Proliferation Of Oligodendrocyte Precursor Cells Promoted By Astrocytes Through Connexion CX47/Ca²⁺/PERK1/2 Pathway

AS the aging problem of the society has been intensified, the incidence of neurodegenerative diseases such as Alzheimer’s disease,dementia and other neurological degenerative disease, is increasing gradually and has a bad effect on the health in old patients and their family life quality. Demyelination is the typical pathological character of neurodegenerative diseases,the remyelination efficiency decreases as age increases. If the myelin resistance and regeneration were increased,it may provide a new way to treat neurodegenerative diseases and protect neurons.The study about occurrence and regulation of oligodendrocytes was the key points of myelination and remyelination research.Both proliferation of oligodendrocytes precursor cells(Oligodendrocyte precursor cell,OPC)and their migration to the damaged area and axonal maturation are the critical point during the period of remyelination. Astrocytes(Astrocyte,AST), the most abundant and widely spreaded cells in the central nervous system,play an important role in regulating the proliferation and differentiation of oligodendrocyte precursor cells.As studies have shown that astrocyte canprovide a good environment beneficial for the proliferation,migration,differentiation of oligodendrocyte precursor cells, having an irreplaceable role in promoting remyelination. In this study, the vitro co-culture model of astrocytes and oligodendrocytes precursor cells was established and the transcriptome sequencing RNA sequencing,RNA-Seq)analysis was used to explain the genomic alterations of OPC under different culture statuses in order to find the key point and their corresponding metabolism for promoting oligodendrocyte precursor cell’s proliferation, and molecular biology experiments were also used to verify the assumed metabolic pathways, providing a theoretical basis for the treatment of neurodegenerative diseases.Part One1.Materials and methods1.1 SD rats(1-3d), clean born, provided by Chongqing medical university experimental animal center, was used to culture oligodendrocytes precursor cells and astrocytes in vitro. And the cells were labeled by primary antibody anti-GFAP(Glial fibrillary acidic protein,GFAP) and anti-PDGFɑR( Platelet-derived growth factor-ɑreceptor,PDGFɑR) separately in order to detect their biological characteristics and purity.1.2 The cells were divided into three groups: the ordinary cultured oligodendrocytes precursor cells, the oligodendrocytes co-cultured withastrocyte in transwell plate, the oligodendrocytes precursor cells co-cultured with astrocytes through direclyt contacting. The OPC growth condition in different periods was observed by microscope and photographed 5 times in randomly selected areas.1.3 Apollo staining and DNA staining of oligodendrocyte precursor cells under three group were carried out by EDU(5-Ethynyl-2’-deoxyuridine,EDU) experiments. The OPC image was detected and obtaind by fluorescence microscopy. The newly borned OPCs was counted by Image J software in order to determine their proliferation ability.1.4 Cell cycle distribution of oligodendrocyte precursor cells purified under three different groups, were detected by flow cytometry.1.5 oligodendrocyte precursor cells purified under three different groups were sent to BGI sequencing company for transcriptome sequencing and interpreting differentially expressed genes and their related metabolic pathways associated with the proliferation.1.6 Differentially expressed genes in directly contacting co-culture group were selected randomly for RT-PCR( Reverse transcription-polymerse chain reaction,RT-PCR) in order to validate sequencing results2 Results2.1 The oligodendrocyte precursor cells were labeled by anti-PDGFɑRand astrocytes were labeled by anti-GFAP,both the kind positive cells were up to more than 95% of the total cells. Oligodendrocyte precursor cells were small, oval, having 2-3 neurites, long and thin; Both astrocytes and their nucleus were big,AST, having abundant cytoplasm, coarse neurites and more branches.2.2 Results from microscopy indicate that astrocytes can promote the proliferation of OPC through two methods: secreting cytokines and gap junctional intercellular communication. The number of OPCs increase in the first 2-3 days and the proliferation effect of co-culture group through directly contacting were most obvious(P <0.01).2.3 Results of EDU staining showed that the number of newborn oligodendrocytes precursor cells of the co-culture group were more than cultured alone, and having significant differences, and the most obvious proliferative effect were in directly