Optogenetic Regulation Of SOCE Activates NFATcl And NFATc3 In Regulating The Differentiation Of Neural Progenitor Cells

Neural progenitor cell?NPCs?are the special unipotent stem cells in the Central Nervous System?CNS?,which possess the abilities of self-renewal and differentiation into astrocyte?Ast?,oligodendrocyte?OL?and neuron?Neo?.NPCs serve as not only the neurogenesis cells in the early developmental stage of mammal CNS,but also the storage cells in the mature CNS,of which a small amount were stored in the specific regions of adult brain.When irreversible cell death and loss occur after CNS is damaged or irritated by diseases,NPCs are reactivated and involved in the damage repair process.Therefore,NPCs transplantation therapy has been applicated in clinic for the treatment of CNS injuries.However,under natural differentiation conditions,approximately 75%of NPCs will differentiate into Ast while only about 15%of NPCs differentiates to Neo,and about 10%of NPCs differentiate into OL.The excessive Ast will form glial scar to interfere the repair of damaged regions.Additionally,the low differentiation ratio of Neo and OL can also severely limit the recovery of neural function.Thus,how to regulate the directional differentiation of NPCs into Neo or OL still have great significances in the field of NPCs research.It is found that the differentiation regulation of NPCs is a very complex signal transduction network,which consist of external and internal signal pathway.However,the expression level of NPCs internal transcription factor was found to play a dominant role in the directional differentiation of NPCs.And overexpression of three important transcription factors of Mash1/Olig2/Hes1 in NPCs can directly promote the directional differentiation of NPCs into Neo/OL/Ast.It suggests that the directional differentiation of NPCs can be achieved via regulating the expression level of transcription factors.The nuclear factor of activated T cells?NFAT?is a large family of transcription factors,including five subtypes:NFATc1-c5.Current studies found that the function of NFAT transcription factor is not only limited to the immune system,but also regulates the important physiological processes in CNS,such as neurogenesis,neural cell differentiation,etc.Tranque,et al.found that after pharmacologically blocking the function of NFAT protein,the proliferation,migration and differentiation of NPCs were significantly affected;Wegner,et al.reported that down-regulation of NFAT transcription factor in OL cell lines inhibits the expression of Olig2,and inhibited differentiation initiation of OL.It indicates that the NFAT transcription factor family may be involved in the regulation of important cellular functions in NPCs,even including regulation of Olig2 expression,which lead to NPCs-OL directional differentiation.However,at present,there are very few research reports assessing NFAT signal in directional differentiation of NPCs,and this hypothesis requests further experimental evidences.Moreover,the activation and deactivation of the NFAT signal are calcium-reliant and regulable.When the cell is under resting condition,phosphorylated NFAT protein enrich in cytoplasm.After stimulation,the calcium([Ca²⁺])concentration in the cytoplasm show a rapid increase,accompanied with activation of calmodulin?CaM?.Then,CaM activate downstream CaN to dephosphorylate NFAT protein.After that,the NFAT protein enter nucleus and regulate the transcription process.It is worth noting that the maintenance of NFAT translocation requires continuous[Ca²⁺]signal stimulation.When the[Ca²⁺]level are decreased,the NFAT protein is rapidly rephosphorylated by the kinase and transported out of the nucleus.Therefore,the function of NFAT transcription factor is not only closely related to[Ca²⁺]concentration in cytoplasm,but also requires sustained high stimulation of[Ca²⁺]signal.Study found that store-operated Ca²⁺entry?SOCE?is one of the largest external calcium influx channels in non-excitable cells,and also the dominant way of intracellular[Ca²⁺]elevation.The SOCE channel is composed of two major protein families,the STIM protein on the endoplasmic reticulum and the Orai protein located on the cell membrane.Nieswandt,et al.found that when the STIM protein in mouse thymic T cells was knocked out,NFAT signal was significantly attenuated or even disappeared.It suggests that the[Ca²⁺]mediated by the SOCE channel is the main source of NFAT activation.Theoretically,by regulating the activation and deactivation of SOCE channel in NPCs,it can indirectly regulate its downstream[Ca²⁺]/CaN/NFAT pathway.Moreover,the hypothesis that regulating the SOCE/NFAT pathway to control the real-time differentiation and fate determine of NPCs is worthy of further exploration.This study utilized the latest