MiR-124 And Small Molecules Synergistically Regulate The Conversion Of Neuronal Cells From Rat Reactive Astrocytes

Irreversible neuron loss caused by central nervous system(CNS)injuries usually leads to persistent neurological dysfunction.The development of the technology of reprogramming somatic cells into neurons provides new hope for solving this difficulty in neuronal regeneration and repair.Reactive astrocytes(RAs),because of their high proliferative ability,proximity to neural lineage and involved in the formation of glial scars,are ideal starting cells for neuronal regeneration.miR-124,which plays an important regulatory role in the neuronal development and differentiation,is much lower in RAs than that of neurons.If the expression of miR-124 in RAs is increased,it is likely to regulate RAs reprogramming into neurons.Additionally,small molecules that target cell signals also provide a powerful mediation pathway for reprogramming somatic cells into neurons.Although several small molecules that can reprogram astrocytes(As)into mainly glutamatergic or GABAergic neurons have been identified in previous studies,in view of the different neuronal subtypes involved in CNS injuries or diseases,whether other signals can be regulated by small molecules to mediate the reprogramming of RAs into other subtype-specific neurons is not yet known.In this study,we conducted the following experiments:1.Isolated primary As from neonatal rat cortex and induced them into RAs with TGF-β1 stimulation in vitro.The RA markers GFAP and S100 were detected by immunostaining to determine the purity of RAs.2.Transfected the purified RAs with a miR-124 mimic to create RAs overexpressing miR-124.q RT-PCR analysis was used to determine the highest transfection efficiency.q RT-PCR,Western blot and immunostaining were used to detect the expression changes of GFAP and S100 after miR-124 overexpression in RAs.3.RAs overexpressing miR-124 were treated with ruxolitinib,SB203580 and forskolin to induce the conversion of RA-to-neuron.Immunostaining was used to detect the neuronal markers and subtypes of induced neuronal cells(iNs).4.The proliferation of iNs was detected by Ed U assay,and the apoptosis of iNs was detected by immunostaining and Annexin V assay.5.The conversion efficiency of RAs treated with different sets of miR-124,ruxolitinib,SB203580 and forskolin was evaluated by immunostaining,and the best induction scheme was determined.6.To examine the differences between RAs,iNs and rat neurons at the genetic level,RNA-seq analyses and q RT-PCR validation were performed on these cells,and GO and KEGG functional enrichment analyses were performed for the differentially expressed genes of iNs and RAs.7.The molecular mechanism of miR-124 regulating the differentiation of RA-to-neuron was explored.Western blot was used to detect the inhibitory effects of miR-124 on SOX9 and NFIA,characteristic proteins of RAs,and the expression of NOTCH downstream effector HES1 related to neuronal differentiation.Subsequently,the relationship between Sox9,Nfia and Hes1 in RAs was detected by q RT-PCR,Western blot and Co-IP assay after RAs were transfected with si-Sox9,si-NFIA or si-Hes1.The results were as follows: 1.The purified and cultured cells were mainly rat RAs,and more than 95% of the cells expressed RA markers GFAP and S100.2.The highest transfection efficiency was with 50 n M miR-124 for 48 h.The overexpression of miR-124 significantly downregulated the expression of RA markers GFAP and S100,and inhibited the reactive characteristics of RAs.3.iNs induced for 7 days showed a typical neuron-like morphology and expressed neuronal markers TUJ1(64.4%),DCX(41.5%),MAP2(38.7%)and NEUN(87.3%);iNs induced for 21 days expressed the neurosynaptic junction marker SYN1(0.7%),and the iNs were predominantly cholinergic neurons(25.9%)and glutamatergic neurons(22.3%).4.Only a few iNs proliferated during the RA-to-neuron conversion process,indicating that most iNs might be directly transdifferentiated from RAs without passing through the stage of neural progenitor cells(NPCs).5.The synergistic effects of miR-124 combined with ruxolitinib,SB203580 and forskolin best promoted the RA-to-neuron conversion.6.The iNs’ gene expression profile was closer to that of rat neurons than initial RAs,and the upregulated genes in iNs were significantly enriched in neuron development,neuron differentiation and axonogenesis,while the downregulated genes were significantly enriched in astrogliosis and scar formation properties of RAs.7.In RAs,miR-124 downregulated the expression of HES1 by targeting the SOX9-NFIA-HES1 axis,thereby promoting the RA-to-neuron differentiation.In conclusion,we present a novel approach to inducing rat RAs into mainly cholinergic and glutamatergic neurons through co-regulation involving miR-124,ruxolitinib,SB203580 and forskolin in vitro,which provides new clues for the research of inhibiting glial scars formation and promoting neuronal regeneration repairing after CNS damage.