Astrocytes Increase Exosomal Secretion Of Oligodendrocyte Precursor Cells To Promote Their Proliferation Via Integrin?4-mediated Cell Adhesion

As the global population ages,the number of individuals with age-related neurodegenerative diseases will increase significantly.Numerous degenerative diseases are associated with demyelinating changes.Oligodendrocytes(OLs)are the only myelinating cell in the central nervous system.Oligodendrocyte precursor cells(OPCs),as a kind of stem cell differentiated into OLs,is crucial for remyelination at the damaged site.Promoting the growth of OPC may be a potential treatment for degenerative diseases.Evidence has been increasing that astrocytes(ASTs)are considered to play an important role in the proliferation of OPC.OPC direct-contact culture with AST(AST-OPC group)significantly promoted OPC proliferation compared with AST culture supernatant with OPC co-culture(CO-OPC group).However,the molecular basis is still poorly understood.We used transcriptome sequencing and bioinformatics to analyze genes and functions that were significantly differentially expressed in two groups of OPC.The results showed that the differentially expressed genes(DEGs)in the two groups of OPC were mainly enriched in genesrelated to cell adhesion and exosomes.Intergrin ?4(ITGB4),as an important cell adhesion molecule,was significantly differentially expressed in these two groups of OPC.After specific knocking down ITGB4 with siRNA,the exosomal secretion was significantly reduced and the proliferation of OPC was significantly decreased in the AST-OPC group.And exosomes can alleviate the inhibitory effect of ITGB4 knockdown on OPC proliferation.In summary,our study demonstrated that AST can promote the exosomal secretion and proliferation of OPC via ITGB4-mediated intercellular adhesion,which may represent a potential treatment for neurological diseases caused by demyelination.The study is divided into three parts:Part I: AST promotes the proliferation of OPC by increasing cell adhesionIn this part,by constructing two co-culture models of OPC and AST,the proliferation capacity of OPC under different culture conditions was detected by microscopy,flow cytometry and EdU.Transcriptome sequencing was performed to analyze OPC gene expression differences under different culture conditions.DAVID was used for GO functional annotation and KEGG pathway enrichment analysis.The main results are as follows:1.The proliferation capacity of OPC in the AST culture supernatant with OPC co-culture(CO-OPC group)and the OPC direct-contact culturewith AST(AST-OPC group)were compared.The results of microscopy and EdU showed that the number of OPC and the proportion of new cells in the AST-OPC group was significantly increased compared with the CO-OPC group(p<0.01).Flow cytometry were used to determine the cell cycle of OPC.And the proportion of OPC in S stage increased significantly in the AST-OPC group(p<0.01).2.Transcriptome sequencing was performed to analyze the OPC gene expression under the two groups and screen the DEGs.GO functional annotation and KEGG pathway enrichment analysis revealed that cell adhesion factors were significantly enriched.The above results indicate that AST promotes the proliferation of OPC by increasing intercellular adhesion.Cell adhesion may play an irreplaceable role in the signal transmission of OPC.Part II: AST promotes the proliferation of OPC via ITGB4-mediated cell adhesionIn this part,DAVID functional annotation clustering analysis was used to confirm the significant changes of cell adhesion factors.It was also found that ITGB4(Gene ID: 25724),which was significantly differentially expressed and participated in three cell adhesion signaling pathways at the same time.ITGB4 may be a key target for AST to promote the proliferation of OPC adhesion.Therefore,ITGB4 was knocked down in the AST-OPC group.The proliferation capacity of OPC were detected by EdU and flowcytometry.The main results are as follows:1.GO and KEGG functional annotation clustering analysis revealed that three pathways related to cell adhesion were significantly clustered.ITGB4 expression was significantly different in the two culture conditions and existed in the three pathways at the same time,which may be a key target to promote the proliferation of OPC adhesion.2.After siRNA knockdown of ITGB4(ITGB4 siRNA2 group),the results of qPCR and Western blot showed that the expression of ITGB4 gene and protein in OPC decreased significantly(p<0.001).The number of OPC and the proportion of newborn cells were significantly decreased(p<0.001).The cell cycle of OPC was blocked in G1 phase(p<0.001),and the proliferation of OPC was significantly reduced.The above results indicate that AST can promote the proliferation of OPC by ITGB4.Part III: AST increase exosomal secretion of OPC to promote their proliferation via ITGB4In this part,DAVID was used to perform GO Cellular Component analysis,and the result showed that 20% of DEGs were significantly enriched in GO:0070062~extracellular exosome.It suggested that the exosomal secretion of OPC in the AST-OPC group was significantly enhanced.After siRNA knockdown of ITGB4 in the AST-OPC group,exosomal secretion was detected by NTA,and expressions of exosomal marker protein Alix and ITGB4 were detected by Western blot.After supplementing exosomes in the ITGB4 siRNA2 group(ITGB4 siRNA supplement with exosomes group),the proliferation capacity of OPC was detected by EdU and flow cytometry.The main results are as follows:1.GO Cellular Component analysis showed that 20% of DEGs were significantly enriched in GO:0070062~extracellular exosome.The results showed that the expression of OPC exosome genes in the AST-OPC group was significantly different.NTA was used to detect exosomal concentration in the culture supernatant,and the results showed that exosomal concentration was increased significantly in the AST-OPC group(p<0.0001).2.After siRNA knockdown of ITGB4,NTA showed that exosomal concentration in culture supernatant was significantly reduced(p<0.0001).Western blot analysis of exosomal marker protein Alix and ITGB4 showed a significant decrease(p<0.05).3.After supplement with PKH26 labeled exosomes in the AST-OPC group,the results showed that exosomes could be absorbed by cells.NTA verified that the exosomal concentration in the culture supernatant was increase(p<0.0001).Western blot was used to detect exosomal marker protein Alix and ITGB4,and the results showed that the expression of bothproteins in culture supernatant was increased(p<0.05).4.After supplement with exosomes in the ITGB4 group(ITGB4siRNA2+ group),EdU showed a significant increase in the proportion of newborn cells(p<0.01).An increase proportion of OPC in S phase was detected by flow cytometry(p<0.01).The above results indicate that AST can promote the exosomal secretion and the proliferation of OPC through ITGB4.