| Background and ObjectivesALS is a progressive neurodegenerative disease involving upper and lower motor neurons of brain and spinal cord.ALS patients often die of the paralysis of respiratory muscle in three to five years.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.Proteomic researches based on SOD1-G93 A transgenic mice all aim for partial neural tissues,and there is no large systemic research using iTRAQ technique,as well as no associated research between ARSB and ALS.Our study applies iTRAQ technique to analyze the proteomic of the brain and spinal cord of SOD1-G93 A transgenic mice for the first time,meanwhile exploring the regular pattern of expression and distribution of ARSB in the spinal cords of SOD1-G93 A transgenic mice.We hope to achieve the following purposes: constructing a ALS spectrum of differentially expressed proteins in SOD1-G93 A transgenic mice,providing enlightenment for the subsequent searching for human disease-related proteins of ALS and the sensitive protein markers,elucidating the biological pathways and molecular mechanisms of SOD1-G93 A associated-ALS,establishing the theoretical basis for new therapy targets,revealing the regular pattern about the distribution of ARSB in the spinal cord of SOD1-G93 A transgenic mice,confirming the role of ARSB as the disease-related protein of ALS.MethodsWe 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.Proteomic analysis of iTRAQ(1)Research objectsFour mice: the experimental group including three first generation mice with SOD1-G93 A mutant gene divided into 3 subgroups according to neurological function and age: Preonset group(60-70 days),Onset group(90-100 days),Progression group(120-130 days),and one normal wild type B6 mouse(120-130days)as the control group.(2)Analysis of iTRAQStripping the brains and spinal cords from the four mice to extracting the whole protein.To the eight samples,we carried out the quality detection and enzymatic hydrolysis after the reductive alkylation.Each group of peptides was tagged by different iTRAQ lable and cultured for two hours under room temperature.Each group of tagged peptides was mixed,then preformed liquid phase separation with SCX column,and analyzed by LC-ESI-MSMS based on Triple TOF 5600.(3)Bioinformatics analysisTransforming the original files of mass spectrometry into mgf format(sample.mgf).Applying the protein identification software Mascot 2.3.02 to perform searching in Uniprot_Mus database(72645sequences)with mgf as the original files.According to the level of protein abundance,when the abundance ratio of some protein between two groups is >1.2 or < 0.833(1/1.2),moreover P value of the independent sample t test is < 0.05,the protein will be regarded as a differentially expressed protein.Pairwise comparing between samples and counting the numbers of differentially expressed proteins.Performing Gene Ontology functional annotation,Cluster of Orthologous Groups of proteins annotation,Pathway annotation aiming at all identified proteins.In accordance with the screening principle,we screened the differentially expressed proteins of the brain and spinal cord tissue between the control group and progression group of the same age.We made the picture about abundance ratio distribution and performed Gene Ontology enrichment analysis and Pathway enrichment analysis aiming at these proteins.2.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 group including nine normal wild type B6 mice devided into 3 subgroup(three mice in each subgroup)according to the age :60-70 days,90-100 days,120-130 days 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.Double immunofluorescent staining experiments: 3 spinal cord sections were observed for each segment.For each section,we observed the white matter,the gray matter including the anterior horn,posterior horn and the area around central canal under the fluorescence microscope at 4×,10×,20×,40× magnification,applying NIS-Element F software to analyze co-localization of ARSB positive cells and Fox3 /NeuN(neurons),claudin-11/oligodendrocyte Specific Protein(oligodendrocytes),GFAP(astrocytes),IBA-1/Microglia(microglia)positive cells.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.Results1.Results of iTRAQ Proteomic analysisOur study is the first research applying iTRAQ technique for proteomic analysis of the SOD1-G93 A transgenic mice.The outcomes are as following:(1)The analysis results of proteins expressed differentiallyThere are 5412 proteins identified.We performed pairwise comparison among the four mice,and counted the numbers and kinds of.When the mice in SOD1-G93 A progression group were compared with the mice in the control group at same age,there are 249 proteins expressed differentially in brain,including 163up-regulated proteins and 86 down-regulated proteins,and there are 611 proteins expressed differentially in spinal cord,including 334 up-regulated proteins and 277down-regulated proteins.There are 107 proteins expressed differentially both in brain and spinal cord,and ARSB is one of them.(2)The analysis results of bioinformaticsWe evaluated the 5412 proteins identified with bioinformatics: GO annotation which analyzes the involved biological processes,molecular function and sub-cellular localization;COG annotation which predicts and classifies the probable functions ofthese proteins;Pathway analysis which illuminates the main biochemical metabolism and signal transduction pathways these proteins participate in.We performed GO enrichment and Pathway enrichment analysiss on the proteins expressed differentially in brain and spinal cord of SOD1-G93 A transgenic mice,and the results show that: of the proteins expressed differentially in brain,the cellular component term enriching most significantly is histone deacetylase complex,the molecular function term enriching most significantly is actin binding,and the biological process term enriching most significantly is amine biosynthetic