Neuron Survival And Apoptosis Regulated By Phosphatidylcholine-specific Phospholipase C And Integrin β4

BACKGROUND AND OBJECTIVEThe development of the vertebrate central nervous system(CNS)involves the precisely orchestrated regulation of neural cell proliferation,cell fate specification, migration and synaptogenesis.There have recently been many reports about neural cell proliferation,differentiation and apoptosis.However,the exact mechanism of neural cell survival and apoptosis regulation is not well understood.Phosphatidylcholine-specific phospholipase C(PC-PLC)might play a key role in the signal transduction.The PC-PLC hydrolyses phosphatidylcholine to DAG. DAG is the second message in the cells and activates protein kinase C(PKC)and mitogen-activated protein kinase(MAPK).Under various physiological and pathological conditions,the signal transduction via PC-PLC is important in cell proliferation,differentiation and apoptosis.In the previous studies,we found that PC-PLC was involved in the proliferation,apoptosis and differentiation of human umbilical vascular endothelial cells(HUVECs)and human marrow stromal cells (hMSCs).In another report,it has been shown that PC-PLC modulates neural cell death caused by oxidative glutamate toxicity.But the role of PC-PLC in the survival of neuron under normal condition is not clear.Integrins,a large family of heterodimeric receptors,withαandβsubunits,play fundamental roles in cellular interactions,motility and signaling during mammalian development.Recently,several lines of evidence suggest that integrins are abundantly expressed in neurons and have been implicated in many phases of neural development.In contrast to otherβsubunits,integrinβ4 has an exceptionally large cytoplasmic domain,more than 1000 amino acids,and associates with cytoskeletal and signaling molecules.The important roles of integrinβ4 in cellular signal transduction have been noted.In Schwann cells,integrinβ4 participates in the initiation of myelin sheath formation but in oligodendrocytes is not expressed. Increasing evidence suggests that integrinβ4 has cell type-specific functions. However,the roles of integrinβ4 in neuronal survival and apoptosis during the development of the central nervous system are not well known.How did PC-PLC and integrinβ4 work in the survival and apoptosis of the neuron? Were some important signal molecules(ROS,NADPH oxidase and Rb) involved in this process? These problems need to be solved.Based on the backgrounds mentioned above,the objective of this study was as follows:To investigate the roles of PC-PLC and integrinβ4 in neuron survival and apoptosis,and the molecular mechanisms mediated by PC-PLC and integrinβ4.The researches would provide a theoretical basis for studying the molecular mechanisms of neuron survival and apoptosis during the development of CNS.Our data would provide a theoretical basis for the neurological diseases theraphy in clinic.STUDY CONTENTS1.The primary culture and identification of mouse neuron.2.Analysis of survival mediated by PC-PLC in neuron using its specific inhibitor D609.3.Analysis of some important signal molecules and mechanism in neuron survival mediated by PC-PLC.4.Investigating the role of integrinβ4 in neuron survival and apoptosis by using RNAi.5.Analysis of some important signal molecules and mechanism in neuron survival mediated by integrinβ4.METHODS:1.Mouse neuron culture and identification:1)Mouse neuron were isolated and cultured as described[Sunanda et al,1998].2)Analysis the expressions of neural specific marker NSE and GFAP by immunofluorescence assay. 2.Cell viability analysis:1)Cell viability was determined by MTT-assay.2)Cell viability was evaluated by trypan blue exclusion testing.3.Cell apoptosis measurement:1)Observation of cell morphological changes by Phase Contrast Microscope.2)Analysis of DNA nuclear fragmentation by acridine orange staining.3)TUNEL assay to determine the apoptotic index.4)LDH assay to determine whether cells underwent necrosis.4.PC-PLC activity assay was performed as described by Wu[Wu et al.,1997].5.RNA interferenceThe expression of integrinβ4 was inhibited by its specific small interfering RNA.6.Analysis ROS level by the fluorescence probe,DCHF.7.SOD activity assay was performed by the SOD detection kit.8.NADPH oxidase activity was examined as described[Li et al,2002].9.Analysis of expression and distribution of proteins:1)Examination of the changes in Rb and integrinβ4 protein level and distribution by Immunocytochemistry.2)Analysis the levels of integrinβ4 by Western blot assay.RESULTS:1.Primary cultured cells were mainly neurons.The primary cultured neurons were obtained from embryo day 14. Morphological of cells could be observed on phasecontrast microscopy.Cells exhibited a typical neuronal appearance and formed extensive networks(Fig. 1-1-1).Meanwhile,we examined the expression of NSE and GFAP in primary cultured neuron by immunofluorescence staining and found 98%of cells as neurons(Fig.1-1-2).2.Viability of neurons remarkably suppressed by D609. 