The Role Of SUR1 Subunit Of K_ATP Channels In The Degeneration Of Nigral Dopaminergic Neurons In Parkinson's Disease

Parkinson's disease?PD?,the second age-related human neurodegenerative disease after Alzheimer's disease?AD?,is a common and complex neurodegenerative disorder characterized by a movement disorder with classical motor symptoms such as muscular rigidity,resting tremor,bradykinesia,postural reflex disorders and etc.PD patients also exihibit non-motor symptoms like cognitive impairment,psychiatric symptoms,autonomic dysfunction?such as constipation?,pain and fatigue in the early stage of the disease.The main pathological feature of PD is the selective loss of dopaminergic?DAergic?neurons in the substantia nigra?SN?with the broad expression and formation of alpha-synuclein??-syn?aggregation,known as Lewy bodies?LBs?.However,the exact pathogenesis mechanisms of PD remain unclear.Genetic factors,environmental factors,aging,inflammation,oxidative stress,mitochondrial dysfunction and abnormal iron metabolism have been proved to be involved in the development of PD.Recent studies have also found that the selective activation of ATP-sensitive potassium channels(K_ATP channels)in the remaining nigral DAergic neurons might be participated in the pathogenesis of PD.K_ATP channels,inwardly rectifying K+ channels,were primitively existed in cardiac muscle.Functional K_ATP channels,a hetero-octameric membrane protein complexes,are formulated by four inward-rectifier potassium channel 6?Kir6,either Kir6.1 or Kir6.2?subunits and four ABCC?ATP-binding cassette,subfamily C?family member sulfonylurea receptor?SUR,as SUR1,SUR2 A,or SUR2B?subunits with regulatory activity.Three types of K_ATP channels,SUR1/Kir6.2,SUR2B/Kir6.2 and SUR1/SUR2B/Kir6.2,were expressed in the nigral DAergic neurons.The m RNA levels of the SUR1 subunit of K_ATP channels in nigral DAergic neurons from the PD patients were approximately two-fold higher compared with those from the control group by quantitative m RNA expression profiling techniques.Our previous study also showed that the m RNA levels and the protein expression of the SUR1 subunit were up-regulated in the SN of 3-month-old A53 T ?-synclein transgenic mice and 6-month-old A53 T ?-synclein transgenic mice,but there was no significant difference in the expression of Kir6.2 subunit and SUR2 B subunit.K_ATP channels were also regarded as metabolic sensors,as their open probability is regulated by the ATP/ADP ratio.K_ATP channels in highly vulnerable DAergic neurons in the SN are particularly sensitive to metabolic stress in animal models by PD-trigger factors such as MPTP?1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine?or rotenone.Due to pathophysiological metabolic stress,the possibilities of K_ATP channels opening were increased,which lead to membrane potential hyperpolarization and excitability of DAergic neurons decreased.In awake PD patients,the DAergic neurons in the SN display high levels of burst activity,which could accelerate the degeneration of DAergic neurons,but the burst firing of DAergic neurons in the SN of Kir6.2 knockout mice was reduced by half after Kir6.2 knockout.However,whether the up-regulation of SUR1 subunit was involved in the spontaneous discharge activity of DAergic neurons in the SN,and whether knocking down the SUR1 subunit could protect DAergic neurons,are still largely unknown.To explore the effects of high expression of SUR1 subunit on the DAergic neurons functions in the SN,A53 T ?-synclein transgenic mice and C57BL/6 mice were applied in the experiments.Extracellular single unit electrophysiological recording in vivo was used to detect the effects of K_ATP channels on the spontaneous firing activity of DAergic neurons in the SN.Whether DAergic neurons discharge activity changing with age was also observed in the SN of A53 T ?-synclein transgenic mice.In addition,SUR1 interference lentivirus was injected into A53 T ?-synclein transgenic mice and MPTP-induced PD mice to explore the effects of SUR1 knockdown on the DAergic neurons functions in the SN by immunofluorescence,high performance liquid chromatography and western blots technology.The experimental results are as follows:1.Sixteen DAergic neurons were recorded in diazoxide group.The spontaneous frequency was decreased from 2.39 ± 0.92 Hz to 0.89 ± 0.74 Hz after K_ATP channels opener diazoxide?100 ?mol/L?treatment in 6 out of the 16 DAergic neurons,and the average decrease was 87.9 ± 8.7%?P<0.05?.Further firing pattern analysis did not show that the coefficient of variation?CV?was changed after the injection of diazoxide?P>0.05?.While the K_ATP channels opener diazoxide decreased the firing rate from 2.69 ± 1.97 Hz to 2.17 ± 1.77 Hz in 10 out of 16 DAergic neurons,the variation of discharge frequency was less than 2 standard deviations,which were regarded as non-reactive cells.2.Seven DAergic neurons were recorded in NMDA group.The spontaneous firing rate was increased from 1.46 ± 