The Protective Roles And Mechanisms Of Kir6.2-containing Atp-sensitive Potassium Channel Opening In Mpp~+-induced Cytotoxicity In Mesencephalic Neuron

Parkinson's disease (PD), a common progressive neurodegenerative disorder, is characterized by selective degeneration of dopamine neurons in the substantia nigra (SN) resulting in debilitating motor signs including tremor, bradykinesia and rigidity. It has a lifetime risk of 2%, making it the second most common neurodegenerative disease after Alzheimer's disease. Current PD medications treat symptoms, mainly by L-DOPA administration; none halt or retard dopaminergic neuron degeneration. Although various hypotheses, including genetic factors, mitochondrial dysfunction, oxidative stress, excitotoxicity, neuroinglamation and apoptosis have been proposed to be involved in the pathogenesis of PD, the exact mechanisms governing dopaminergic loss remain unclear. The main obstacle to developing neuroprotective therapies is a limited understanding of the key molecular events that provoke neurodegeneration.Mitochondrial oxidative stress has been implecated in a range of degenerative disease. Oxidative stress infers an imbalance between the formation of cellular oxidants and the antioxidative processes. Mitochondria are not only major source of reactive oxygen species (ROS) generation in aerobic cells, but they are also sensitive target for the damaging effects of oxygen radicals. Oxidative stress may account for the nigral defect of complex I activity, in that complex I is highy vulnerable to oxidative damage and inhibition of complex I leads to increased ROS formation. Meanwhile mitochondria participate in the regulation of both energy metabolism and cell death. Excessive ROS production impairs mitochondria membrane system, loss of mitochondrial membrane potential (ΔΨm) and activation mitochondrial permeability transition pore (PTP), resulting in release of pro-apoptotic proteins which subquently initiated mitochondrial apoptotic pathway. Mitochondria dysfunction is a prominent feature in apoptosis and release of pro-apoptotic proteins from the mitochondrial intermembrane space has been considered to be a critical event that occurs during apoptosis. Restoring mitochondrial function helps to provide neuroprotective effects on neurogenetive disorders.KATP channels, which link cell metabolic state to excitability, consist of discrete pore-forming and regulatory subunits and are activated by a decrease in ATP/ADP ratio. Specific combinations of various Kir6.x and SUR subunits comprise KATP channels in different cells in the brain. Kir6.2 subunit constitutes the neuronal KATP channels, while Kir6.1 subunit constitutes the glial KATP channels. Our previous studies have demonstrated that KATP channel openers (KCOs) exerted neuroprotective effects on various PD models induced by haloperidol, 6-OHDA, rotenone, and 1-methyl-4-phenylpyridinium (MPP+) through anti-excitotoxicity, anti-apoptosis, inhibiting neuroinflammatio, as well as maintaining mitochondrial function. However, there are no direct evidences to support the contribution of KATP channels expressed in neurons to MPP+-induced cytotoxicity; whether KCOs exert neuroprotective effect in vitro and the involved mechanisms is unclear.In the present study, we investigated that Kir6.2-containing KATP channels participated in MPP+-induced neuronal injury and opening of Kir6.2-containing KATP channels excert neruoprotective effect in vitro and the underlying mechanisms were focused on inhibiting ROS production.AIM: To investigate the protective roles and mechanisms of Kir6.2-containing ATP-sensitive potassium channel opening in MPP+-induced cytotoxicity in mesencephalic neuron.METHODS: Mesencephalic primary neuron cultures were prepared from the ventral mesencephalic tissues of embryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice and cultures were used after 7 days in vitro (DIV). Tyrosine Hydroxylase immunocytochemistry quantification of Tyrosine Hydroxylase immunoreactive (THir) neuronal counts and processes; Staining with Hoechst 33342 was used to determine neuronal apoptosis; Assary of lactate dehydrogenase (LDH) released in neuronal media was used to determine neuronal injury; Intracellular ROS and mitochondrial membrane potential (ΔΨm) were detected by molecular probe DCFH-DA and JC-1, respectively; Western blotting was taken for analyses of cytochrome c (cyto c).RESULTS: 1) MPP+ induces cytotoxicity in Kir6.2+/+ and Kir6.2-/- primary mesencephalic neurons especially dopaminergic neurons in a concentration-dependent manner but Kir6.2 subunit deficiency reduces MPP+-induced cytotoxicity in mesencephalic neurons; 2) Pretreatment with KCOs protects primary mesencephalic neurons against MPP+-induced cytotoxicity depending on Kir6.2-KATP channels; 3) Pretreatment with KCOs suppresses MPP+-triggered ROS elevation, prevents MPP+-inducedΔΨm loss and pro-apoptotic factor release in primary mesencephalic neurons. CONCLUSION: 1) Pretreatment with KCOs protects DA neurons against MPP+-induced primary mesencephalic neurons especially dopaminergic neuronal injury via reduce baneful ROS generation, stabilize mitochondrial membrane potential and decrease cytochrome c release from mitochondria. 2) Kir6.2 knock out attenuate MPP+-induced neuronal injury, which provides direct evidence that Kir6.2-containing KATP channels participated in MPP+-induced neuronal injury.