Heterogeneity Of NG2-glia In The CPZ-induced Demyelination And Remyelination Process

Objective: Oligodendrocyte precursor cells(OPCs),also known as NG2-glia due to their expression of proteoglycan(NG2),are the fourth glial cell in the central nervous system(CNS)distinct from astrocytes,oligodendrocytes(OLs)and microglia.They are evenly distributed in all areas of the adult central nervous system.Recently it is shown by lots of researches that NG2-glia is a highly heterogeneous cell population that not only lies in the three developmental sources and region heterogeneity,but also shows heterogeneity even in the same region.Currently,it is considered that the main function of NG2-glia is to differentiate into OLs under the physiological and pathological conditions,so as to promote myelin formation and repair.Besides,as the largest number of nerve cells with proliferation ability in the adult brain except germinal zones,NG2-glia can respond to mechanical injury,neurodegenerative diseases and demyelination lesions through the cell morphology change,enhance of movement and migration ability and proliferation and differentiation potential changes.As a result,many new features of NG2-glia have been demonstrated,such as formation of synapses with neurons,participating in the formation of the glial scar after trauma or frostbite,devouring myelin debris and involvement in immune response.Therefore,understanding the reactivity and possible heterogeneity of NG2-glia in response to injury and pathological changes,is of great significance for us to search for new clinical therapeutic targets for neurological diseases.However,studies on the reactivity and heterogeneity of NG2-glia are still limited,and it remains to be further explored.Methods: Single-cell Transcriptome Sequencing(10X Genomics)was carried out to analyze the OPCs from three phases during the classical model of demyelination—CPZ-induced demyelination model(3 weeks after CPZ feeding/demyelination,5 weeks after CPZ feeding/ demyelination peak and 2 weeks normal feeding after 5 weeks’ CPZ feeding/remyelination).Then a variety of bioinformatics methods were used to perform differential expression analysis and Pseudo-time analysis and so on,trying to find emerging clusters with specific features.Aiming at specific subgroups’ marker molecules,the sequencing analysis results were firstly verified by comprehensive use of research methods such as cell biology,molecular biology and morphology,which defined the emergence and evolution rules of subgroups in different stages of disease model,and then the function of new subgroups will be further investigated.Result: The single cells suspension from the PDGFR-H2B-EGFP mice’s whole brains of three stages and control groups was prepared,and then GFP+ cells were sorted by using flow cytometry.The single-cell RNA sequencing analysis showed that there were four major types of cells,a total of 15 subgroups,including oligodendrocyte lineage cells(OPCs,COP/NFOL,MFOL1/2,MOL),microglia-like cells(cluster 4,cluster 14)and also pericytes or vascular and leptomeningeal cells(VLMC)(cluster 9,cluster 11).Through the combined analysis of the experimental groups with the control group,we found that under the toxic stimulation of CPZ,there were several reactive new subgroups(cluster 1,3,5,7,13).Among them,cluster 13 highly expressed molecules involved in interferon response and antigen presentation,which might be a cluster of reactive OPC related to immune response;although cluster 1,cluster 3,cluster 7 are different in classification,the functions of specific upregulation molecules of each cluster are mostly related to vesicle secretion and membrane raft assembly.And the immunofluorescence staining and in situ hybridization of their specific markers were then performed and the existence of these reactive new clusters in the CPZ-induced demyelination model was verified.