2.1 The results from MTT assay experiments showed that when the cells were exposed to D609 2-8μg/ml for 8h,the viability remarkably declined from 100% to 68.78-20.46%in the concentration-dependent manner(p＜0.01,Table 1-2-1).2.2 Morphological changes of neuron could be observed on phasecontrast microscopy.When the cells were exposed to D609 8μg/ml for 8h.neuritis degenerated and many apoptotic bodies appeared(Fig.1-2-1).2.3 Meanwhile,acridine orange staining showed severe apoptotic changes in treated neurons,including nuclear condensation,DNA fragmentation and apoptotic body formation(Fig.1-2-2).3.The related mechanisms mediated by PC-PLC.3.1 The PC-PLC activity,assay showed that there were at least two isoforms of PC-PLC in mouse neuron.They were the Ca²⁺-dependent PC-PLC and the Ca²⁺-independent PC-PLC.Following the treatment with D609 8μg/ml for 8h. the activities of the both PC-PLC decreased significantly(p＜0.01,Fig.1-3-1).3.2 Immunocytochemistry assay showed that when the cells were exposed to D609 8μg/ml for 6h,the integrinβ4 level was increased(p＜0.05,Fig.1-3-2). Meanwhile,the Rb level was increased obviously(p＜0.05,Fig.1-3-3).3.3 Following the treatment with D609 10μg/ml for 6h,the intracellular ROS level was decreased significantly(p＜0.01,Fig.1-3-4).4.siRNA-mediated down-regulation of integrinβ4 in neurons.4.1 We detected the levels of integrinβ4 in both the mouse cerebral cortex and primary cultured neurons at embryo day 14 gestation(E14)by immunofluorescence staining.Integrinβ4 was abundant in the developing cerebral cortex and especially rich in primary cultured neurons(Fig.2-1-1).4.2 Laser scanning confocal microscopy of neurons transfected with non-silencing rhodamine red-labeled siRNA revealed a transfection efficiency of siRNA between 30%and 50%,so siRNA-mediated gene silencing was effective(Fig. 2-1-2).4.3 Immunofluorescence staining and Western blot analysis revealed that the level of integrinβ4 in neurons transfected with integrinβ4 specific siRNA was downregulated significantly(Fig.2-1-5).And siRNA downregulated integrinβ4 in a concentration-(Fig.2-1-3)and time- manners(Fig.2-1-4).5.siRNA-mediated down-regulation of integrinβ4 triggered apoptosis in neurons.5.1 The results obtained from MTT assay and trypan blue exclusion testing showed that the viability of the cells remarkably declined,from 95.3%to 13.9%,after transfection with integrinβ4 siRNA,20-60 nM,for 24,48 and 72 h;control siRNA had no effect on neuron survival(Fig.2-2-1).5.2 We investigated apoptosis in neurons transfected with integrinβ4 siRNA for 48 h. Morphological changes of apoptosis could be observed on phasecontrast microscopy.Neuritis degenerated and many apoptotic bodies appeared. Meanwhile,acridine orange staining showed severe apoptotic changes in treated neurons,including nuclear condensation,DNA fragmentation and apoptotic body formation(Fig.2-2-2).5.3 We further assessed neuron apoptosis by TUNEL technique.After down-regulation with integrinβ4 siRNA,the proportion of apoptotic cells was markedly increased,to 85.5%(p＜0.01,Fig.2-2-3),whereas only a few apoptotic cells(no more than 6.8%)were detected in cells transfected with control siRNA.5.4 Following the treatment with integrinβ4 siRNA or control siRNA for 48 h,there was no significant difference in the LDH release among normal and siRNA treated groups,which suggests that knockdown of integrinβ4 did not cause necrosis in neurons(Fig.2-2-4).6.The survival and apoptosis mechanisms mediated by integrinβ4 in neuron.6.1 During the neuronal apoptosis induced by down-regulation of integrinβ4,the intracellular ROS level was increased significantly(p＜0.01,Fig.2-3-1).6.2 There were no significant differences in the activities of MnSOD and Cu-Zn SOD among normal and siRNA-treated groups(Fig.2-3-2);however,cells treated with integrinβ4 siRNA showed significantly increased activity of NADPH oxidase(p＜0.01,Fig.2-3-3).6.3 We investigated the administration of DPI,a specific inhibitor of NADPH oxidase,to validate the involvement of NADPH oxidase in this process.We found that the apoptotic ratio was significantly attenuated(p＜0.01,Fig.2-3-4). Meanwhile,DPI attenuated the increased level of ROS(p＜0.01,Fig.2-3-5).CONCLUSION:1.PC-PLC had an important role in regulating neuron survival under normal condition.2.Suppressing PC-PLC activity blocked neuronal survival.Integrinβ4 and Rb protein could be involved in this process mediated by PC-PLC.3.The modest intracellular ROS level might be necessary for neuron survival.4.Integrinβ4 might be a key factor in neuronal survival and apoptosis.5.The ROS controlled probably by NADPH oxidase might be the main element in the survival and apoptosis mediated by this integrin subunit.6.The modest expression of integrinβ4 might be necessary for neuron survival.