activation in gh-treated sod1g93a transgenic mice. reduced expression of bcl- has been observed in the spinal cord of transgenic mice expressing sod with the g93a mutation and in human patients having als without sod mutations. bcl- is an important inhibitor of most types of apoptotic cell death, and may reduce motor neuron losses by obstructing the activation of apoptosis induced by cu/zn-sod mutation. in addition, it has been demonstrated that bcl- expression is reduced in symptomatic sod1g93a transgenic mice.  our results demonstrate that gh has a protective effect in mutant sod-1-expressing motor neuronal death. we confirmed these results with in vitro and in vivo studies in mutant sod cells and mice, respectively. our results further indicate that the protection afforded by gh to mutant sod- expressing motor neurons could be through a novel non-mitochondrial mediated pathway that is different from the established pathway. however, more trials are needed in order to further clarify the neuroprotective mechanism of gh action. 
survival analysis of motoneuronal loss was done using cresyl violet, gfap, and bcl- staining. gh decreased cresyl violet positive motoneuronal loss with strong bcl- and less gfap immunoreactivity. amyotrophic lateral sclerosis is characterized by selective degeneration of motor neurons. growth hormone concentrations are low in the cerebrospinal fluid of patients with als; however, its association with motoneuronal death is not known. amyotrophic lateral sclerosis growth hormone mutated sod1issue-copyright-statement© the author 2015 in sod- transgenic mice, rotarod motor performance was evaluated. gh-treated mice showed prolonged survival with improved motor performance and weight loss. our results demonstrate that gh has a protective effect on mutant sod-1-expressing motor neurons. we tested the neuroprotective effects of gh on human sod-1-expressing cultured motor neurons and sod1g93a transgenic mice. in cultured motor neurons, cytotoxicity was induced by a gnso, or homocysteine, and the effects of gh were determined by mtt, bax, parp cleavage pattern, hoechst nuclear staining, mapk, and pi3k assay.
 amyotrophic lateral sclerosis is characterized by selective degeneration of motor neurons. the gene encoding cu/zn superoxide dismutase is responsible for 20% of familial als cases. growth hormone concentrations are low in the cerebrospinal fluid of patients with als; however, its association with motoneuronal death is not known. we tested the neuroprotective effects of gh on human sod-1-expressing cultured motor neurons and sod1g93a transgenic mice.  in cultured motor neurons, cytotoxicity was induced by a gnso, or homocysteine, and the effects of gh were determined by mtt, bax, parp cleavage pattern, hoechst nuclear staining, mapk, and pi3k assay. in sod- transgenic mice, rotarod motor performance was evaluated. survival analysis of motoneuronal loss was done using cresyl violet, gfap, and bcl- staining. gh prevents motorneuronal death caused by gsno and homocysteine, but not that by a it activates mapk and pi3k. gh-treated mice showed prolonged survival with improved motor performance and weight loss. gh decreased cresyl violet positive motoneuronal loss with strong bcl- and less gfap immunoreactivity.  our results demonstrate that gh has a protective effect on mutant sod-1-expressing motor neurons. keywords amyotrophic lateral sclerosis growth hormone mutated sod1issue-copyright-statement© the author 2015 