optogenetics technology to stably expressed light-sensitive SOCE proteins in NPCs.470 nm blue light stimulation?BLS?was applicated to regulate the activation and deactivation of SOCE pathways in NPCs in real time and accurately.The intracellular[Ca²⁺]level was detected by Fluo-3 after BLS.The activation of SOCE pathway and the nuclear translocation of NFAT signal after BLS were detected by PCR and ICC.Finally,ICC was used to detect the differentiated cell type of NPCs after BLS,and the distinct regulatory function of NFAT subtypes in NPCs.This study aims to construct the in vitro cell model for the regulation of NPCs directional differentiation by optogenetic techniques,and to provide experimental evidences for highlight of NFAT channel in NPCs differentiation.This experiment is divided into four parts:Part 1.Construction and evaluation of optogenetic lentivirus plasmid:rLOVsoc and its transfection efficiency in NPCs in vitro.The original LOVsoc plasmid was presented by professor Yubin Zhou?Addgene,#101245?.The primary structure is pTriEx-mCh-LOV2-hSTIM1.We replace the hSTIM1fragment with the rat-derived rSTIM1 fragment by molecular cloning technique.And the mCh-LOV2-rSTIM1 fragment was amplified and recombined into LeGO-iT2 lentivirus vector to form the rLOVsoc plasmid,which were used in this experiment.In vitro culture of NPCs was made from the brain cells of SD neonatal rats within postnatal 3 d.ICC showed that more than 90%of the cells were strongly positive for Nestin,suggesting that the purity of NPCs was high.Then,by lentiviral packaging,the recombinant plasmid of rLOVsoc was transfected into NPCs in vitro.Statistical results displayed that the percentage of NPCs with red fluorescent signal was 92%,indicating that the transfection rate was high.Western Blot showed that after two weeks of rLOVsoc transfection into NPCs,the mCherry protein band was still positive.It suggests that the recombinant protein of mCh-LOV2-STIM1 was stably expressed in the transfected cells,which laid the foundation for further experiment.Part 2.The effects of different light intensity on the function regulation of NPCs and the optimized light-induced differentiation mode of NPCs in VitroFirstly,vector plasmid LeGO-iT2 was packaged as lentivirus to transfect NPCs.After BLS 0-60 min,there was no significant increase in the percentage of trypan blue-positive cells and no decrease in Nestin fluorescence intensity among groups,suggesting that BLS stimulation alone would not affect the function of NPCs.Then photogenetic rLOVsoc lentivirus was used to transfect NPCs.After BLS 0-60 min,the percentage of trypan-blue-positive cells increased significantly after BLS 30 min.The positive fluorescence intensity of Nestin decreased gradually with BLS time prolonged,especially at BLS 30 min,there were almost no Nestin positive cells in the fields.It suggests that after BLS 30 min,the differentiation of all NPCs have initiated within 3 d.In combination with the results of trypan blue,the BLS 30 min will be used as the appropriate stimulation model,which can not only effectively induce the differentiation initiation of NPCs,but also avoid inducing cell damage and even death.Part 3.After BLS,the activation of intracellular[Ca²⁺]oscillation induces NFATc1 and NFATc3 nuclear translocation,which participates in the directional differentiation of NPCs into OL.First,we used[Ca²⁺]imaging technology to detect Fluo-3 marked intracellular[Ca²⁺]levels in resting and dynamic conditions.The results showed that after BLS 30 min stimulation,intracellular[Ca²⁺]increased locally near the aggregation of recombinant protein,while BLS 60 min stimulated the extreme increase of[Ca²⁺]fluorescence signal in whole cytoplasm.The[Ca²⁺]curve showed that BLS 30 min induced high and low"Calcium oscillation"signal waveforms,and BLS 60 min induced"Calcium overload"damage waveforms after rapid rise.PCR results showed that after BLS 30 min,the STIM1,Orai1,and CaN mRNA bands were significantly increased,along with the increase of downstream NFATc1,NFATc2,NFATc3 mRNA bands.It suggests that BLS activated SOCE pathway and also CaN protein to regulate the expression of NFATc1-3.Furthermore,ICC was used to verify whether the NFAT1-3 subtypes were involved in the nuclear translocation process.Results showed that after BLS stimulation,the NFATc1 and NFATc3fluorescence signal increased mainly in the nucleus region,while the NFATc2 fluorescence signal increased in the cytoplasm,not in the nucleus.It suggested that only NFATc1 and NFATc3 protein were translocated into nuclear after BLS.The cell types of NPCs differentiation showed that the percentage of GFAP positive cells in NPCs differentiated cells decreased significantly,and the percentage of OLIG positive cells increased significantly after BLS.In the