Association Between Ubiqutin And SOD1 In The Animal Model Of Family Amyotrophic Lateral Sclerosis

Objective: Amyotrophic lateral sclerosis (ALS) is one of the most common adult-onset neurogenerative diseases, having a prevalence of ⁵ per 100,000 individuals. ALS is a lethal disease and characterized by selective degeneration of motor neurons in the anterior horn of spinal cord, brain stem and cerebral cortex, which clinical features are delayed onset, progressive weakness and atrophy of limbs'muscles, fasciculation, hypermyotonia, tendon hyperreflexia, the occurrence of pathologic reflex and dysarthria. There is no effective remedy and the most patients died of respiratory failure 3-5 years later. From its speciality of episode and heredity, ALS can be divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS). FALS is less than 10% of the total ALS patients, while SALS is more than 90%. The pathogenesis of ALS is not clear at present, however in the FALS there are approximate 20% patients are mainly caused by the mutation of copper/zinc superoxide dismutase (Cu/Zn SOD) gene, which site is at 21q22.1～22.2. In the patients and transgenic mice of FALS, some research indicated that the mutant SOD1 is misfolded and can not be degradated by the ubiquitin-proteasome system(UPS), which result cause the accumuliation of SOD1 and formation of the SOD1 immunoreactive inclusions. However, the gain toxic function of abnormal aggregation of SOD1 can contribute the functional change of motor neurons and pathogenesy of ALS. Meanwhile, ubiquitin immunoreactive inclusions in motor neuron cytoplasm is the pathological hallmarks of ALS; the formation and accumulation of ubiquitinated aggregates in motor neurons are thought to be involved in the toxic gain of function of mutant SOD1. It has also been shown that the ubiquitin-proteasome system plays a role in the clearance of the toxicity of mutant protein; moreover, ubiquitination of substrate protein is the priming signal of UPS. In our study, we will qualitatively investigate the interaction between ubiquitin and the mutant SOD1 with the progression of amyotrophic lateral sclerosis (ALS) in the SOD1^G93A transgenic mice, and reveal if the ubiquitination of mutant SOD1 is disfunctional.Methods: SOD1^G93A transgenic mice, the classical familial amyotrophy lateral sclerosis model, was used as the experimental animals in this study; if the offspring mice identified to contain SOD1^G93A gene were the SOD1^G93A transgenic mice, otherwise were the littermate controls. The experiment was divided into three groups:the presymptomatic group, the symptomatic group and the end group; each group included the corresponding littermate controls; at every stage, there were 6 mice in each group. The expression SOD1 in the lumbar spinal cord was observed at presymptomatic, symptomatic and end stages of SOD1^G93A transgenic mice using western blot; the distribution and colocation of ubiquitin and SOD1 in the lumbar spinal cord was observed at presymptomatic, symptomatic and end stages of SOD1^G93A transgenic mice and their corresponding littermate controls using laser confocal microscope, whereas the interaction between ubiquitin and SOD1 was determined by immunocoprecipitation technique.Results:1 The Western blot showed:There were two SOD1 bands in the lumbar spinal cord of SOD1^G93A transgenic mice, the smaller molecular weight band belonged to the endogenous SOD1 of the mice, the higher molecular weight band belonged to human mutant SOD1; however there was only one band which belonged to the endogenous SOD1 of the mice in the littermate controls.2 The laser confocal microscope showed:In the control groups, there were normal morphous and quantitative motor neurons in the anterior horn of spinal cord, the proteins of ubiquitin and SOD1 distriduted proverbially and uniformly in the cytoplasm were colocated widely; In the presymptomatic groups, the numbers of motor neurons in the anterior horn of spinal cord were multiple, the proteins of ubiquitin and SOD1 rich in the cytoplasm distriduted uniformly and were colocated widely; In the symptomatic groups, the numbers of motor neurons in the anterior horn of spinal cord were reduced, the proteins of ubiquitin and SOD1 colocated widespread in the cytoplasm aggregated punctately and the merge of them was observed in the aggregation; In the end groups, the numbers of motor neurons were reduced remarkably, the essential morphous was vanished in the anterior horn of spinal cord, the proteins of ubiquitin and SOD1 aggregated punctately schisticly and irregularly in the anterior horn of spinal cord and the merge of them was aslo observed in the aggregation.3 The immunocoprecipitation showed:The scalariform bands detected in every sample were no difference between experimental group and control group, that indicated ubiquitinated SOD1 existed in the samples; we detected SOD1 interacted with ubiquitin with 10% SDS-PAGE gelatin system and found a lampros band at the position of 17KD marker, and there were no difference between experimental group and control group, that indicated ubiquitin only interacted with the mice endogenous SOD1.Conclusion: The ubiquitination of mutant SOD1 was disfunctional in SOD1^G93A transgenic mice.