The Modulatory Effects Of ATP-sensitive Potassium Channels On Neuroinflammtion And Adult Mice Neurogenesis

ATP-sensitive potassium channel (K-ATP channel) is a special classof potassium channel, which links cell metabolic state to excitability.K-ATP channels consist of discrete pore-forming and regulatory subunitsand are activated by a decrease in ATP/ADP ratio. Recently, it isdemonstrated that K-ATP channel especially mitochondrial K-ATP(mitoK-ATP) channel is the important neuroprotective target, and may bethe endogenous protective mechanism after ischemic, hypoxia oroxidative stress-induced injury. Moreover, K-ATP channel participatesin the initiation and progress of Parkinson's disease (PD), and may be thenovel target to investigate the pathogenesis and therapeutics. K-ATPchannel opener iptakalim (IPT), which can freely cross the blood-brainbarrier and has little effect on peripheral blood pressure, exerts significantneuroprotection in vivo and in vitro. Thus, IPT is considered as apromising neuroprotective agent targeting K-ATP channel. However,besides the direct protective effects of K-ATP channel openers on neurons,little is known about effects on other type neural cells.Neurodegerative diseases include Parkinson's disease (PD),Alzheimer's disease (AD), Huntington's disease (HD) and so on,characterized by neural cell degeneration. It has been demonstrated thatexcitotoxicity, neuroinflammation, oxidative stress, apoptosis andmitochondrial dysfunction play critical role in neurodegeneration. According to the etiology of neurodegenerative disorders, people areengaged in looking for the neuroprotective agents that can retard theprogress of neuron death or improve the neural regeneration. However,since the pathogenesis of neurodegerative disease is complicated, most ofthe neuroprotective agents failed to exert ideal clinical curative effectsbecause of the single pharmacological target. Therefore, seeking for thenovel neuroprotective agents that can block multiple steps in the progressof neurodegeneration may offer prospective clinical therapeutic benefits.Based on the mechanisms and time course of neuronal injury, theco-application of anti-excitotoxicity, anti-neuroinflammtion andimproving neurogenesis was considered as a prospective approach forneuroprotection. Our previous studies have demonstrated that K-ATPchannel opener exhibited anti-excitotoxicity and anti-apoptosis effects.As a promising endogenous target for neuroprotection, it remainsunknown whether K-ATP channel participate in the regulation ofneuroinflammation and neurogenesis. Microglial activation andsubsequent neuroinflammation have been demonstrated to be early signsthat often precede and trigger neuronal death in neurodegenerativediseases. Activated microglia can release a great deal of neurotoxicfactors, such as TNF-α, PGE₂, IL-1βand reactive oxygen species (ROS),which are believed to contribute to progressive damage in PD. Thus,inhibition of microglial activation and subsequent neuroinflammationmay offer prospective clinical therapeutic benefits forinflammation-related neurodegenerative disorders. On the other hand,dysregulation of adult neurognesis is considered as a critical contributorto the pathogenesis of neurodegenetative disorders and psychiatricdisorders. Promoting adult neurogenesis has been regarded as aprospective strategy for cell replacement therapy for functional recoveryin degenerative diseases.However, little is known about the compositon and function of K-ATP channels in microglia and neural stem cells. In the present study,we first investigated the neuroprotective effects of K-ATP channelopeners against rotenone. Then, primary cultured microglia were usedto explore the roles of K-ATP channels in microglial activation. Finally,we studied the effects of K-ATP channel opener on neurogenesis in vivoand in vitro.Partâ… The neuroprotective effects and mechanisms of K-ATPchannel openers against rotenone-induced neurodegenerationAIM: To investigate the neuroprotective effects and mechanisms ofK-ATP channel openers on rotenone-induced degeneration ofdopaminergic neurons in rats.METHODS: 1) IPT (1.5, 3.0 mg·kg^-1·day^-1, p.o.) or diazoxide (1.5mg·kg^-1·day^-1, p.o.) alone was administered to rats for 3 days, and thenfrom the 4th day IPT or diazoxide was pretreated one hour before theinjection with rotenone (2.5 mg·kg^-1·day^-1, s.c.) on a daily basis for 4weeks. The catalepsy test and rotard test were used for the assessment ofthe effects of drugs on rotenone-induced parkinsonian symptoms; 2)Immunostaining was taken for TH, OX-42, ED1 and GFAP expression inrat substantia nigra; 3) mRNA levels of TNF-αand COX-2 in ratsubstantia nigra and striatum were analyzed by RT-PCR; 4) ELISA wasused to determine the content of TNF-αin substantia nigra and peripheralblood; 5) BrdU was used to inverstigate the neurogenesis inhippocampus.RESULTS: 1) K-ATP channel openers IPT and diazoxide bothexerted neuroprotection on rotenone-treated rats, including attenuatedweight loss, mortality and parkinsonian symptoms; 2) IPT and diazoxideattenuated rotenone-induced degeneration of dopaminergic neurons in ratsubstantia nigra; 3) IPT and diazoxide exerted inhibitory effects on rotenone-induced microglial activation in rat substantia nigra; 4) IPT anddiazoxide down-regulated mRNA levels of TNF-αand COX-2 in ratsubstantia nigra and striatum; 5) IPT and diazoxide alleviatedrotenone-induced suppression