Mitochondrial Transfer From Astrocytes Protected Dopaminergic Neurons In PD Model

Part Ⅰ The establishment of hiPSCs and the differentiation of dopaminergic neurons and astrocytesObjectives:Induced pluripotent stem cells(iPSCs)can differentiate into all kinds of cells,including dopaminergic neurons(DA neurons)and astrocytes.Thus iPSCs offer an effective tool for modeling Parkinson’s diseases.In this study,we employed human peripheral blood mononuclear cells(PBMCs)for reprogramming into hiPSCs.We introduced four specific genes encoding transcription factors by Sendai virus and then generated hiPSCs.Next,we applied hiPSCs differentiated DA neurons and astrocytes to model Parkinson’s disease and study the protected function of astrocytes in PD.Methods:Plated the PBMCs in 5x10^5/well density,and then introduced four specific genes encoding transcription factors(Oct3/4,Sox2,c-Myc and Klf4)by CytoTune iPS 2.0 Sendai Reprogramming Kit;Changed medium every day until small colonies appeared and grown large enough for passage.After around one week,we cut the colonies into small pieces and re-plated upon MEF;Changed medium every day;We chose the colonies with smooth edge and followed the previous and modified paradigm to derived DA neurons and astrocytes from hiPSCs.Results:(1)About 3 days after transfection with Sendai virus and replating PBMCs,the colonies appeared above the fibroblast feeder and grown up;(2)the ALP stain was employed after the iPSCs were passaged to four or five generation.(3)the colonies were stained for the specific stem cells’ marker TRA1-60,OCT3,Nanog,SOX2,SSEA4 by immunochemistry stain;(4)Flow cytometry was used to detect specific marker SSEA4 and TRA1-81,and the data showed a positive result in generated colonies;(5)Immunostaining were employed to identify dopaminergic neurons.The neuronal marker Neuron-specific Class III p-tubulin(TuJ1)and dopaminergic neuron marker Tyrosine hydroxylase(TH)were detected in differentiated dopaminergic neurons.(6)Astrocyte-specific marker Glial fibrillary acidic protein(GFAP)and S100β were examed by immunostaining,and the positive staining existed in our generated astrocytes.Conclusion:The introduction of the four transcription factors Oct3/4,Sox2,c-Myc,and Klf4 can reprogram the PBMCs into induced pluripotent stem cells effectively,and the iPSCs expressed the specific markers of embryonic stem cells.The iPSCs can differentia into dopaminergic neurons and astrocytes by exposing to a set of growth factors and small molecular sequently.The generated dopaminergic neurons and astrocytes expressed their own specific markers.Part II:Mitochondrial transferred from astrocytes to DA neurons in PD model rescue the aerobic respirationObjectives:Mitochondria are the energy center of cells,and the maturity and stability of mitochondrial is necessary for maintaining the normal physiological state of cells.Mitochondrial dysfunction is one of the pathogenesis of Parkinson’s disease.Astrocytes are the most abundant glial cell type in the central nervous system and are considered to serve as the supportive component in the brain.Astrocytes have been reported to effectively rescue mitochondrial dysfunction in the differentiation of iPSCs-derived dopaminergic neurons in the previous study.In this study,we employed iPSCs-derived dopaminergic neurons to mimic Parkinson’s disease and establish neuron-astrocyte co-culture system,which allows for the illumination of the interaction between DA neurons and astrocytes,as well as the underlying mechanism.Methods:we applied neurotoxin rotenone to established PD model in iPSCs-derived dopaminergic neurons.Astrocytes were plated above dopaminergic neurons at a 1:1 ratio.Immunostaining was used to measure the number and the dendrite length of TH positive neurons in the belonging two groups:(1)rotenone-treated neurons;(2)neurons co-cultured with astrocytes;Astrocytes were labeled with Mito-Tracker(green)before co-cultured with dopaminergic neurons.Immunostaining was used to detect mitochondrial transfer.Astrocytes were labeled with Mito-Tracker(green)and dopaminergic neurons were labeled with celltrace(red),flow cytometry was employed to test and distinguish the green staining,red staining,and double staining population;Cell Counting Kit-8 was used to measure the viability of dopaminergic neurons;Celltiter kit was employed to determine the intracellular ATP of dopaminergic neurons;Plating astrocytes on cell culture insert,and then put these inserts onto 12-well plate seeded with dopaminergic neurons.After 24 hours of co-culture,we tested the neurons’ viabilities and intracellular ATP by Cell Counting Kit-8 and Celltiter kit.Result:(1)Immunostaining showed that the dendrite length of TH positive neurons in rotenone treated group was shorter than that in co-culture group.(2)Laser scanning confocal microscopy observed the labelled mitochondrion from astrocytes existed in dopaminergic neurons,indicated that the mitochondrial transfer from astrocytes to dopaminergic neurons;(3)Flow cytometry confirmed the mitochondrial transfer between astrocytes and dopaminergic neurons by separated the green staining,red staining and double staining populations;(4)Cell counting kit-8 showed that the cell viability decreased when treated with rotenone,while the viability increased when the neurons co-cultured with astrocytes.