1.45 Hz to 3.40 ± 1.29 Hz after NMDA receptor agonist NMDA?50 ?mol / L?treatment,and the average increase of the firing rate was 65.14 ± 3.34%?P<0.05?.Further firing pattern analysis showed that the CV value was increased after the injection of NMDA?P<0.01?.The K_ATP channels inhibitor glibencliamine and NMDA were co-applicated in 5 DAergic neurons.The spontaneous firing rate induced by the co-application of glibencliamine and NMDA was increased from 2.90 ± 1.63 Hz to 4.30 ± 2.48 Hz,and the average increase of frequency was 55.3 ± 40.1% compared with that before drug?P>0.05?.After the block of the K_ATP channels,the CV value was changed significantly after NMDA receptor activation by firing pattern analysis?P>0.05?.3.The spontaneous firing rate of DAergic neurons,both in 10 DAergic neurons of 3-month-old A53 T ?-synclein transgenic mice and 8 DAergic neurons of 6-month-old A53 T ?-synclein transgenic mice,was significantly higher than that of WT mice at the same age?P<0.05?.More irregular discharges and fewer cluster discharges were observed in the 3-month-old A53 T ?-synclein transgenic mice,compared with that of WT mice at the same age.Compared with that of 6-month-old WT mice,more irregular discharges and a large number of clustered discharges were observed in 6-month-old A53 T ?-synclein transgenic mice.4.SUR1 lentiviral vector and empty letiviral vector were injected stereotaxically into the SN of WT mice and A53 T ?-synuclein transgenic mice for 60 days,the levels of SUR1 protein were decreased by 40.93% and 34.72%,respectively?P<0.05?.The levels of TH protein and the number of TH-positive cells in the SN of the A53 T ?-synuclein transgenic mice carrying empty lentivirus were decreased by 29.36% and 19.86%?P<0.05?,respectively,compared with that of the control group.The expression of TH protein and the number of TH-positive neurons in the SN of the A53 T ?-synuclein transgenic mice with SUR1 knockdown were increased by 49.11% and 23.41%,respectively?P<0.05?,compared with the A53 T ?-synuclein transgenic mice carrying empty lentivirus group.5.After five-day MPTP treatment,the levels of the TH protein in the SN were decreased by 33.17%,which was statistically significant compared with that of the control group?P<0.01?.The m RNA levels and the levels of SUR1 protein in the SN of MPTP-treated mice were increased by 54.21% and 52.76%,respectively?P<0.05?.There was no significant change in SUR2 B or Kir6.2 subunits?P> 0.05?.6.SUR1 lentiviral vector and empty letiviral vector were injected stereotaxically into SN for 21 days and MPTP was injected intraperitoneally for 5 days.The levels of TH protein and the number of TH-positive neurons in the SN of the MPTP-treated mice carrying empty lentivirus were decreased by 56.27% and 48.42%,respectively,compared with that of the control group?P<0.001,P<0.01?.Compared with that of the MPTP-treated mice carrying empty lentivirus,the levels of TH protein and the number of TH-positive neurons in the SN in MPTP-treated group with SUR1 knockdown were increased by 100.87% and 62.95%,respectively?P<0.01,P<0.05?.The results of HPLC showed that the DA,DOPAC and HVA content in the MPTP-treated mice carrying empty lentivirus were reduced by 89.41%,86.65% and 73.16%,respectively,compared with that of the control group?P<0.001?.Compared with that of the MPTP-treated mice carrying empty lentivirus,the contents of DA,DOPAC and HVA in the MPTP-treated group with SUR1 knockdown were increased by 360.00%,109.00%,and 86.32%,respectively?P<0.05,P<0.01,P<0.05?.7.SUR1 lentiviral vector and empty letiviral vector were injected stereotaxically into the SN for 21 days and MPTP was injected intraperitoneally for 5 days.The levels of SOD1 protein and the Bcl-2/Bax ratio in the SN were detected by western blots.Compared with that of the control group,the levels of SOD1 protein and the Bcl-2/Bax ratio in MPTP-treated group carrying empty lentivirus were decreased by 40.64% and 58.47%,respectively?P<0.001,P<0.01?.The levels of SOD1 protein and the Bcl-2/Bax ratio in the MPTP-treated group with SUR1 knockdown were increased by 38.48% and 102.83%,respectively?P<0.05?,compared with that of the MPTP-treated mice carrying empty lentivirus.In summary,the K_ATP channels might participate in the occurrence of burst firing of DAergic neurons in the SN.With the high expression of SUR1 subunit in the SN,the firing rate and the occurrence of burst firing of DAergic neurons were increased significantly in the process of PD.In PD transgenic mice and MPTP-induced PD mouse models,SUR1 knockdown could antagonize the degeneration of DAergic neurons in the SN.This study shows that the degeneration of DAergic neurons was caused by the selective upregulation of SUR1 subunit of K_ATP channels in the SN.Knockdown of SUR1 subunit could partially antagonize the damage of DAergic neurons and play a protective role on neurons,which will provide an important target for the development of therapeutic drugs for PD in the future.