The Impacts Of ?7 Nicotinic Acetylcholine Receptors Activation On Nerve Cell Apoptosis In Parkinson's Disease

Parkinson's disease(PD)have been relatively common disorder with the aging pohenomena becoming prominent increasingly,and the incidence has ranked the second around the world.PD is characterized by the degeneration of dopaminergic neurons at the substantia nigra compacta(SNc),and the decrease of dopamine(DA)in the striatum,accompanied by the pathological accumulation of Lewy body(LB)and the formation of Lewy neuritis(LN)_Detailed pathogenies are connected with environmental,age,genetic,oxidative stress,and protein clearance dysfunction.Currently L-dopa is the gold standard treatment for PD motor problems,particularly in the early disease stages.However,it does not improve the other symptoms,nor reduce the inevitable disease progression.Novel therapeutic strategies for Parkinson's disease are therefore critical.Epidemiological studies confirmed the smoking habits as a factor working against the development of PD.As the nicotine is the important chemical component of tobacco,it activates nicotinic acetylcholine receptors(nAChRs),and reduces the inevitable disease progression.Neuronal nAChRs are pentameric ligand-gated ion channels composed of diverse combinations of a and ? transmembrane subunits. a7-nAChRs are uniquely localized throughout the brain,although there is substantial overlap in their distribution withother nAChR subtypes.It has a very highcalcium permeability which allows them to regulate numerous calcium-dependent cellular mechanisms important for optimal CNS function such as anxiety,attention,learning,memory,movement and sensory gating.Our previous study found that the activation of ?7-nAChRs has a protective effect on PD animal models,but the specific mechanism is unclear.In this study,we will investigate the effect of activating a7-nAChRs on apoptosis of nerve cell induced by 1-methyl-4-phenylpyridine ion(MPP+),in order to provide a reliable idea and experimental basis to find effective targets for the treatment of PD.In the first part of the present study,human neuroblastoma cell line(SH-SY5Y)was used to study the protection mechanisms of a7-nAChRs activation against MPP+-induced apoptosis;In the second part,astrocytes were used to study the mechanisms of ?7-nAChRs activation against MPP+-induced astrocyte apoptosis.This study not only extends the pharmacological mechanisms of a7-nAChRs,but also provides a new idea for studying the effective drug therapies of PD.Part I Activation of a7-nAChRs inhibits MPP+-induced apoptosis of SH-SY5Y cellsAIM:To purify the protective effect of a7-nAChRs activation on MPP+-induced apoptosis of SH-SY5Y cells,and to elucidate the protective mechanism of a7-nAChRs activation on neuron apoptosis.METHODS:After exposure to MPP+,?7-nAChRs non-selective agonist nicotine(NIC),a7-nAChRs selective agonist PNU-282987 and a7-nAChR selective antagonist methyllycaconitine(MLA)for 24 h,SH-SY5Y cell viability was measured by the methyl thiazolyl tetrazolium(MTT)and lactate dehydrogenase(LDH).The morphological features of apoptotic cells were monitored by fluorescence microscopy after staining with Hoechst 33342.The ratio of apoptotic cells was detected using an FITC annexin-V/PI cell apoptosis kit.Western blotting(WB)was applied to detect the leavels of apoptosis-related proteins,mitogen-activated protein kinases(MAPKs),p53 and nuclear factor-KB(NF-?B)signaling pathways.SH-SY5Y cells were transfected specific siRNA to decrease the expression of CHRNA7,this way was applied to investigate the effect of a7-nAChRs expression on apoptosis.RESULTS:(1)Pretreatment with NIC(10?M)and PNU-282987(10?M)before MPP+(500 ?M)addition significantly inhibited the decrease of SH-SY5Y cell viability and reduced the release of LDH compared to MPP+ treatment alone;these effects were abolished by MLA(100 nM).(2)Hoechst33342 fluorescent staining further confirmed the anti-apoptotic effect of a7-nAChRs activiation on MPP+-induced apoptosis,untreated SH-SY5Y cells exhibited regular and round-shaped nuclei,by contrast,condensation and fragmentation of nuclei,which are characteristics of apoptotic cells,were evident in SH-SY5Y cells treated with MPP+for 24 h.Pretreatment with NIC and PNU-282987 reduced MPP+-induced apoptotic cells;The effect was blocked by MLA.(3)Flow cytometry showed that NIC and PNU-282987 significantly have reduced the number of positive cells of Annexin V/PI markers,which were blocked by MLA.(4)Treatment of cells with NIC and PNU-282987 befor MPP+addition downregulated the expression of Bax and upregulated the level of Bcl-2,these changes were reduced by antagonist MLA.