(5)Celltiter kit indicated that under the rotenone challenge,the ATP within dopaminergic neurons descended sharply.On the contrary,the ATP of neurons increased after co-cultured with astrocytes.(6)Cell culture inserts with seeded astrocytes were put into 12-well plate with neurons,cell counting kit-8 suggested that this co-culture system don’t rescue the cell viability decreased by rotenone.(7)Cell titer kit measured the intracellular ATP after rotenone and co-culture in the system described above.Data indicated that co-culture with astrocytes plated in cell culture insert cannot increase intracellular ATP of neurons treated with rotenone.Conclusion:co-culture with astrocytes increased the cell viability and intercellular ATP of dopaminergic neurons in PD model,indicated that astrocytes protect dopaminergic neurons in PD model.Health mitochondrion can transfer to dopaminergic neurons to rescue the damages respiratory function.When the co-culture system separated astrocytes form neurons by cell culture inserts,the neuroprotective effect disappeared.Part III:rotenone promote the neuronal uptake of mitochondria by activation of p38 MAPK pathwayObjectives:Previous studies have revealed mitochondrial transfer existed between several cell types,which can restore the damaged respiratory function,and rescue injured cells.Moreover,the mechanism of mitochondrial transfer has also been elaborated in studies.In this study,we aimed to explore the mechanism underline mitochondrial transfer in our neuron-astrocyte co-culture system.Methods:co-culture of Mito-Tracker(green)labelled astrocytes and rotenone-treated dopaminergic neurons for 24h.Distinguish two kinds of cells by TH and phalloidin staining.Collected the astrocytic condition medium,centrifuged to remove debris.It called ACM.Ultracentrifugation was employed to remove the mitochondrion in the ACM,producing the mitochondria-depleted ACM(dm ACM);Flow cytometry detected the mitochondria in ACM and dmACM.JC1 dye was cultured with mitochondria in ACM and dmACM to measure the mitochondrial membrane potential.We used the O2 sensor kit to test and compared the 02 consumption in ACM and dmACM.Collected the ACM to culture the dopaminergic neurons under rotenone challenge,and then cell counting kit-8 and celltiter kit were applied to measure the cell viability and intracellular ATP.The protein level of MIRO1、TNFaIP2、p-p38 in PD model was measured by western blot.After treatment of p38 MAPK special inhibitor SB203580,tested the level of p-p38 by western blotting,and cell counting kit-8 examed the cell viability in PD model,in which the p38 MAPK signaling pathway was inhibited.The SB203580 treated dopaminergic neurons were labeled with Celltrace(red)and astrocytes were labeled with Mito-tracker(green),flow cytometry was used to detect the double staining population after co-culture,and immunostaining was employed to observe whether the Mito-tracker(green)labeled mitochondria existed in dopaminergic neurons.Result:(1)Laser scanning confocal microscopy didn’t observe the intercellular tunneling nanotubes(TNT)between astrocytes and dopaminergic neurons in the co-culture system;(2)Flow cytometry detected extracellular mitochondria particles in ACM and dmACM.The data showed that the number of extracellular mitochondria particles in ACM were significantly more than dmACM.(3)JC1 staining showed that the mitochondrial membrane potential in ACM was higher than dmACM.(4)The oxygen consumption of ACM was also higher than dm ACM.(5)ACM can rescue the rotenone-treated dopaminergic neurons while dmACM didn’t show any neuroprotective effect.(6)ACM can increase the intracellular ATP while dmACM can’t.(7)The protein level of MIRO1,TNFaIP2 showed no significant change in the PD model,while p-p38 increased.This data indicated that the p38 MAPK signaling pathway was activated in rotenone-induced PD model.(8)When the PD model was treated with SB203580,the inhibitor of the p38 MAPK signaling pathway,the p-p38 level was suppressed significantly.(9)The neuroprotective effect of ACM disappeared when the neurons were treated with SB203580.(10)Flow cytometry was applied to analysis the co-culture system consisted of Mito-tracker(green)labeled astrocytes and celltrace(red)labeled neurons.When neurons were treated with SB203580,the double staining percentage decreased from 16.81%to 7.53%.(11)The transferred mitochondria in dopaminergic neurons were observed by Laser scanning confocal microscopy.The data suggested that when the neurons were exposed to SB203580,the number of mitochondria existed in neurons declined sharply.Conclusion:The health mitochondria can be released from astrocytes to the medium,and then be taken in by neurons in PD model.The transferred mitochondria restored the damaged respiratory function of neurons and played a neuroprotective effect.In our rotenone-induced PD model,the p38 MAPK signaling pathway was activated and promoted the uptake of mitochondria by neurons.