The Expression Characteristic Of Ubiquitin In Lumbar Spinal Cord Of Family Amyotrophic Lateral Sclerosis Animal Model

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that involves selective loss of motoneurones in the spinal anterior horn, brainstem and primary motor cortex, reflecting progressive muscular weakness, atrophy, fasciculation, hyperreflexia, pathologic reflex positive, finally died of respiratory failure. Approximately 90%～95% of ALS cases are sporadic, the remaining 5%～10% of patients are familial. Approximately 20% of familial cases are caused by mutations in the superoxide dismutase (SOD1) gene. Although the disease pathogenesis is still unknown at present, the presence of ubiquitylated inclusions in the remaining motor neurons is a neuropathological feature of both ALS patients and animal models of the disease.Ubiquitin is prevalent in eukaryotic cells and a sequence highly conservative small molecule spherical protein, its molecular weight about 8.5 K Da, composed of 76 amino acid residues. Ubiquitin is covalently attached to target proteins in cells. Ubiquitin and ubiquitin-activating enzymeE1, ubiquitin-conjugating enzymeE2, ubiquitin-protein ligaseE3, de-ubiquitinating enzymes, proteasome and its substrates (proteins) constitute ubiquitin- proteasome system(UPS). UPS is the major route for protein degradation in eukaryotic cells, which can degraded 80% of intracellular proteins. Many researches have suggested that UPS also plays an important role in the misfolded protein or other mutein degradation processes. The disorders of ubiquitin-proteasome pathway can lead to accumulation abnormal proteins in cells, and further cause cell disorders and denaturation. UPS dysfunction can occur in any part of UPS pathway, such as the imbalance in the quantity of ubiquitin, dysfunction of E1, E2 or E3 in ubiquitylation process, disorder of proteasome degradation or deubiquitination. Experimental results indicated that the ubiquitin-protein ligasen dorfin localized in the inclusions of ALS.Dorfin physically bound and ubiquitylated various SOD1 mutants, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Therefore, there is a close relationship between the ubiquitin-proteasome system and amyotrophic lateral sclerosis.At present, there are not domestic research reports about ubiquitin in amyotrophic lateral sclerosis. This experiment apply familial amyotrophic lateral sclerosis animal model (SOD1^G93A transgenic mice) and observe ubiquitin in different stages of ALS expression, aims to explore the role of ubiquitin in the pathogenesis of ALS.Methods: Breeding familial amyotrophic lateral sclerosis animal models SOD1^G93A transgenic mice. Reference Weydt, p. description SOD1^G93A transgenic mice were divided into pre-symptomatic stage(60 days), disease onset stage ,end stage.The control group animals were no SOD1 gene mutations mice in the same nest with experimental mice.1 After anesthesia, animal tissues were fixated via heart perfusion by 4% paraformaldehyde for 20min. The lumbar spinal cord of mice cut were fixated in 4% paraformaldehyde for 48 hour. Then they were dehydrated by gradient ethanol, transparentized by xylene, embedded by paraffin and made into conventional histological sections futher (5μm thick). 5μm thick paraffin sections were used to immunohistostain for detection of ubiquitin-protein expression and distribution.2 After anesthesia, then decapitate, extract the lumbar spinal cord of mice immediately, put it into liquid nitrogen freezing , - 80℃storage. Extract total protein using the kit to detected the express quantity of ubiquitin in different periods SOD1^G93A transgenic mice by Western-blotting.3 After anesthesia, extract the lumbar spinal cord of mice immediately, liquid nitrogen freezing , - 80℃storage. RNA was extract, through reverse transcription and gene amplification, to detected the express quantity of ubiquitin mRNA in different periods SOD1^G93A transgenic mice. 4 The experiment data was demenstrate as mean±standard deviation ((x|)±s). Statistical analyses were performed using one-way ANOVA followed by Student's t-test with SPSS 13.0 statistical software. Differences were considered significant at P < 0.05.Results:1 Ubiquitin immunohistochemical indicated, ubiquitin was extensive eveniy distributed in the lumber spinal cord anterior horn neurons of control mice , cytoplasm and nuclei evenly expression. In the lumber spinal cord ventral horn neurons of SOD1^G93A transgenic mice, ubiquitin was gradually gathered with the disease progress, to form aggregate.2 Total protein of SOD1^G93A transgenic mice's lumber spinal cord Western-blotting showed, as the disease progress,the quantity of 8.5K Da single ubiquitin and molecular weight 17K Da above poly-ubiquitin protein level were no obvious increase(F =0.241,1.972,P>0.05), no statistically significant.3 Ubiquitin mRNA of SOD1^G93A transgenic mice's lumber spinal cord reverse transcription, PCR gene amplification showed, the quantity of UBA in different SOD1^G93A transgenic mice period compared with control group had difference(F =9.938,P<0.05).There was no obvious difference for UBA between SOD1^G93A transgenic mice in pre-symptomatic stage and mice in the control group(P = 0.08 > 0.05). SOD1^G93A transgenic mice in disease onset stage compared with mice in pre-symptomatic stage, UBA expressed significantly increased(P = 0.04 < 0.05). SOD1^G93A transgenic mice in end stage compared with mice in pre-symptomatic stage, UBA expressed significantly increased, a statistically significant(P = 0.01 < 0.05). There was no obvious difference for UBB(F=0.637,P=0.612>0.05) in the different stage of disease.Conclusions:In the disease progress process of familial amyotrophic lateral sclerosis animal models SOD1^G93A transgenic mice , ubiquitin was gradually gathered, forming aggregates. But western blotting found that free ubiquitin protein levels and ubiquitin conjugated protein levels did not increase obviously. UBA mRNA levels increased, but UBB mRNA levels did not increase obviously, so ubiquitin protein synthesis was not increased. Protein ubiquitination was abnormity in protein degradation process by UPS.