process;of the proteins expressed differentially in spinal cord,the cellular component term enriching most significantly is cell projection,the molecular function term enriching most significantly is structural molecule activity,and the biological process term enriching most significantly is cellular component disassembly.There are 11 pathways in the proteins expressed differentially in brain,of which the one enriching most significantly is pathway and the one enriching most proteins is tight junction pathway.There are 14 pathways in the proteins expressed differentially in spinal cord,of which the one enriching most significantly and most proteins is ribosome pathway.This study confirms that the reported protein markers of ALS previously involving NfM,NfH,Cystatin C,C3,S100 b,galectin-3 are also the proteins expressed differentially in the spinal cord of SOD1-G93 A transgenic mice.Compared with the control group,NfM,NfH,S100 b expressed downregulatedly and Cystatin C,C3,galectin-3 expressed upregulatedly in the spinal cord of ALS mice with SOD1-G93 A mutation.2.Results of verification study of ARSBThis study is the first research about association between ARSB and ALS,we got the following results:(1)The iTRAQ results of ARSBARSB is a kind of protein expressed differentially both in the brain and spinal cord of SOD1-G93 A transgenic mice,the level of ARSB in the spinal cord shows as the following rules: the ratio of abundance between progression group and control group is 2.127,i.e.,control group<experimental group,and in experimental group,preonset group < onset group < progression group.The associated pathways of ARSBare metabolic pathways,Glycosaminoglycan degradation and Lysosome pathway.GO analysis illustrates that the involved biological process terms are central nervous system development and response to estrogen stimulus,the involved molecular function terms are metal ion binding,N-acetylgalactosamine-4-sulfatase activity and arylsulfatase activity,the involved cellular component terms are Golgi apparatus,lysosome and rough endoplasmic reticulum.(2)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.(3)The results of double immunofluorescent staining experimentsThe results revealed that co-localization of ARSB positive cells and FOX3/NeuN,IBA1 positive cells in the spinal cord grey matter of all mice,i.e.,ARSB protein distributes in the neurons and microglias of spinal cord grey matter of all mice.(4)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 differentanatomical 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.When segment exists as an independent factor,it has no impact on the percentage of ARSB positive cells(P=0.16),but if pairwise comparing,and then compared thoracic to the waist,the percentage of ARSB positive cells in thoracic segment is higher than in lumbar segment significantly(P=0.03).When age 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),and the percentage of ARSB positive cells rises with the increasing age.The feature is particularly obvious in the experimental group.There is an interaction between the disease factor and the anatomical region factor(P=0.02),and the change of one factor will influence the effect of another factor.ConclusionsThis 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:1.For the first time we constructed a spectrum of proteins expressed differentially in SOD1-G93 A transgenic mice,illustrated the number and category of these proteins,and revealed the functions and related pathways of 5412 proteins.2.When the mice in SOD1-G93 A progression group were compared with the mice in the control group at same age,there are 249 proteins expressed differentiallyin brain,including 163 up-regulated proteins and 86 down-regulated proteins.Of these proteins,the cellular component term enriching most significantly is histone deacetylase complex,the molecular function term enriching most significantly is actin binding,and the biological process term enriching most significantly is amine biosynthetic process.There are 11 pathways in the proteins expressed differentially in brain,of which the one enriching most significantly is pathway and the one enriching most proteins is tight junction pathway.When the mice in SOD1-G93 A progression group were compared with the mice in the control group at same age,there are 611 proteins expressed differentially in spinal cord,including 334 up-regulated proteins and 277 down-regulated proteins.Of these proteins,the cellular component term enriching most significantly is cell projection,the molecular function term enriching most significantly is structural molecule activity,and the biological process term enriching most significantly is cellular component disassembly.There are 14 pathways in the proteins expressed differentially in spinal cord,of which the one enriching most significantly and most proteins is ribosome pathway.There are 107 proteins expressed differentially both in brain and spinal cord.3.We confirmed that the reported disease-related proteins of ALS previously including NfM,NfH,Cystatin C,C3,S100 b and galectin-3 are also the proteins expressed differentially in the spinal cord of SOD1-G93 A transgenic mice.Compared with the control group,NfM,NfH,S100 b expressed downregulatedly and Cystatin C,C3,galectin-3 expressed upregulatedly in the spinal cord of ALS mice with SOD1-G93 A mutation.4.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 according tothe following rules:experimental group>control group,anterior horns>region around the central canal>posterior horns,thoracic segment>lumbar segment,rising with the increasing;there exists an interaction between the disease factor and the anatomical region factor on the percentage of ARSB-positive cells in spinal cord;ARSB positive cells colocalize with the neurons and microglias in grey matter of all mice.6.ARSB is a candidate disease-related protein of ALS. |
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