Expression And Distribution Of Arylsulfatase B Are Closely Associated With Neuron Death In SOD1 G93A Transgenic Mice

Background and Objectives:Amyotrophic lateral sclerosis(ALS)is a fatal chronic motor neuron disease,its characterizes are the progressive and selective death of the upper and lower motor neurons at the cerebral,spinal and/or brain stem regions.ALS patients often die of the paralysis of respiratory muscle in three to five years.ALS was firstly described by the French neurologist Jean Martin Charcot in 1869,therefore,ALS is sometimes also known as Charcot disease in honor of the first person to describe the disease by Jean-Martin Charcot in the commemoration of his discovery.ALS has insidious onset and poor prognosis.Currently there is no specific method of diagnosis and treatment.If a sensitive disease-related protein is found,it will not only contribute to diagnosis ALS earlier,track the progress of the disease,and evaluate the post-treatment reaction,but also help us to better understand the molecular pathogenesis of ALS.According to current information,most of researches on disease-related proteins of ALS are repeating and validation researches.The known proteins only explained the partial pathogenesis of amyotrophic lateral sclerosis(ALS).Therefore,this study was aimed at search the novel proteins possibly involved in ALS.In this study,we analyzed the expression and distribution of the candidate protein arylsulfatase B(ARSB)in the different segments,anatomic regions and neural cells of spinal cord at the different stages of the wild type and SOD1 G93 A transgenic mice using the fluorescent immunohistochemistry and the western blot.The results revealed that the ARSB extensively expressed and distributed in the entire spinal cord,the expression and distribution of ARSB was significantly different in the different regions of spinal cord,the anterior horn of grey matter(AHGM)was significantly more than that in the posterior horn of grey matter(PHGM)significantly more than that in the central canal。The expression of ARSB significantly increased in the other anatomic regions besides the thoracic PHGM significantly decreased at the progression stage,occurredthe redistribution from the AHGM and the PHGM to the CC at the onset and progression stages,no any alteration of ARSB expression and distribution occurred between the different neural cells in the SOD1 G93 A mice compared with the wild-type mice.The increase of ARSB expression and distribution followed with the increased of neuron death.Our data suggested that the abnormal expression and distribution of ARSB were closely associated with the neuron death in the SOD1 G93 A transgenic mice.Methods:We constructed the B6SJL-Tg(SOD1*G93A)1Gur/J transgenic mice model,which reproduced by mating with normal C57BL/6J mice.We applied PCR technique to detect the positive SOD1-G93 A transgenic mice,and the study was divided into two parts:1.Verification study of ARSB expressed differentially in ALS mice(1)Research objects of Western blotTwelve mice: the experimental group including nine first generation mice with SOD1-G93 A mutant gene divided into 3 subgroups(three mice in each subgroup)according to neurological function and age: Preonset group(60-70 days),Onset group(90-100 days),Progression group(120-130 days),and the control group including three normal wild type B6 mice(60-70 days).(2)Western blotStripping the spinal cords from the twelve mice to extracting the whole protein,and detecting their concentration.Western blot compared the expression level of ARSB in the spinal cords of the four mice from the experimental group and the control group each time,repeating for three times.Quantity One analysis software was applied to analyzing the relative optical density.(3)Research objects of immunofluorescence experimentsEighteen mice: the experimental group including nine first generation mice with SOD1-G93 A mutant gene divided into 3 subgroups(three mice in each subgroup)according to neurological function and age: Preonset group(60-70 days),Onset group(90-100 days),Progression group(120-130 days),and the control groupincluding nine normal wild type B6 mice devided into 3 subgroup(three mice in each subgroup)according to the age in Wild type group as the control group.(4)Immunofluorescence experimentsFrozen sections were made after stripping the spinal cords from the eighteen mice,and these sections were divided into cervical,thoracic and lumbar segments for each mouse in each group.Single immunofluorescent staining experiments : 9 spinal cord sections were observed for each segment.For each section,we observed the distribution of ARSB positive cells in the white matter and the gray matter under the fluorescence microscope at 4×,10×,20×,40× magnification.In the spinal cord gray matter where ARSB positive cells existed,we took five view fields from the two anterior horns,the two posterior horns and the region around central canal under the fluorescence microscope at 20 × magnification,and counted the numbers of ARSB positive cells and calculated the rate of positive cells with Image-Pro Plus 6.0 software.(5)Statistical analysis and image processingWe applied SPSS17.0 statistical software package to manipulate datas.All datas were expressed by "mean±standard deviation" and analyzed with factorial design analysis of variance,pairwise comparing with Bonferroni method.P<0.05 was considered significant statistically.All images are processed with NIS-Element F and Photoshop software.Results:1.Results of verification study of ARSBThis study is the first research about association between ARSB and ALS,we got the following results:(1)The results of Western blotSame as the iTRAQ results of proteomic analysis,the results of Western blot showed that the expression level of ARSB in the spinal cord of control mice is less than in the spinal cord of experimental mice,and in the experimental group,the expression level of ARSB increased along with the progress of the disease.(2)The results of single immunofluorescent staining experimentsARSB positive cells are widely present in the spinal cord gray matter of all mice,however,ARSB positive cells were not observed in spinal cord white matter of all mice.Distribution feature of ARSB positive cells in the spinal cord of experimental group shows as following: in the cervical segment,the region around the central canal,the anterior horns,and the posterior horns——wherever ARSB localizes——the percentage of ARSB positive cells in experimental group at the different disease phage(preonset,onset,progression)is significantly different,and it increases with the progress of the disease;in the lumbar segment,the percentage of ARSB positive cells in mice at progression phage is significantly different in the different anatomical regions(the region around the central canal,the posterior horns),and the anterior horns > the posterior horns > the region around the central canal.Distribution feature of ARSB positive cells in the spinal cord of control group shows as following: in the cervical and thoracic segment,the percentage of ARSB positive cells in mice at preonset phage is significantly different in three different anatomical regions,and the anterior horns > the posterior horns > the region around the central canal;in the lumbar segment,the percentage of ARSB positive cells in the posterior horns is significantly different at the different age(60-70 days、90-100 days、120-130 days),and it rises with the increasing age.When disease exists as an independent factor,it impacts on the percentage of the ARSB positive cells in spinal cord of mice extremely significantly(P=0.00).In any anatomical segment of spinal cord,the average percentage of ARSB positive cells in experimental mice is higher than in normal mice at the same anatomical region.When anatomical region exists as an independent factor,it impacts the percentage of ARSB positive cells in spinal cord of mice(P=0.01),and the distribution feature of ARSB positive cells in different anatomical regions shows that: the anterior horns> the region around the central canal>the posterior horns.Conclusions:This study is the first large systemic research which is based on SOD1-G93 A transgenic mouse model,applying Western blot technique to search disease-related proteins of ALS,and the conclusions are as following:1.For the first time we constructed a spectrum of proteins expresseddifferentially in SOD1-G93 A transgenic mice,illustrated the number and category of these proteins.2.For the first time we expounded the features about the expression and distribution of ARSB in different anatomical regions,segments and age in the spinal cord of SOD1-G93 A transgenic mice and control mice: the average percentage of ARSB positive cells in experimental mice is higher than in normal mice,and it increases with the progress of the disease;ARSB positive cells are widely present in the spinal cord gray matter of all mice,when the disease,anatomical regions,segments and age exist as independent factors,the percentage of ARSB positive cells in spinal cord of the mice will be influenced.3.ARSB is a candidate disease-related protein of ALS.