The Regulation Of Parkin's S-nitrosylation On DMT1 And Its Implication In Parkinson's Disease

Parkinson's disease?PD?is a common neurodegenerative disease.The main pathological change is the loss of dopaminergic neurons,iron deposits,and the accumulation of?-synuclein??-syn?in the remaining neurons which forms the Lewy body in the substantia nigra.Nitrative stress can cause S-nitrosylation?SNO?of various proteins,such as parkin protein,which participate in the pathogenesis of neurodegenerative diseases.Parkin is an E3 ubiquitin ligase that mediates the ubiquitination degradation of the iron transporter divalent metal transporter 1?DMT1?.In the previous study,we observed that increased expression of DMT1 played an important role in the iron accumulation in the substantia nigra.However,the underlying mechanism is largely unknown.Since nitrification stress is a common pathological manifestation in PD,it is of great significance to clarify the regulation of DMT1 by S-nitrosylation of parkin and its underlying mechanism,which could elucidate the cause of nigral iron accumulation.In the present study,the levels of nitric oxide?NO?,SNO-parkin and DMT1 were observed in NO donor,nitrosoglutathione?GSNO?-treatment SH-SY5Y cells,as well as the iron uptake function.Next,these indexes were also measured in1-methyl-4-phenylpyridinium?MPP⁺?-treated SH-SY5Y cells.We also observed the changes of SNO-parkin and DMT1 protein levels in the substantia nigra in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine?MPTP?-treated mice.To further elucidate the molecular mechanism,the effect of S-nitrosylation of parkin on the levels of ubiquitination of DMT1 was studied in SH-SY5Y cells.The involvement of p53 in the regulation of SNO-parkin on DMT1 was also studied.This study could provide a basis for elucidating the causes of increased expression of DMT1 in the substantia nigra during the process of PD.The results were as follows:1.After SH-SY5Y cells were transfected with parkin for 24 hrs,the levels of DMT1were decreased by 53.9%,compared with that of the control?P<0.01?.When parkin-transfected SH-SY5Y cells were treated with GSNO,the S-nitrosylation of parkin appeared 20 hrs and reached its peak level at 24 hrs.2.The parkin-transfected SH-SY5Y cells were treated with GSNO for 24 hrs,and the levels of DMT1 were increased by 277%,compared with the control group?P<0.001?.3.The parkin-transfected SH-SY5Y cells were treated with GSNO and MPP⁺for 24 hrs,the NO levels were increased by 46.5%?P<0.01?and 60.2%?P<0.001?,respectively,compared with the control group.In parkin-transfected cells treated with NOS inhibitor L-NNA,the levels of NO were decreased by 35.1%compared with the control group?P<0.001?.4.The parkin protein could be nitrosylated in parkin-transfected SH-SY5Y cells after treatment with MPP⁺for 24 hrs.And the levels of DMT1 were increased by 203%,compared with the control group?P<0.01?.In parkin-transfected cells treated with L-NNA,the levels of DMT1 were decreased by 43.1%,compared with the control group?P<0.01?.5.The parkin-transfected SH-SY5Y cells were treated with GSNO and MPP⁺for 24 hrs,respectively,the iron uptake ability of the cells increased significantly,compared with the control group,the difference was highly statistically significant?P<0.001?.In parkin-transfected cells treated with L-NNA,the iron uptake ability of the cells was decreased significantly,compared with the control group?P<0.001?.6.Myc-DMT1-or siDMT1-transfected SH-SY5Y cells were treated with 100?M FeSO₄ for 24 hrs,respectively.Overexpression of DMT1 by Myc-DMT1 aggravated the decrease in cell viability induced by iron,which was highly statistically significant compared with the control group?P<0.001?.Inhibition of DMT1 expression by siDMT1attenuated iron-induced cell death,compared with the control group,the difference was statistically significant?P<0.05?.7.In the PD mouse model induced by MPTP,the S-nitrosylation of parkin in the substantia nigra appeared 2 hrs after the last injection of MPTP and reached its peak level at 4 hrs.At the same time,the levels of DMT1 in the substantia nigra were significantly increased at 4 hrs,compared with the control group?P<0.001?.However,no changes were observed in the levels of SNO-parkin and DMT1 in the striatum?P>0.05?.8.After co-transfection of Flag-WT-parkin,HA-Ub and Myc-DMT1 in HEK293T cells for 24 hrs,the ubiquitination of DMT1 was significantly increased,while the ubiquitination was significantly decreased after GSNO pretreatment.When the mutants with Flag-C241A-parkin,Flag-C260A-parkin and Flag-C323A-parkin were co-transfected in HEK293T cells,and the ubiquitination did not change significantly after pretreatment with GSNO,which was not affected by S-nitrosylation.However,the three mutants,Flag-C241A-parkin,Flag-C260A-parkin and Flag-C323A-parkin,were transfected into HEK293T cells,respectively,the ubiquitination of DMT1 was significantly reduced after GSNO pretreatment.The similar results were obtained when Flag-WT-parkin transfected.9.After SH-SY5Y cells were transfected with parkin for 24 hrs,the levels of p53 were decreased by 26.5%,compared with the control group?P<0.001?.When parkin-transfected SH-SY5Y cells were treated with GSNO and MPP⁺for 24 hrs,the levels of p53 were 314%and 64.2%higher than that of the control group,respectively?P<0.001?.In parkin-transfected cells treated with L-NNA,the levels of p53 were decreased by 57.9%,compared with the control group?P<0.01?.10.After SH-SY5Y cells were transfected with p53 siRNA,the levels of DMT1 were decreased by 54.6%,compared with the control group?P<0.01?.In summary,GSNO or MPP⁺treatment could lead to an increase in intracellular NO levels in parkin-transfected SH-SY5Y cells,resulting in S-nitrosylation of parkin,which affecting the function of its ubiquitin ligase.Therefore,the ubiquitination degradation of the substrate DMT1 was decreased,thereby enhancing the iron uptake capacity of the cells.This study suggests that S-nitrosation of parkin leads to abnormal regulation of DMT1 expression,which might be a therapeutic target for PD.