meantime,this phenotype was accompanied by a decrease in Hes1 mRNA expression and an increase in Olig2 mRNA expression.It indicates that NFAT nuclear translocation induce the directional differentiation of NPCs into OL by regulating the expression of Olig2.However,this phenotype could be reversed by VIVIT application?specific blocker of CaN/NFAT pathway?Part 4.NFATc1 and NFATc3 exert distinct regulatory functions in NPCs through[Ca²⁺]/CaN/NFAT pathwayBy means of Crispr-cas9 technique,we constructed NFATc1 and NFATc3 knock-down?KD?plasmids.The results of Western Blot showed that they both effectively interfere the expression of NFAT protein.When the NFATc1 KD plasmid was packaged as lentivirus to transfect NPCs.TUNEL results showed no significant increase in percentage of apoptotic cells in the KD,compared with the control group.However,Nestin ICC results showed that there were still plenty Nestin-positive neural sphere in the KD group at 3d differentiation.It suggests that NFATc1 knockdown inhibits the differentiation initiation of NPCs.ICC detection showed a significant increase in the proportion of TUJ1 positive cells in the KD group,while the ratio of GFAP and Olig positive cells were significantly down regulated.It indicates that NFATc1 plays an important role in the differentiation of NPCs into glial cells.When the NFATc3 knock-down plasmid was packaged as lentivirus to transfect NPCs,results showed that the percentage of TUNEL positive cells in KD group was significantly higher than that in the control group,suggesting that NFATc3 KD induced apoptosis.However,the results of Nestin ICC showed that there were no obvious Nestin positive cells in the KD group after BLS 30min stimulation,which was similar to the control group.Furthermore,staining of differentiated cells showed that the percentage of TUJ1 positive cells was significantly decreased and almost invisible in the fields,which proved that NFATc3 played an important role in the differentiation of NPCs into Neo.When both NFATc1 and NFATc3 were knocked down at the same time,all NPCs died within 24 h.Part 5.Experiments of[Ca²⁺]signal in regulating the injury and differentiation of OLsOxygen and glucose deprivation?ODD?experiments were used to induce hypoxia injuries in cultured OLs in vitro,which increased the concentration of extracellular adenosine and thereby stimulated the excessive activation of[Ca²⁺]channel IP3R2 to verify the function of[Ca²⁺]signal in regulating the injury and differentiation of OLs.Results showed that the expression of IP3R2 protein was significantly increased after OGD stimulation,accompanied with the enhancement of adenosine 1 adenosine receptor?A1AR?fluorescence signal,the attenuation of transcription factor Olig2 fluorescence signal and the elevation of intracellular[Ca²⁺]resting level.However,this phenotype can be blocked by Caffeine?AR non-selective blocker?application.Subsequently,a high concentration of adenosine was used to induce the intracellular[Ca²⁺]acute response.[Ca²⁺]curve exhibited[Ca²⁺]overload signal.After Caffeine application,the fluctuation of[Ca²⁺]curve was stably maintained near baseline,which was similar to the control group.Further exploration of OL's differentiation showed that OGD stimulation significantly inhibited the intensity of CNPase fluorescence signal in OLs precursor cells and the fluorescence signal intensity of MBP in mature OLs.These results suggest that OGD-induced[Ca²⁺]overload mediates the occurrence of OLs injury and OLs differentiation.However,by pharmacological blockade of[Ca²⁺]overload signal to maintain[Ca²⁺]homeostasis can reverse OGD damage in OLs and protect OL's differentiation and maturation.Conclusion:In this study,we constructed recombinant rLOVsoc plasmid and transfected NPCs,to establish the cell model of real-time regulation of NPCs-OL directional differentiation in vitro,and illustrated the mechanism:After stimulation,rLOVsoc plasmid induced“Calcium oscillation”signal,activated CaN in the downstream,promoted nuclear translocation of NFATc1 and NFATc3 proteins,and finally up-regulated the expression of transcription factor Olig2 to regulate the differentiation of NPCs into OL.This was also the first study to provide experimental evidences for the highlight of NFAT signal in NPCs:NFATc1 participates in the differentiation initiation of NPCs and regulates the differentiation of NPCs into glial cells,while NFATc3 inhibits apoptosis in NPCs and regulates the differentiation of NPCs into Neo.When both NFATc1 and NFATc3 proteins were inhibited,the survival of NPCs could not be maintained.In addition,experiments of[Ca²⁺]signal in regulating OL's injury and differentiation further proved that[Ca²⁺]signal plays an important role in NPCs-OLs directional differentiation and also the early differentiation of OLs.