The Expression Of Caspase-12 Gene Affected By Ovariectomy In Lumber Spinal Cord Of SOD1G93A Transgenic Mice

Objective:Amyotrophic lateral sclerosis(ALS),a chronic progressive neurodegenerative disease manifested as muscle weakness,muscle atrophy and pyramidal signs,is mainly involving the upper and lower motor neurons.Numerous studies have verified that the expression level of endoplasmic reticulum stress-related protein was up-regulation in spinal cord anterior horn motor neurons of ALS patients and SOD1 transgenic mice.Another study found that pro-apoptotic and anti-apoptotic proteins which involved in endoplasmic reticulum stress were imbalance in presymptomatic SOD1 transgenic mice.These findings suggest that endoplasmic reticulum stress is associated with the onset and progression of ALS.Caspase-12 is a member of the cysteine protease family,which is located in the outer membrane of the endoplasmic reticulum.It is reported that caspase-12 deficient mice are partially resistant to apoptosis induced by ER stress but not by other apoptotic stimuli.Caspase-12 is a key molecule which causes endoplasmic reticulum stress apoptosis and it is not activated in death receptor-mediated and mitochondrial dysfunction-mediated apoptosis pathway,suggesting that caspase-12 is involved in the mechanism of endoplasmic reticulum stress-mediated apoptosis but not non-endoplasmic reticulum stress-mediated apoptosis.Our previous study demonstrated ovariectomy can advance the onset age of SOD1G93 A transgenic mice,indicating that estrogens play a neuroprotective role in ALS.Many studies have shown estrogen can intervene the endoplasmic reticulum stress by the estrogen receptor.In the present study,we sought to detect the expression change of caspase-12 in SOD1-G93 A transgenic mice ovariectomized group(OVX)and positive control group(NO-OVX)and investigated the possible relationship between caspase-12 and estrogen in the process of ALS.Methods:1 Animal Model and groupBreeding male familial amyotrophic lateral sclerosis transgenic mice animal models(SOD1G93A).The ten SOD1G93 A mice of five weeks were randomly divided into two experimental groups(n=5):ovariectomized group(OVX),the positive control group(NO-OVX).The negative control group(CON)were non-transgenic mice have same duration with experimental one.2 MaterialsWe removed the eyeball and draw blood after 10% chloral hydrate 350 mg /kg intraperitoneal injection of anesthesia.Then we quickly decapitated and stripped out the brain tissue and spinal cord,placed in Eppendorf tubes and frozen in liquid nitrogen,transferred to the ultra-low temperature freezer for later use(Materials of OVX group and NO-OVX group were obtained at each symptomatic stage,CON group was obtained with NO-OVX group simultaneously).3 Gene expression profilesThe gene expression profiling of lumbar spinal cord were analyzed by gene chip technology.4 qRT-PCRThe expression levels of caspase-12 in lumbar spinal cord were analyzed by qRT-PCR.5 Statistical analysesAll statistical analyses were analyzed using SPSS 13.0.Differences in mean values between groups were assessed for significance using one-way analysis of variance(ANOVA)and LSD test was used to compare data between any two.P<0.05 was considered statistically significant.Result: 1 Microarray results 1.1 Cluster analysisCluster analysis of genes obtained from SOD1G93 A transgenic mice in the 3 groups.Cluster thermograph acquired after Cluster analysis and samples of each group clustered together.Gene expression were obvious different among OVX group,NO-OVX group and CON group.1.2 Genetic screening resultCompared the gene expression profile between SOD1G93 A transgenic female mice OVX group and NO-OVX group,the screening conditions were P?0.05 and fold-change values ? + 2 or ?-2.19 genes were up-regulated and 2 genes were down-regulated.Differentially expressed genes are shown in Table 1.Compared the expression levels of caspase-12 between OVX group and NO-OVX group,the fold-change value was 2.2458.2 The expression levels of caspase-12 in OVX,NO-OVX and CON groups.Compared the NO-OVX group with the CON group,the expression level of caspase-12 was up-regulation in lumer spinal cord of OVX group.The difference was statistically significant.Compared the NO-OVX group with the CON group,the expression levels of caspase-12 were no significant difference.Conclusions:1 The gene level of caspase-12 was enhanced in lumber spinal cord of ovariectomized SOD1G93 A transgenic mice.2 Estrogen may play a neuroprotective role by inhibiting endoplasmic reticulum stress-mediated apoptosis in SOD1G93 A transgenic mice.