Screening Of Autism Spectrum Protein Biomarkers And Its Related Mechanisms

Autism Spectrum Disorders is neurodevelopmental disorder characterized by social deficits,repetitive/stereotypical behaviors and interests,as well as communication problems.An increase in the prevalence of autism is being reported worldwide with social,behavioral and economical burdens,and recent epidemiological studies have indicated that at least one in every 100 people has some form of autism.Even more,autism related disorders are increasing at an alarming rate and have now affected 2% of US school-aged children.The boys had a higher prevalence than girls and the boy to girl's ratio on average is 4.2:1..The etiology of autism is complex and there is no perfect diagnosis or treatment.In order to find disease biomarkers and explore the mechanism of disease,this study was designed upto three different levels: Peripheral blood(autistic children and healthy control peripheral blood),peripheral blood mononuclear cells(autistic children and healthy controls peripheral blood Mononuclear cells)and animal models(VPA-induced autism model mice).Firstly,differential proteomics studies of peripheral blood mononuclear cells(PBMCs)from autism patients and healthy controls were conducted by iTRAQ based quantitative proteomic analysis.Compared with healthy controls,autistic children showed significant changes in 53 proteins which were mainlyassociated with mitochondria,metabolic pathways,endoplasmic reticulum(ER)stress and protein folding,endocytosis,immunity and inflammation.Bioinformatics analysis showed that endoplasmic reticulum stress,unfolded protein response(UPR),acute phase response(APR),inflammatory response and endocytosis/phagocytosis may be involved in the pathophysiological processes in autism.Among the differentially expressed proteins,20 proteins belong to mitochondrial proteins,17 of which are up-regulated in children with autism.The Western blot(WB)verification results are consistent with proteomic results,suggesting that PBMCs may be “activated” in children with autism.In addition,we tested ELISA for the detection of plasma,and four types of pro-inflammatory disorders associated with autism have also been reported in the literature.Cytokines(IFN-?,IL-1?,IL-6,and TNF-?)were found to be up-regulated in plasma.Compared with the results of previous plasma quantitative proteomics,we detected the expression levels of three proteins(C3,CALR,and SERPINA1)consistent with plasma proteins found in PBMCs which increased the specificity of detection.These proteins may serve as potential biomarkers for the diagnosis of autism.Secondly,we collected transcriptome data of autism brain tissue through a public database,and used SAM method to screened the characteristic genes of autism.We found that 379 autism-related genes were found in the GEO database GSE28521,of which 364 genes showed differential expression..(6 genes up-regulated and 358 downregulated).We analyzed the differentially expressed proteins by GO analysis and found that these differential proteins were mainly distributed in mitochondria,myelin sheaths,cytoplasm and tonoplasts,and were mainly involved in the tricarboxylic acid cycle,neurotransmitter transport,synaptic vesicle exocytosis and other biological processes.The molecular functions mainly associated include postsynaptic density,ATP binding,protein binding etc..The prediction of hemoglobin in the differential proteins revealed that 59 proteins may be detected in both the brain and blood,and both are downregulated.Through literature analysis,it was found that 10 proteins have already been reported in the literature to be associated with occurrence of autism.We found that 6 proteins i.e RARS,ACTL6 B,PRKAA1,SLC25A12,LIMK1,and BDNF were significantly down-regulated as evidenced by EILSA experiment.Consistent with the prediction and literature reports;ARHGEF4,POLR3 C,SLC25A27 and other three proteins showed low expression trend,but statistical analysis showed no significant difference;while SLITRK5 showed up-regulation(p-value = 0.0185).Finally,through the iTRAQ experiment with valproic acid-induced autism rats,102 peptides were found with a wriggling number greater than or equal to 2 and a pvalue of less than 0.05..Located in the myelin sheath,exosomes,mitochondria,membranes,axons and cytoplasm,in particular,we have enriched 37 mitochondrial associated proteins,of which 27 were up-regulated and 2 were down-regulated.Through GO process analysis,we found that the biological processes of these proteins in vivo mainly include TCA cycle,glycolysis process,citrate metabolism process,reaction to drugs,coupled proton transport of ATP hydrolysis,etc.Molecular functions enrichmetn showed that these proteins are mainly involved in protein binding,ATP binding,ATPase activity,and protein kinase binding in vivo.Based on these finding,we can conclude that proteins reported here not only help to further understand the pathological mechanisms of the disease but may also serve as biomarkers for early detection after further in depth studies.