(5)Treatment with MPP+increased the levels of both phosphorylated JNK and p38,decreased the levels of phosphorylated ERK;Pretreatment with NIC and PNU-282987 suppressed MPP+-induced increases in the levels of phosphorylated JNK and p38,improved MPP+-induced decreases in the levels of phosphorylated ERK;The antagonist MLA significantly blocked the changes in phosphorylation levels of JNK,p38 MAPK and ERK induced by PNU-282987;MLA had no significant effect NIC-induced decreases in the levels of phosphorylated JNK and p38,but the suppressive effect of NIC on ERK phosphorylation was only partially reversed by MLA,(6)Treatment with MPP+increased the levels of both NF-?B p65 and phosphorylated p53;Pretreatment with NIC and PNU-282987 suppressed MPP+-induced increases in the expression of NF-?B p65 and phosphorylated p53;The suppressive effect of NIC on NF-?B p65 and phosphorylated p53 were reversed by MLA;The suppressive effect of NIC on the expression of NF-?B p65 was not reversed by MLA,but phosphorylated p53 was reversed by MLA.(7)Downregulation of CHRNA7 by siRNA did not affect the cell viability and the release of LDH,but it abolished the above benefical effects of activating a7-nAChRs.Downregulation of CHRNA7 abrogated NIC and PNU-282987-induced changes of Bax and Bcl-2,also suppressed the inhibitory effect of NIC and PNU-282987 on apoptotic cells.CONCLUSION:Activation of a7-nAChR does not directly affect the toxic effects of MPP+ on neurons,but relieves MPP+-induced SH-SY5Y cell apoptosis by regulating p53 signaling pathway and MAPK signaling pathway.Part ? Activation of a7-nAChRs inhibits MPP+-induced apoptosis of astrocyteAIM:The objective of this part study was to determine the protective effect of activating ?7-nAChRs on MPP+-induced astrocyte apoptosis and to investigate the protective mechanisms of ?7-nAChRs activation on astrocyte apoptosis.METHODS:Immunofluorescence staining was used to analyse GFAP positive astrocyte in cultured mice primary astrocyte.After exposure to MPP+,?7-nAChRs selective agonist PNU-282987 and ?7-nAChRs selective antagonist methyllycaconitine(MLA)for 24 h,astrocyte cell viability was measured by the methyl thiazolyl tetrazolium(MTT)and lactate dehydrogenase(LDH).Apoptotic cells were monitored by Hoechst 33342 staining,TUNEL and flow cytometry.Western blotting(WB)was applied to detect the leavels of apoptosis-related proteins,mitogen-activated protein kinases(MAPKs),p53 and nuclear factor-?B(NF-?B)signaling pathways.RESULTS:(1)Pretreatment with PNU-282987(100 pM)significantly inhibited MPP+(800 ?M)-induced the decrease of astrocyte cell viability and reduced the release of LDH;these roles were abolished by MLA(100 pM).(2)Hoechst33342 fluorescent staining showed that PNU-282987 significantly reduced MPP+-induced astrocyte apoptosis with condensation and fragmentation of nuclei;the effect was blocked by antagonist MLA.(3)Flow cytometry and TUNEL staining showed that PNU-282987 significantly reduced the number of apoptotic cells in astrocyte;Pretreatment with MLA reversed these phenomenons.(4)Treatment of cells with PNU-282987 before MPP+addition suppressed MPP+-nduced increases the expression of Bax and the ratio of cleaved-caspase3/caspase3 in astrocytes,improved the level of Bcl-2;The antagonist MLA significantly blocked the changes of these proteins.(5)Treatment with MPP+ increased the levels of both phosphorylated JNK and p38 MAPK,reduced the levels of phosphorylated ERK;PNU-282987 reduced the phosphorylation levels of JNK and p38,increased the phosphorylation level of ERK;The effects of PNU-282987 about the phosphorylation levels of JNK,p38 and ERK were reversed by ML A.(6)Treatment with MPP+ increased the levels of both NF-?B p65 and phosphorylated p53 in astrocytes;Pretreatment with PNU-282987 suppressed MPP+-induced increases in the expression of NF-?B p65 and phosphorylated p53;The suppressive effect of PNU-282987 on NF-?B p65 and phosphorylated p53 were reversed by MLA.CONCLUSION:Activation of a7-nAChRs reduce MPP+-induced astrocyte apoptosis by regulating p53,NF-?B and MAPK signaling pathway.In summary,the major contribution of the present study are listed as follows:1.The activation of ?7-nAChRs plays a protective role in MPP+-induced apoptosis of neuron and astrocyte,which provides promising experimental basis for the further study of the effect of a7-nAChRs on neuronal-astrocyte networks.2.It is suggested that the protective effect of activating a7-nAChRs on MPP+-induced apoptosis of nerve cell may be dependent on p53,NF-?B and MAPK signaling pathway,these findings may provide evidence for neuronal protection mechanisms of a7-nAChRs.