of neurogenesis in hippocampus.CONCLUSION: K-ATP channel openers exerted neuroprotectiveeffects on rotenone-induced degeneration of dopaminergic neurons,which were related to the inhibition of microglia activation and theimprovement of neurogenesis.Partâ…¡The expression of K-ATP channels in microglia and itsregulatory effects on microglial activationAIM: To investigate whether K-ATP channels were expressed inprimary cultured rat microglia and to explore the regulatory effects andmechanisms of K-ATP channel openers on microglial activation.METHODS: Western blotting and RT-PCR were taken for theanalyses of K-ATP channel subunits in microglia; phase contrastmicroscopy for cell morphology and immunochemistry for ED1expression were used for determination of microglial activation; Theamount of TNF-αand PGE₂ in the medium were determined with a ratTNF-αELISA kit and rat PGE₂ RIA kit; Mitochondrial membranepotential was detected by molecular probe JC-1, Western blotting wastaken for analyses of p38/JNK MAPK phosphorylation.RESULTS: 1) Microglia express K-ATP channel subunits Kir6.1and SUR2, but not Kir6.2 and SUR1; 2) Pretreatment with K-ATPchannel opener iptakalim or selective mitoK-ATP channel openerdiazoxide suppressed rotenone-induced microglial activation, evidencedby inhibition of microglial amoeboid morphological alteration, declinedexpression of ED1 and decreased production of TNF-αand PGE₂; Theseeffects were reversed by selective mitoK-ATP channel blocker 5-HD; 3) Pretreatment with iptakalim or diazoxide prevented rotenone-inducedmitochondrial membrane potential loss and p38/JNK MAPK activation inmicroglia, which may be the signaling transduction pathway responsiblefor the regulatory effects of K-ATP channel openers on microglialactivation.CONCLUSION: K-ATP channels, which are composed of Kir6.1and SUR2 subunits, are expressed in rat primary cultured microglia.Opening of microglial mitoK-ATP channels inhibited rotenone-inducedmicroglial activation and subsequent release of pro-inflammatory factor,which was related to stabilization of mitochondrial membrane potentialand inhibition of p38/JNK MAPK pathway.Partâ…¢The regulatory effects and mechanisms of K-ATPchannel opener on neurogenesis in C57B1/6J miceAIM: To investigate whether K-ATP channel opener participate inthe regulation of adult neurogensis.METHODS: 1) Western blotting and RT-PCT were used to identifythe existence and subunit composition of K-ATP channel in primarycultured adult neural stem cells (NSCs); 2) [³H]-thymidine incorporationwas used to assess the cell proliferation rate of the primary cultured adultNSCs; 3) Normal C57B1/6J mice were administered with IPT (10mg·kg^-1·day^-1, i.p.) or fluoxetine (10 mg·kg^-1·day^-1, i.p.) for 28 days. Theproliferation of the newborn cells were determined using BrdUimmunohistochemistry and the differentiation of newly formed cells wereanalyzed by double labeling for mature neurons with neuron specificunclear protein (NeuN) and for glia cells with glial fibrillary acid protein(GFAP); 4) Western blotting was taken for analyses of the phosphorylatedlevels of ERK1/2 and CREB; 5) Kir6.2^-/- mice were used to investigate whether Kir6.2 knockout could affect the promotive effects of K-ATPchannel opener IPT on neurogenesis.RESULTS: 1) Western blotting analysis showed that adult neuralstem cells expressed Kir6.1 subunit not Kir6.2 subunit; 2) The in vitroinvestigation by [³H]-thymidine incorporation assay revealed thepromotive effects of IPT on the proliferation of adult neural stem cells;3) Chronic administration with IPT increased neural stem cellsproliferation in the hippocampus of mice. The population of survivingBrdU-positive cells essentially matured into neurons, but the phenotypicexpression pattern remained unchanged between groups; 4) Chronicadministration with iptakalim could activate ERK and CREB; 5) IPTtreatment exhibited no significant difference between wild type mice andKir6.2 knockout mice.CONCLUSION: Kir6.1-composed K-ATP channels were expressedin neural stem cells. Activation of K-ATP channels by IPT couldpromote adult neurogenesis via phosphorating ERK and CREB. IPTpromoted neurogenesis through Kir6.1-composed K-ATP channels.Thus, targeting K-ATP channel may be a novel approach to regulate adultneurogenesis, and iptakalim may be developed as a promising agent forneurogenesis in neurodegenertative disorders.The major contributions of the present study lie in:1. The neurprotective effects of K-ATP channel openers are related tothe anti-neuroinflammatory effect and the protmotive effect onneurogenesis, which provides a novel therapeutic approach forneurodegenerative diseases. 2. K-ATP channels are expressed in rat primary cultured microglia,which are composed of Kir6.1 and SUR2 subunits. Opening ofmicroglial mitoK-ATP channels inhibits microglial activation andsubsequent neuroinflammation. The underlying mechanismsinvolve the stabilization of mitochondrial membrane potential andthe inhibiton of p38/JNK activation. K-ATP channel in microgliamay be a novel target for anti-neuroinflammtion agents.3. Kir6.1-composed K-ATP channels were expressed in adult neuralstem cells and participated in neurogenesis. Activation of K-ATPchannels by IPT could promote adult neurogenesis via phosphoratingERK and CREB. IPT promoted neurogenesis throughKir6.1-composed K-ATP channels. Targeting K-ATP channels maybe a novel approach to regulate adult neurogenesis...