| Background and Objectives:Amyotrophic lateral sclerosis(ALS)is a kind of involvement of the brain and spinal cord of upper and lower motor neurons,including cerebral cortex,motor neurons of anterior horn cells of the spinal cord and brainstem motor nuclei,with progressive and selective motor neuron injury as the main characteristics of the fatal neurodegenerative disease.The onset of the disease is insidious,and the patient often die of the paralysis of respiratory muscle in three to five years,Unfortunately,the prognosis is very poor.Currently,Riluzole is the only approved by the United States FDA in the crowd to confirm the efficacy of amyotrophic lateral sclerosis,but it can only delay the life of the 2-3month,and costly.The pathogenesis of amyotrophic lateral sclerosis is still not completely clear,in recent years many scholars have studied on amyotrophic lateral sclerosis disease-related proteins,but most research is repeated verification,The pathogenic protein have been found can partly explain the pathogenesis of amyotrophic lateral sclerosis.If we can find the sensitive disease related protein,not only can the early diagnosis of ALS,tracking disease progression,evaluation of treatment after the reaction,but also help us to better understand the molecular pathogenesis of ALS,and therefore backward based one step to clarify SOD1-G93 A related ALS mutant biological pathways and molecular mechanisms.Therefore,this study focuses on the search for novel proteins associated with amyotrophic lateral sclerosis.Our study applies i TRAQ technique to analyze proteomics of brain and spinal cord of SOD1-G93 A transgenic mice,and screened proteins with different meanings in brain and spinal cord previously.Then we used immunofluorescence and Western blot to screen out and to analyze the expression and distribution of ALDH1A2 in the spinal cord of SOD1-G93 A transgenic mice and to analyze the co-localization of ALDH1A2 positive cells in neurons.Methods:The normal C57BL/6J mice were mated with SOD1*G93A transgenic mice to establish the animal model.We applied PCR technique to detect the positiveSOD1-G93 A transgenic mice.The expression of ALDH1A2 positive cells in experimental group and control group were analyzed by Western blot technique;The distribution of ALDH1A2 positive cells in the experimental group and the control group were observed and analyzed by using fluorescence immunohistochemistry,and counted the numbers of ALDH1A2 positive cells and calculated the rate of positive cells with Image-Pro Plus 6.0 software.We apply double immunofluorescence labeling technique to mark the Fox3 / NeuN(neurons),claudin-11/oligodendrocyte Specific Protein(oligodendrocytes),GFAP(astrocytes),IBA-1/Microglia(microglia)positive cells,Each of the anatomical regions was subjected to double fluorescence immunohistochemical staining,Image processing technique was used to overlay the positive cells with different staining,and the co-localization of positive cells and nerve cells was observed under microscope.Results:Reviewing the literature,this study is the first research about association between ALDH1A2 and ALS,we got the following results:(1)The iTRAQ results of ALDH1A2The results of iTRAQ analysis in our laboratory were compared with those of normal wild type mice,ALDH1A2 is a kind of protein expressed differentially both in the brain and spinal cord of SOD1-G93 A transgenic mice,the level of expression in the spinal cord of mice is as follows: :the ratio of abundance between progression group and control group is 0.63,experimental group<control group,and in experimental group,preonset group < progression group < onset group.(2)The results of Western blotThe same proteomics analysis of iTRAQ results in Western blot results showed that ALDH1A2 expression levels in control of mouse spinal cord were higher when compared with experimental spinal cord.(3)The results of double immunofluorescent staining experimentsThe ALDH1A2 mainly distributed in the GFAP(Astrocyte cell),IBA-1(Microglial cell),Fox3/NeuN(Neuron cell)and claudin-11(Oligodendrocyte cell)in the WT and G93 A mice.The results indicated that ALDH1A2 could be expressed in four kinds of cells.(4)The results of single immunofluorescent staining experimentsALDH1A2 positive cells were widely distributed in the gray matter and white matter of the spinal cord in the experimental group and the control group.When disease exists as an independent factor,the percentage of ALDH1A2 positive cells in the experimental group is less than the control group,the two groups were statistically significant(P=0.02);When segment exists as an independent factor,the characteristics of the percentage of ALDH1A2 positive cells show: cervical >thoracic > lumbar,three groups was statistically significant(P=0.00),if pairwise comparing,there was no significant difference between the cervical and thoracic segments,but a significant difference between cervical and lumbar segments,thoracic and lumbar segments;When anatomical region exists as an independent factor,white matter of the spinal cord > gray matter of the spinal cord,There was significant difference between the six groups(P=0.00),but when pairwise comparingand between the gray matter and white matter of the spinal cord,there is no significant difference;When age exists as an independent factor,it impacts on the percentage of the ALDH1A2 positive cells:onset group>progression group>preonset group,the three groups were statistically significant(P=0.00),when pairwise comparingand,there is also significant difference;There are interaction between the age and disease(P=0.04),and the change of one factor will influence the effect of the another factor.Conclusions:This study is the first large systemic research which is based on SOD1-G93 A transgenic mouse model,applying iTRAQ technique to search disease-related proteins of ALS,and the conclusions are as following: For the first time we expounded the features about the expression and distribution of ALDH1A2 in different anatomical regions,segments and age in the spinal cord of SOD1-G93 A transgenic mice and control mice: ALDH1A2 positive cells were widely distributed in the gray matter and white matter of the spinal cord in the experimental group and the control group.When disease,age,segment and regional exist as independent factor,the percentage of ALDH1A2 positive cells in the spinal cord of mice could be affected as the following rules:the control groupthe > experimental group,white matter of the spinal cord > gray matter of the spinal cord;There are interactionbetween the age and disease of the percentage of ALDH1A2 positive cells;The ALDH1A2 mainly colocalize with the GFAP,IBA-1,Fox3/NeuN and claudin-11 in the WT and G93 A mice,and were mainly expressed in the cytoplasm.The redistribution of positive cells may occur in some anatomical areas of the spinal cord,but no redistribution in different neurons is detected.ALDH1A2 is a candidate disease-related protein of ALS. |
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