Astrocytic Cx43Mediated O/A Coupling Affects The Differentiation Of Oligodendrocyte Progenitor Cells

In the central nervous system （CNS）, oligodendrocytes （OLs） are responsible formyelin production which is important for normal activity of neurons. OLs are generated bythe proliferation and sequential differentiation of migratory bipolar progenitors known asO-2A cells or oligodendroglia progenitor cells （OPCs） with expression of PDGFαR andNG2. These cells then become multipolar with expression of CNPase and O4and finallyexpress myelin genes such as myelin basic protein （MBP） and proteolipid protein （PLP）. Tounderstand the mechanism of OPC differentiation and myelination is an interesting anddifficult issue in neurosciences. One approach is to examine communications amongdifferent types of glial cells.A typical feature of glial cells is their high expression levels of connexins, themolecular constituents of gap junction channels or hemichannels. Increasing evidencesindicate that connexin-mediated communication between astrocytes （ASTs） and ASTs-OLsmay be important for the development of OPC, including proliferation, differentiation andmyelination. However, a clear demonstration of this process is not yet available.Different from neuronal transmission, signaling is transmited in glia network via Ca²⁺ wave. It has been considered that the whole process of OPC development is directly orindirectly regulated by the changes of the intracellular free Ca²⁺ concentration. Previousstudies were mainly focused on voltage gateded Ca²⁺ channels and G protein coupledmetabotropic receptors which were located on OLs membrane, but little was known aboutthe calcium channels on intracellular organelles such as endoplasmic reticulum （ER）.Among the recptors of the ER membrane, ryanodine receptor family （RyR） is a majorreceptor which could trigger the release of Ca²⁺ into the cytoplasm. The expression of RyRin OLs, however, is poorly investigated and is still debatable. In this study, by applyingprimary OPC cultures, we firstly set up OPC-AST co-culture and detected thedifferentiation of OPCs after Cx43had been down-regulated by Cx43siRNA, then we investigated the effect of RyR Ca²⁺ signaling on differentiation of OPCs by comparing thechanges of the OLs stage-special makers in OPCs with or without the treatment of RyRblocker.The investigation is composed of two parts:Part1. Effect of astrocytic Cx43on differentiation of OPCIn this part, we firstly set up OPC-AST co-culture system, and then we constructedCx43siRNA plasmid and identified its effectiveness. Finally, ASTs transfected withCx43siRNA were cultured with OPCs to observe the effect of Cx43on differentiation ofOPCs by immunocytochemistry and Western blot analysis.The results were as follows:1. In OPC-AST co-culture, ASTs could promote proliferation but inhibitdifferentiation of OPCs as compared with OPC culture alone, and the effect depended oncell-cell contact.2. The siRNA vector targeting rat Cx43was successfully constructed, and theexpression of Cx43was reduced about70%in ASTs transfected with Cx43siRNAcompared with that of control.3. OPCs planted on ASTs transfected with Cx43siRNA expressed fewer MBP ascompared with ASTs transfected with vector（P<0.05）, while there was no significantdifference of NG2and CNPase expression between these two groups.Based on the above experimental results, we speculate that that astrocytic Cx43mayaffect the differentiation, especially the later stage of differentiation of OPC.Part2.Calcium releasing from RyR channel affect differentiation of OPCsRT-PCR was used to determine the distribution of RyR channels in OLs, and Ca²⁺ imaging was utilized to detect the different patterns of Ca²⁺ signaling during thedevelopment phase of OPCs. After administration of RyR blocker in primary OPC culture,immunocytochemistry and Western blot analysis were applied to observe expression ofstage-special protein of OLs. Finally caffeine was used to further certify the effect of RyRchannel Ca²⁺ signal on differentiation of OPCs.The results were as follows:1. All RyR isoform mRNA had been detected in the brain tissue of rat brain while OLsonly expressed RyR3isoform mRNA. The expression of RyR3was decreased during the differentiation of OPCs.2. Intercellular Ca²⁺ responses to caffeine stimulation were recorded in three typicalcharacteristic waves during OPCs development. Ca²⁺ transient responses of each stage ofOLs were restricted exclusively to identify with the morphological feature that had beenassociated with the immunocytochemical marker of the OLs phenotype （ie. PDGFαR, NG2,CNPase and MBP）.3. Ryanodine （high concentration） treament resulted in inhibition of OPCsdifferentiation while the effect could be attenuated by applying caffeine.These results suggest that Ca²⁺ releasing from RyR channel is quite important for thedifferentation of OPC.