| Background and Objectives:Amyotrophic lateral sclerosis(ALS)is a neurodegenerative disease that involves both upper and lower motor neurons.The pathogenesis of ALS is unknown.Recent studies have suggested that protein abnormalities may play an important role in ALS.However,currently only a few ALS-related proteins explain the pathogenesis of the disease,further screening and identification of other ALS-related proteins is needed.This study first analyses and compares the proteomic alterations in the brain of SOD1G93 A transgenic mice to wild-type SOD1 mice using isotope-labelled relative and absolute quantitative techniques(i TRAQ)and bioinformatics methods.Transcriptomic data from corticospinal neurons(CSN)of SOD1 transgenic mice at different disease stages were analysed to further reveal key proteins associated with disease progression in the brains of SOD1 transgenic mice.Methods:The SOD1 G93 A transgenic mouse model was mated and bred to wild-type mice,and SOD1 G93A-positive offspring mice were first detected and screened by PCR.The following studies were performed:iTRAQ proteomic analysis to identify differential expressed proteins in the brains of SOD1 G93 A mice: SOD1 G93 A transgenic mice aged 68 days(pre-onset),100 days(onset)and 130 days(progression)and wild mice at day 124 were used as normal controls.The brains of the mice were stripped,whole brain proteins were extracted and the left and right brains of each sample were separated and assayed for proteomic analysis using i TRAQ technology.Each sample was assayed in 30 technical replicates,and the mass spectra were annotated with bioinformatics software for protein annotation.Proteins with a fold change greater than 1.2 or less than < 0.833(1/1.2)were selected as differential proteins,and the differential proteins in the brains of SOD1G93 A transgenic mice at different stages were screened for comparison with normal control mice,respectively.Cluster of Orthologous Groups of protein(COG)functional analysis,Gene Ontology functional annotation and KEGG Pathway enrichment analysis were performed for the identified differential proteins.Using weighted gene co-expression network analysis(WGCNA)to identify key proteins associated with disease progression in the CSN of SOD1 transgenic mice:SOD1 transgenic mouse and wild-type mouse CSN transcriptome data were downloaded from the Arrayexpress database and the modules most associated with disease stages in SOD1 transgenic mice were analysed using the WGCNA approach.Based on the results of the correlation analysis between the obtained module genes and the disease stage,the genes with a significant correlation of >0.5 with the disease stage and a correlation of >0.8 with the gene expression profile of the module were selected as the candidate key genes within the module.Finally,the candidate key genes in the module were intersected with the differential proteins in the brain of SOD1 G93 A transgenic mice to identify the key proteins in the brain of SOD1 transgenic mice that may be involved in ALS disease progression.Results:Compared with wild-type mice,203 proteins were significantly upregulated in the brains of SOD1 G93 A transgenic mice in the pre-onset phase,187 proteins were upregulated in the onset phase and 357 proteins were upregulated in the progression phase;360 proteins were significantly downregulated in the brains of SOD1 G93 A transgenic mice in the pre-onset phase,118 proteins were downregulated in the onset phase and 165 proteins were downregulated in the progression phase.The number of proteins significantly down-regulated in the brain of pre-onset SOD1 G93 A mice was360,118 in the onset and 165 in the progressive phase.Seven proteins were found to be differentially expressed in the brains of pre-onset,onset and progressive SOD1G93 A mice compared to wild-type mice.These included Beta-globin,Protein IMPACT,Fatty acid binding protein 7(brain),EF-hand domain-containing protein D2,Signal transducer and activator of transcription 1,Protein FAM177A1 and Exocyst complex component 3.COG functional analysis of the identified progressive differential proteins revealed that the main functions include signal transduction mechanisms,cytoskeletion,inorganic ion transport and metabolism,amino acid transport and metabolism,replication,recombination and repair,transcription,energy production and conversion,carbohydrate transport and metabolism,lipid transport and metabolism,posttranslational modification,protein turnover,chaperones,cell cycle control,cell division,chromosome partitioning,nucleotide transport and metabolism,secondary metabolites,biosynthesis,transport and catabolism.Results of gene ontology(GO)enrichment analysis: histone deacetylase complex,secretory granule membrane,zonula adherens,cytoplasmic part,contractile fiber,the extrinsic to external side of plasma membrane,striated muscle thin filament,the paranode region of axon,the juxtaparanode region of axon and myofibril are predominantly enriched in the cellular component;actin binding,cytoskeletal protein binding,arylsulfatase activity,chitinase activity,spermidine synthase activity,hexosaminidase activity,hydrolase activity,hydrolyzing O-glycosyl compounds,pyrimidine nucleoside binding,UTP binding and sulfuric ester hydrolase activity are predominantly enriched in the molecular function;amine biosynthetic process,autophagy,aspartate family amino acid metabolic process,methionine metabolic process,the regulation of protein complex disassembly,Lmethionine salvage from methylthioadenosine,amino acid salvage,barbed-end actin filament capping,the regulation of epithelial cell proliferation involved in prostate gland development and L-methionine biosynthetic process are predominantly enriched in the biological processes.Significantly enriched KEGG pathways include staphylococcus aureus infection,Malaria,cysteine and methionine metabolism,hematopoietic cell lineage,african trypanosomiasis,adherens junction,tight junction,drug metabolism-other enzymes,sulfur metabolism,bacterial invasion of epithelial cells,leukocyte transendothelial migration.Results of WGCNA analysis: A total of 29 cases of CSN data from SOD1 transgenic and wild-type mice were extracted from the E-MTAB-7876 dataset,containing CSN transcriptome data from SOD1 transgenic and wild-type mice at 30 days old,60 days old,90 days old and 105 days old.19 modules were identified by WGCNA,of which blue modules were positively correlated with disease stage The blue module was positively correlated with disease stage and the grey60,ochre and darkturquoise modules were negatively correlated with disease stage.The blue and darkturquoise modules with correlation to disease stage were selected for candidate hub gene and enrichment analysis.The blue module was mainly enriched in biological processes such as tricarboxylic acid cycle,mitochondrial function and RNA metabolism,while the darkturquoise module was mainly enriched in forebrain neuronal differentiation,sensory organ morphogenesis and endocrine hormone secretion.The candidate genes in the blue and darkturquoise modules were extracted separately and intersected with the differential proteins in the brains of progressive SOD1 G93 A mice,and seven of the 944 candidate genes in the blue module were found to be differentially expressed in the brain proteins.Eukaryotic Translation Initiation Factor 5A(EIF5A),CAMP Regulated Phosphoprotein 19(ARPP19),tetraspanin 3(TSPAN3),Basic Leucine Zipper And W2 Domains 1(BZW1),RNA Polymerase II Subunit H(POLR2H),Proteasome 26 S Subunit,Non-ATPase 13(PSMD13),NCK Adaptor Protein 1(NCK1)in the brains of SOD1 transgenic mice was positively correlated with the degeneration of CSN.Conclusions:In this study,we revealed that multiple differential proteins are involved in the pathogenesis of different disease stages in SOD1 G93 A transgenic mice,and systematically elucidated the functions and signalling pathways involved in the differential proteins.7 differential proteins were also found to be involved in the entire disease process,and these findings suggest that different proteins play different roles at different times of the disease in SOD1 G93 A transgenic mice.In addition,we identified key proteins associated with disease progression in the brain of SOD1 transgenic mice that may be intimately involved in the process of neurodegeneration in the brain of ALS,which provides potential research targets for future ALS studies. |
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