Study On The Mechanisms Underlying The Upregulation Of K_ATP Channel SUR1 Subunit In Dopaminergic Neurons In Parkinson's Disease

Parkinson's disease?PD?is the most prevalent neurodegenerative disorder.The incidence rate of the disease in the Chinese population over 65 years old was 1.7%,and in the population over 70 years old was 3.4%.There are about 10 million PD patients in China,which has become the most suffering country in the world.PD is pathologically characterized by a selective loss of dopaminergic?DAergic?neurons in the substantia nigra?SN?and the aggregation of alpha-synuclein??-Syn?forming Lewy bodies?LBs?in the remaining DAergic neurons throughout the SN,whereas the DAergic neurons in the neighboring ventral tegmental area?VTA?are less affected.The underlying mechanism is largely unknown.In recent years,it has been found that the selective activation of the SUR1/Kir6.2 type ATP sensitive potassium channel(K_ATP channel)on DAergic neurons in the SN rather than DAergic neurons in the VTA is the main cause of neuronal death in the pathological state of PD,implicating an important role of K_ATP channels in PD.The K_ATP channel is composed of two subunits of inwardly rectifying K⁺channel?Kir6.1/Kir6.2?and ATP binding protein family member SUR?SUR1/SUR2A/SUR2B?.The composed octamer protein structure is widely distributed in a variety of excitable cells,such as islet?cells,cardiomyocytes,smooth muscle cells,skeletal muscle cells,and neurons.The K_ATP channels in the DAergic neurons mainly include SUR1/Kir6.2,SUR2B/Kir6.2 and SUR1/SUR2B/Kir6.2.In the autopsy of PD patients,although the subunits of SUR1,SUR2 and Kir6.2 were expressed in the remaining DAergic neurons in the brain,only the SUR1 mRNA expression levels were shown 2-fold increase.The mRNA levels of SUR2 and Kir6.2 did not change.In addition,in PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine?MPTP?,the mRNA levels of the SUR1 subunit in the SN were 2-fold higher than that in the VTA region.Whether the selective upregulation of SUR1 leads to the DAergic neurons damage and its underlying mechanisms are still unknown.Although studies on pancreatic?-cells have revealed that FOXA1 and FOXA2 can directly bind with and activate the promoter of the SUR1 gene,the regulation of SUR1 on neurons has not been reported.Therefore,this experiment was designed to investigate the mechanism of selective upregulation of SUR1 in DA neurons in PD and its role in neuronal death.In this experiment,cell culture,gene transfection,quantitative PCR,Western blots,and dual luciferase reporter genes were used to observe the effects of highly expressed?-Syn on the expression of various subunits of the K_ATP channel,and the transcription factors FOXA1 and FOXA2.At the same time,the binding of FOXA1 and FOXA2 to SUR1 promoter was observed.Furthermore,in the PD transgenic mice carrying the human A53T mutant?-Syn(?-SynA53T^+/-mice or?-SynA53T^+/+mice),the expressions of K_ATP channel and various subunits and transcription factors FOXA1 and FOXA2 in the SN was observed during disease progression.Lentivirus interfering vectors carrying SUR1 were stereotactically injected into the SN of mice,and then MPTP was given intraperitoneally to observe the effect of SUR1 knockdown on the impairment of DA neurons in the SN caused by MPTP.The experimental results were as follows:1.After transfected with WT-?-Syn and A53T-?-Syn for 24 hrs in MES23.5 cells,the mRNA levels of SUR1 subunit were increased by 35%and 31%,respectively,compared with the control group.The difference was statistically significant?P<0.05?.However,the mRNA level of SUR2B or Kir6.2 subunit did not change?P>0.05?.2.In 15 m and 20 m?-SynA53T^+/-mice,the expression levels of SUR1 in the SN were increased by 41%and 21%,respectively,which was statistically significant compared with the control group?P<0.05?.However,the protein levels of SUR2B did not change?P>0.05?.3.In 3 m,4 m and 6 m?-SynA53T^+/+mice,the mRNA levels of SUR1 subunits in the SN were increased by 21%,80%and 49%,respectively,with a statistically significant difference?P<0.01,P<0.05,P<0.01?.SUR1 protein levels were increased by 108%,48.5%and 65%,respectively,compared with the control group?P<0.05?.SUR2B mRNA levels in 3 m and 4 m mice were also increased,with a 30%and 21%increase,respectively?P<0.05,P<0.001?.There was no change in SUR2B mRNA detected in 6 m mice?P>0.05?.Furthermore,there were no significant difference in SUR2B protein levels and Kir6.2 mRNA levesl in 3 m,4 m or 6 m mice?P>0.05?.4.After transfected with WT-?-Syn and A53T-?-Syn for 24 hrs in MES23.5 cells,the transcription factor FOXA1 mRNA levels were increased by 36%and 49%,respectively,compared with the control group?P<0.05,P<0.01?.The mRNA levels of FOXA2 were increased by 35%and 41%,respectively,compared with the control group?P<0.01?.5.Double luciferase assay showed that the relative fluorescence intensity of GV230-FOXA1 group and GV230-FOXA2 group was significantly higher than that of the empty group,and the difference was highly statistically significant?P<0.001?.6.In 3 m,4 m and 6 m?-SynA53T^+/+mice,the mRNA levels of transcription factor FOXA1 were increased by 36%,90%and 240%,respectively,compared with the control group?P<0.05?.The mRNA levels of FOXA2 increased by 58%,25%and 160%respectively compared with the control group?P<0.05,P<0.05,P<0.01?.7.SUR1 lentiviral vector and empty lentiviral vector were injected stereotaxically into the SN of mice for 21 days,and MPTP was injected intraperitoneally for 5 d.Western blots showed that the expression of TH protein in the SN was reduced by 27%in the MPTP group?P<0.05?.Compared with the MPTP group,the expression of TH protein was increased by 23%in the mice with SUR1 knockdown?P<0.05?.In summary,in the progresses of PD,the expression of SUR1 subunit was increased in the DAergic neurons,which was due to the increased expression of the transcription factors FOXA1 and FOXA2.FOXA1 and FOXA2 could upregulate the mRNA level of the SUR1 subunit by binding to its gene promoter.After knocking down the SUR1 gene in the SN region of mice,it could partially antagonize the neurotoxicity induced by MPTP and protect DAergic neurons.This experiment clarified the role of SUR1/Kir6.2 K_ATPTP channels in the degenerative changes of nigral DAergic neurons in PD and will provide a new target for PD drug therapy.