The Research Of The Ras Homologous Protein Enriched In Brain And Rapamycin Mammalian Target Protein In The SOD1-G93A Transgenic Mice

Objective:Amyotrophic lateral sclerosis(ALS)is the disease whichinjures the motor neuron,leading to chronic progressive muscle weaknessand muscle atrophy,while do not involve the nervous system degenerativediseases.Clinical studies have showed that the patients often died ofrespiratory muscle paralysis3-5years after the onset of illness. AmongALS patients,approximately90%to95%are sporadic, and5%to10%are familial,while about20%of familial ALS could be due to the genemutation of the Cu/Zn superoxide dismutase(SOD1).The current studysuggests that neuron apoptosis caused by various pathogeny, includingabnormal protein aggregation,glutamate excitotoxicity,oxidativestress,immunological factors,autophagy abnormalities and axonaltransport barriers,leads directly to the disease.After further study,theautophagic dysregulation has been increasingly concerned.There are many autophagy pathways in the cell.With the regulationof these pathways,the cell is able to adapt to the external and internalchanges and survive in adverse conditions,so as to ensure the normalbody functions.Of the numerous pathways regulating autophagy,the mostimportant is the one related to the Ras homologous protein enriched inbrain(Rheb)and mammalian target of rapamycin protein(mTOR).Rheb isa member of RAS GTP binding protein superfamily,hich is involved invarious aspects of the body’s regulation.The studies have demonstratedthat Rheb could regulate mTOR via TSC,a protein has a relationship withTuborous sclerosis.mTOR is a threonine/serine kinase,which has animportant role in cell growth.For example,it can regulate the transcriptionof genes,involve in protein translation,regulate the ribosome synthesis and inhibit cell autophagy.The studies have confirmed the presence of theabnormal aggregation of SOD1in familial ALS,so it can be inferred thatautophagy is aberrant and the cell inevitably fails to clean the internalabnormal material.Whether the Rheb-mTOR pathway is capable ofregulating aberrant autophagy needs further investigation.So far,SOD1-G93A transgenic mice is an ideal pathological animalmodel of the familial ALS.The aim of this research is to find thecorrelation between Rehb and mTOR in the SOD1mice,and to explorethe role of the two proteins in the development of ALS.Methods: The male SOD1-G93A transgenic mice were used in theexperiment,and divided by symptoms into three groups which were60-day group(pre-symptomatic group), stage group and end-stagegroup;while100-day negative mice were selected as control group.Eightmice were in each group.Anesthetized by intraperitoneal injection of10%chloral hydrate (350mg/Kg body weight), mice were sacrificed forlumbar marrow which was quickly put in liquid nitrogen for frozen, thenstored at-80℃;After intracardial injection of4%paraformaldehyde,themice’s lumbar spinal cord was peeled,and fixed with4%paraformaldehyde.The morphology of neurons was observed byimmunohistochemical techniques and the quantity of the Rehb andmTOR was assayed by western blot in lumbar spinal cord of each groupof mice.Spss17.0statistical software was used to analyze the data.Results:1.The concentration of the Rheb in lumbar spinal cord ofSOD1-G93A transgenic mouse: Immunohistochemistry indicated that thenumber of neurons which the immunocompetence was reduced in theanterior horn of lumbar myeloid in SOD1-G93A transgenicmice,compared with negative control group.The western blot analysisshowed the Rheb content significantly decreased in both the SOD1-G93Atransgenic mouse group than negative control group.With thedevelopment of the disease,the Rheb content in lumbar spinal cord of SOD1-G93A transgenic mice went down,and there is a significantdifference among the groups.2.The concentration of the mTOR in lumbar spinal cord ofSOD1-G93A transgenic mouse: Immunohistochemistry indicated that thenumber of neurons which the immunocompetence was reduced in theanterior horn of lumbar myeloid in SOD1-G93A transgenicmice,compared with negative control group.The western blot analysisshowed the mTOR content significantly decreased in both theSOD1-G93A transgenic mouse group than negative control group.Withthe development of the disease,the mTOR content in lumbar spinal cordof SOD1-G93A transgenic mice went down,the end-stage is significantreduced than the60-day group.3.The correlation between the Rheb and mTOR in the SOD1-G93Atransgenic mice:The correlation coefficient of them was0.804,both werepositively correlated.Conclusion: Morphological observation of lumbar spinal cordanterior horn cells and measurement of the Rehb and mTOR content inSOD1-G93A transgenic mice demonstrated that the Rehb and mTORcontent of lumbar spinal cord is on the decrease in the progress of disease.The results confirmed that the autophagy regulatory was hampered in thelumbar spinal cord neurons of SOD1-G93A transgenic mice,which maylead to SOD1accumulation and cell apoptotic,as one of the ALSaetiological agents.