Proteomics Analysis Of The Fmr1 Knockout Mice Reveals The Upregulation Of Complement System Proteins In The Prefrontal Cortex Associated With Mental Disorder Symptoms

IntroductionFragile X syndrome(FXS)is the most common form of inherited intellectual disability,resulted from loss of FMRP.In addition to mental retardation,FXS is also accompanied by several psychiatric disorders including anxiety,social withdrawal,inattention and irritability.Among them,anxiety and social withdrawal are considered as core features of the FXS phenotype.Anxiety aggravates the illness of FXS and leads to drug dependencies,which increase the difficulty of treatment.Therefore,Therefore,it is important to explore the molecular mechanism of mental symptoms in FXS.Previous studies have shown that hippocampus,amygdala and prefrontal cortex are the key brain areas for anxiety.Studies on FXS animal models also showed that alterations of neural excitabilities in hippocampus and amygdala were related to overreaction and anxiety.However,the involvement of prefrontal cortex in FXS with anxiety remains to be further investigated.FMRP,as a multifunctional RNA binding protein,regulates specific mRNA stability and protein translation,which are believed to be involved in the regulation of brain development and synaptic function.Previous proteomic studies on some brain regions(such as hippocampus and cerebral cortex)from FXS animal models have shown that changes of multiple genes resulted from loss of FMRP were involved in the synaptic abnormalities and learning and memory disorders.We speculate that FMRP absence may lead to changes of the levels of some important proteins in the prefrontal cortex,which should participate in the development of FXS with anxiety.ObjectiveIn this study,the proteomic and bioinformatics analyses of the prefrontal cortex of Fmr1 KO mice are used to identify the abnormal proteins and signal pathways caused by loss of FMRP,which will reveal the potential role of loss of FMRP in FXS with anxiety by dysregulation of the gene expression in prefrontal cortex.Methods1.Western blots were used to verify the presence of FMRP in the prefrontal cortex of Fmr1 KO mice and wild-type mice.2.Tandem mass tag(TMT)-based quantitative proteomics analysis was used to detect the protein levels of the prefrontal cortex from Fmr1 KO mice and wild-type mice.3.The gene functions and pathways resulted from significantly-altered genes were identified using Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.4.Western blots were used to verify the levels of protein complement Clq,complement C3,and SERPING1 in the prefrontal cortex of Fmr1 KO mice and wild-type mice.5.The qPCR experiments were performed to detect the mRNA levels of C1q,C3 and Serping1 genes.Results1.Western blots showed that FMRP protein presented in the prefrontal cortex of wild-type mice,but not in Fmr1 KO mice.2.The TMT-based quantitative proteomic analyses identified a total of 5886 proteins with 47 upregulated proteins and 53 downregulated proteins in the Fmrl KO mice.3.The GO classification and enrichment analyses showed that there were more than 30 significantly-altered proteins which are involved in the regulation of immune and stress.4.The KEGG pathway analyses suggested that the significantly-altered proteins were involved in 9 enriched signaling pathways,and among them,the most significant pathway was the coagulation complement system with three significantly-altered proteins,Clq,C3 and SERPING1.5.Western blot data showed that the levels of the three coagulation complement cascade components Clq,C3 and SERPING1 were upregulated in prefrontal cortex tissues of Fmr1 KO mice compared with that of the wild-type mice.6.The qPCR results indicated that,compared with the wild-type mice,the mRNA levels of C1q and C3 genes in the prefrontal cortex were increased upon loss of FMRP,but no significant change was observed for Serping1 gene.ConclusionThis study provides a proteomic profile of prefrontal cortex in response to loss of FMRP with more than 100 significantly-altered proteins.Of them,more than one third proteins participate in the regulation of immune stress function and these significantly-altered proteins are enriched in 9 KEGG pathways,and coagulation complement system represents the most significant pathway.The present study suggests that FMRP might play an important role in the emotional regulation through immune stress and coagulation complement system in the prefrontal cortex,which provides a new clue for the future investigation of the pathogenesis of the FXS with anxiety symptoms.