contacting co-culture group,(P <0.01).2.4 Flow cytometer analysis(Flow cytometer,FCM) showed that more oligodendrocytes precursor cells were placed in S phase after co-cultured with astrocytes,the percentage of S phrase of OPCs of directly contacting co-culture group was more than the other two. indicating that astrocytes can promote proliferation of OPC through DNA replication after co-culturing(P <0.05).2.5 transcriptome sequencing analysis showed that the expression of connexin(Connexin,CX)CX47 in directly contacting co-culture group wassignificantly up-regulated and most differentially expressed genes after co-culturing were associated with cell proliferation(P <0.01).2.6 The RNA expression times of different groups carried out by RT-PCR were in accord with RNA transcriptome sequencing, indicating that the RNA transcriptome sequencing results were accurate.Part Two1.Materials and methods1.1 SD rats(1-3d), clean born, provided by Chongqing medical university experimental animal center, was used to culture oligodendrocytes precursor cells and astrocytes in vitro. The experiments has four groups: directly contacting co-culture group、Blank interference of connexin group、CX47 interference group、Erk1/2(Extracellular regulated protein kinases,ERK1/2) blocked group. specific CX47 interfering RNA were selected by immunofluorescence(immunofluorescence,IF),RT-PCR,WB(Western Blot,WB), ERK1 / 2blocking effect was detected by WB.1.2 oligodendrocyte precursor cells in the four group were stained by Fluo-3. relative content of calcium ions of oligodendrocyte precursor cells were measured by flow cytometry and confocal analysis.1.3 ERK1 / 2 phosphorylation level of four group cells analylized by Western-blot.1.4 Apollo staining and DNA staining of oligodendrocyte precursorcells in four groups were carried out by EDU experiments. The OPC image was detected and obtaind by fluorescence microscopy. The newly borned OPCs was counted by Image J software in order to determine their proliferation ability.1.5 Absorbance of OPCs were measured by CCK 8 experiments in order to compare the cell viability of four groups.1.5 Cell cycle distribution of oligodendrocyte precursor cells purified under four different groups, were detected by flow cytometry.1.6 The expression of Cyclin A, Cyclin D1, Cyclin E were abtained by Western-blot analysis.2 Results2.1CX47 specifical interfering RNA sequences were CCGAGAAGACTGTCTTCTT; phosphorylation level of ERK1 / 2 in ERK1 / 2 blocked group decreased significantly than other groups(P<0.05).2.2 Both confocal analysis and flow cytometry showed that the indrawal calcium decreased significantly while CX47 was interferenced(P<0.05).2.3 Western-blot results showed that ERK1 / 2 phosphorylation in CX47 interference group was decreased(P <0.01).2.3 EDU experiments show that the number of newborn cells were decreased and CCK8 experiments show that the cell viability weredecreased while CX47 was interferenced and ERK1/2 was inhibited by U0126,indicating the reduced proliferative ability.(P <0.05)2.4 Flow analysis showed that cell cycle of OPC was arrested in G1 phase and S phase was significantly shortened while CX47 was interferenced and ERK1/2 was inhibited by U0126(P <0.05).2.5 Western-blot results showed that the expression of Cyclin D1,Cyclin E were decreased, the expression of Cyclin A was increased,while CX47 was interferenced and ERK1/2 was inhibited.(P <0.01).Conclusion1. astrocyte can promote the proliferation of OPC primarily in two ways: the secretion of cytokines, connection(Gap junction, GJ)communication, but the latter is more significant.2. In this study, molecular biology experiments and transcriptome sequencing was combined together in order to comprehensively analyze the co-culture state of astrocytes and oligodendrocytes precursor cells.In the analysis,CX47 was up-regulated and was the key target in promoting the proliferation of OPC.3. CX47 can promote the proliferation of oligodendrocyte precursors by regulating calcium signaling, which affects ERK1/2 phosphorylation.While CX47 was silienced by RNA interference, calcium influx was decreased, expression of phosphorylated ERK1/2 was decreased,the proliferation of OPCs were decreased.4. CX47 can promote the proliferation of oligodendrocyte precursors by regulating calcium signaling, and then increasing the phosphorylation level of ERK1/2,which affects cell cycle-related proteins.After specific RNA interference gene silencing CX47 and ERK1 / 2 was blocked by U0126, expression of phosphorylated ERK1/2 was decreased,expression of protein Cyclin A in S phase was declined, expression of proteins Cyclin D1, Cyclin E in G1 phase was increased,the proliferation of OPCs were decreased.