Protective Effects Of Cannabinoid Type ? Receptor On MPP~+-induced Neurotoxicity In DA Neurons

Parkinson's disease(PD)is one of the most common degenerative diseases in the central nervous system.The pathological features of PD are progressive degeneration of dopamine(DA)neurons in the substantia nigra(SN)and thus resulting in the depletion of DA in the striatum.Oxidative stress,mitochondrial dysfunction,inflammation,genetics,and environmental factors are involved in the pathogenesis of PD.However,the etiology and pathogenesis of PD have not been elucidated.Growing evidences indicate that abnormal iron accumulation in the SN is involved in the pathogenesis of DA neuron degeneration.Iron deposition in the SN may produce a large amount of hydroxyl radicals through the Fenton reaction,which in turn enhance the oxidative stress in this region,thereby inducing cytotoxicity to DA neurons.Recently,studies indicated that activation of cannabinoid type II receptor(CB2R)can reduce the loss of DA neurons and alleviate the motor symptoms in PD model mice.CB2R activation mediated neuroprotection may be attributed to the inhibition of glial cells activation,and the reduction of pro-inflammatory factors.It was reported CB2R are also expressed on DA neurons.However,the effects of CB2R on DA neurons is still unknown.Previous studies showed that the activation of CB2R may inhibit the phosphorylation of the divalent metal-ion transporter-1(DMT1),thereby reducing the iron uptake by cells.We hypothesize that the CB2R may trigger a neuroprotective effect by inhibiting iron transport in the DA neurons.To test this hypothesis,we investigated the protective effects of CB2R on MPP~+-induced cytotoxicity in primary cultured DA neurons directly and its possible mechanism,we selected ventral mesencephalon(VM)neurons and SH-SY5Y human neuroblastoma cells.The cells were pre-treated with specific CB2R agonist JWH-133 and the specific CB2R antagonist AM630 for 30 min,and then 1-methyl-4-phenylpyridinium(MPP~+)was co-treated for 24 h.The changes in mitochondrial transmembrane potential of the cells(??m)were detected by flow cytometry.The protein expressions of DMT1,CB2R and tyrosine hydroxylase(TH)were detected by western blots.The iron content was detected by laser scanning confocal microscopy.The results are as follows:1.Compared with the control,the CB2R expression in SH-SY5Y cells was significantly down-regulated in MPP~+group(P<0.001).Pre-treatment with JWH-133inhibited MPP~+-induced decrease in the protein expression of CB2R(P<0.01).Co-treatment with CB2R antagonist AM630 inhibited JWH-133-induced up-regulation of CB2R protein(P<0.01).2.Compared with the control,the??m in the MPP~+-treated SH-SY5Y cells was significantly decreased(P<0.001).This effect was alleviated by JWH-133 pre-treatment(P<0.01).Co-treatment with AM630 inhibited JWH-133-induced increase in??m(P<0.05).3.Compared with the control,the TH protein expression in the MPP~+-treated SH-SY5Y cells was significantly down-regulated(P<0.05).Pre-treatment with JWH-133inhibited MPP~+-induced down-regulation of TH protein expression(P<0.05).Co-treatment with AM630 inhibited JWH-133-induced up-regulation of TH protein expression(P<0.01).4.Compared with the control,the TH protein expression in the MPP~+-treated VM neurons was decreased(P<0.01).Pre-treatment with JWH-133 inhibited MPP~+-induced down-regulation of TH protein expression(P<0.05).Co-treatment with AM630 inhibited JWH-133-induced up-regulation of TH protein expression(P<0.05).5.The iron content in SH-SY5Y cells was detected by laser scanning confocal microscopy.Calcein was used as a fluorescence indicator.Compared with the control,the fluorescence quenching in the MPP~+-treated SH-SY5Y cells was significantly increased.Pre-treatment with JWH-133 slowed down the MPP~+-induced increase in the fluorescence quenching,indicating that the iron entry into SH-SY5Y cells were inhibited.Co-treatment with AM630 blocked the JWH-133-induced inhibition of the fluorescence quenching.6.The iron content in VM neurons was detected by laser scanning confocal microscopy.Compared with the control,the fluorescence quenching in the MPP~+group was accelerated.Pre-treatment with JWH-133 slowed down the MPP~+-induced increase in the fluorescence quenching,indicating that the iron entry into the VM neurons were inhibited.Co-treatment with AM630 blocked the JWH-133-induced inhibition of the fluorescence quenching.7.Compared with the control,the protein expression of DMT1 in MPP~+-treated SH-SY5Y cells was increased(P<0.05).Pre-treatment with JWH-133 inhibited MPP~+-induced down-regulation of DMT1 protein expression(P<0.05).Co-treatment with AM630inhibited JWH-133-induced down-regulation of DMT1(P<0.01).8.Compared with the control,the protein expression of DMT1 in MPP~+-treated VM neurons was increased(P<0.01).Pre-treatment with JWH-133 inhibited MPP~+-induced down-regulation of DMT1 protein expression(P<0.05).Co-treatment with AM630inhibited JWH-133-induced down-regulation of DMT1(P<0.05).In summary,these data showed that activation of CB2R by JWH-133 alleviates MPP~+-induced decrease in TH protein expression in SH-SY5Y cells and VM neurons.JWH-133also alleviated the decrease in??m in SH-SY5Y cells,indicating that CB2R activation attenuated MPP~+-induced cytotoxicity.JWH-133 also inhibited MPP~+-induced iron influx and up-regulation of DMT1 protein expression.These may attributed to CB2R mediated neuroprotection in DA neurons.