Poly-L-ornithine Reverses The Inhibitory Effect Of Fibronectin On Oligodendrocyte Differentiation

Background:Oligodendrocytes are responsible for myelination in the central nervous system(CNS).Myelin sheath is the multi-layered membrane wrapping around the axon.It helps in efficient electrical signal conduction and provides metabolic support for axons.In demyelinating diseases,myslin is damaged,even lost from axons,which ultimately results in neuronal death.At present,there are no effective treatments for demyelinating diseases.A large number of studies have found that the extracellular microenvironment in demyelinating diseases has changed to be nonpermissive for the regeneration of myelin sheath.One of those microenvironment changes is the accumulation of fibronectin.Fibronectin affect oligodendrocytes development,however,its effects on myelin regeneration are still controversial.We decided to work out the function of fibronectin during the development and regeneration of oligodendrocytes,and looking for novel ways to promote myelin regeneration.Methods:(1)Primary culture of OPCs.The OPCs were purified twice for enrichment.(2)Immunoflourescence technique was used to detect the expression levels of NG2,Olig2 and Ki67 in OPCs proliferation phase.Then the cell purity,cell number and proliferation rate were analyzed.During the differential stages,the expression levels of CNP and MBP were also measured by immunoflourescence technique and analyzed afterward.(3)In differentiation stages,cell morphology analysis(including fractal dimension,ring-like structure,network structure,membrane structure,essence myelin sheath membranous structure)were used to evaluate differentiation degrees of OLs.Quantitative detection of the corrected total fluorescence(CTCF)and the mean fluorescence intensity(MFI),and the area of myelin membranes were also measured for determining differentiation degrees.(4)Real-time PCR was used to detect the mRNA expression levels of CNP and MBP in different stages,as well as the expression of different integrin subunits.(5)RNA-seq was used to detect the expression levels of different integrin subunits during early and late differentiation.(6)Western blot technique was used to measure the protein expression levels of CyclinD1,Cleaved caspase3,Erk1/2,Akt,CNP and MBP.Results:(1)In the proliferation stages,FN increases the number of total cells and OPCs,as well as the ratio of Ki67+ cells.FN also promotes the protein levels of CyclinDl,p-Akt and p-S6K,but shows no effects on the protein levels of Cleaved-caspase3.(2)In the differentiation stages,OLs with FN treatment showed less branches in cell processes,less ring-like structures,less membranous structure,lower levels of CNP,MBP proteins and mRNAs.FN also reduced the MBP immunofluorescence intensity and total area of the myelin membrane.FN reduced the protein levels of p-Erk 1/2 in early differentiation stages,and p-Akt,p-CREB protein expression in late differentiation stages.(3)In the proliferation stages,PLO did not affect the number of OPCs and the ratio of Ki67+ cells.Similarly,the protein expression levels of CyclinDl,Cleaved-caspase3,p-Akt and p-S6K were not affected.(4)PLO promotes OL differentiation.After PLO treatment,the cell branch processes,ring-like structures and membranous structures increased significantly,as well as expression levels of CNP,MBP proteins and mRNAs.PLO also increased the MBP immuofluorescence intensity and area of the myelin membrane.PLO increased p-Erk 1/2 protein expression during early differentiation,and p-Akt,p-CREB protein expression in late differentiation.(5)The combination of PLO and FN promoted the proliferation of OPCs,similar to the effect of FN alone.(6)The combination of PLO and FN promotes OLs differentiation,similar to the effect of PLO alone,showing that PLO can reverse the negative effect of FN on OPC differentiation.(7)RNA-seq and q-PCR were used to determine the expression levels of integrins during different stages of oligodendrocyte development.Conclusions:(1)FN promotes the proliferation of OPCs.The possible mechanism is that FN can activate the PI3K/Akt/S6K signaling pathway,which leads to cyclin D1 activation and cell proliferation.(2)FN has inhibitory effects on OL differentiation,via Erk1/2 signaling pathway in the early stages of differentiation,and PI3K/CREB signaling pathway in the mid-late differentiation stages.FN can affect different signaling pathways at different stages,which may be caused by the dynamic expression of integrin subunits during oligodendrocyte differentiation.(3)PLO has little effect on OPCs proliferation.The combination of PLO with FN shows the same enhancement on cell proliferation as FN alone.(4)PLO promotes OLs differentiation.When combined with FN,PLO can reverse the inhibitory effects of FN on OLs differentiation.The mechanism is that PLO can reverse the inhibitory effects of FN on Erkl/2(early differentiation)and AKT/CREB(mid-late differentiation)pathways.(5)PLO may be used as a potential therapeutic agent to reverse the myelin regeneration disorder caused by local accumulation of FN in demyelinating diseases.