The Modulatory Effects Of ATP-sensltive Potasslum Channels On Parkinson's Disease

ATP-sensitive potassium channel (K-ATP channel), a unique link betweencellular energetics and electrical excitability, consists of discrete pore-forming andregulatory subunits and is activated by a decrease in ATP/ADP ratio. K-ATPchannels are widely distributed in the brain, but do not belong to a homogenous group.Generally, Kir6.2 subunit forms the pore of the K-ATP channels in most neuronsincluding dopaminergic neurons, while Kir6.1-containing K-ATP channels areexpressed in the astrocytes, microglia and neural stem cells. Many studies havedemonstrated that K-ATP channels play a role in protecting against brain injuryinduced by hypoxia, ischemia or metabolic inhibition. Moreover, K-ATP channelsparticipate in the initiation and progress of Parkinson's disease (PD).Parkinson's disease (PD) is a progressive neurodegenerative disordercharacterized by rigidity, bradykinesia, postural instability and resting tremor. Themajor symptoms are related to the progressive loss of dopaminergic neurons in thesubstantia nigra pars compacta. Its prevalence is increasing such that over the next25 years the number of individuals over 50 years of age with PD can be expected todouble in the world's 10 most populous nations. According to the pathomechanism governing dopaminergic loss, various hypotheses, including excitotoxicity,mitochondrial dysfunction, oxidative stress and inflammation have been proposed tobe involved in the pathogenesis of PD. However, most of the neuroprotective agentsdeveloped according to these hypotheses failed to exert ideal clinical curativeeffects. Advances in our understanding of the etiology and pathogenesis of PDtherefore have the potential to make a significant impact on neurologic practice.Our previous studies have demonstrated that K-ATP channel openers (KCOs)exerted neuroprotective effects on various PD models induced by haloperidol,6-OHDA, rotenone, and 1-methyl-4-phenylpyridinium (MPP+) throughanti-excitotoxicity, anti-apoptosis, anti-neuroinflammtion as well as maintainingmitochondrial function. However, it is a very interesting and unexpected reportfrom our recently studies that genetic inactivation of Kir6.2-containing K-ATPchannels resulted in a selective rescue of substantia nigra (SN) dopaminergic (DA)neurons in the chronic MPTP and probenecid (MPTP/p) mouse model of Parkinson'sdisease. These findings challenged the traditional opinion that Kir6.2-containingK-ATP channel activation played protective roles in brain, but they also suggest thatthe remaining glial and neural stem cellular Kir6.1-containing K-ATP channels mayplay an important potential role in protecting against MPTP-induced brain injury inthe Kir6.2 knockout mice. In recent years, we realised that glial cells especiallyastrocytes are critical participants in every major aspect of the brain, includingproviding mechanical and metabolic support for neurons, regulating the metabolismof neurotransmitter, protecting neurons from oxidative stress and excitotoxicity andpromoting adult neurogenesis. Our previous studies have revealed that opening ofK-ATP channels in astrocytes protects against MPP+-induced astrocytic apoptosis viainhibition of mitochondria apoptotic pathway and regulating the MAPK signaltransduction pathways and opening of microglial K-ATP channels inhibits rotenone-induced neuroinflammation and protects DA neurons. Whether glialK-ATP channels are involved in the regulation of DA neuron injury in the MPTP/pPD mouse model is still unknown.Therefore, the aim of present studies is to investigate the role of K-ATP channels,especially glial Kir6.1-containing K-ATP channels in PD. We first explore the rolesand the involved mechanisms of glial and neural stem cellular K-ATP channels inMPTP/p PD mouse model. Then, the neuron-glia co-culture systems were used tostudy the regulatory effects of K-ATP channels on astrocyte function. Further, weestablished the chronic mild stress (CMS) model to investigate the role of K-ATPchannels in neurogenesis. Finally, the proteomic studies were used to obtain moredefinitive proofs at the molecular levels of the role of K-ATP channels in Parkinson'sdisease.Part I Effects of K-ATP channels on MPTP/p mousemodel of Parkinson's diseaseAIM: To investigate the role and the mechanism of K-ATP channels onMPTP-induced degeneration of dopaminergic neurons in MPTP/p PD model usingKir6.2 deficiency mice.METHODS: Kir6.2+/+ and Kir6.2-/- mice were treated with chronic MPTPintoxication protocol: 20 mg·kg-1 MPTP in saline was injected subcutaneously, and250 mg·kg-1 probenecid in DMSO was injected intraperitoneally every 3.5 d over aperiod of 5 weeks. Iptakalim (IPT, 10 mg·kg-1·day-1, p.o.) was administered to miceone hour before the first injection with MPTP on a daily basis for 6 weeks. Micewere killed 1 week after the final injection of MPTP. For acute MPTP neurotoxicity,Kir6.2+/+ and Kir6.2-/- mice were injected with a single dose for acute MPTP effects:20 mg·kg-1 MPTP in saline was injected subcutaneously, and 250 mg·kg-1 probenecid in DMSO was injected intraperitoneally only once. IPT (10 mg·kg-1·day-1, p.o.)wasadministered to mice one hour before the injection of MPTP on a daily basis for 3days. Mice were killed 3.5 days after the injection. Immunohistochemistry wastaken for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP),5-bromodeoxyuridine (BrdU) and ionized calcium-binding adapter molecule1(Iba-1)expression. The total numbers of TH-positive neurons in the substantianigra pars compacts (SNpc), GFAP-positive cells and Iba-1-positive cells densities inthe substantia nigra (SN), and BrdU-positive cells in the subventricular zone (SVZ)and subgranular zone (SGZ) were obtained stereologically using the opticalfractionator method. The level of D-Ser and serine racemase (SR) in TH neuronsand astrocytes were determined by TH and D-Ser, TH and SR, GFAP and D-Ser,GFAP and SR double immunofluorescence, respectively. The levels of Connexin 43(Cx43) and TNF-αwere determined by Western blot and ELISA, respectively.RESULTESULTS: 1) Chronic MPTP treatment decreased DAergic neurons in SNpc,reduced TH-immunopositive fibers in the dorsal striatum in Kir6.2+/+ mice, but not inKir6.2-/- mice. IPT could reverse chronic MPTP-induced loss of TH-positive SNneurons, and remarkably alleviated the reduction of striatal TH fiber density inKir6.2+/+ mice. A single MPTP injection-induced loss of DA neurons in SN and THfibers in striatum in Kir6.2-/- mice, and IPT treatment could reverse the single MPTPinjection-induced loss of DA neurons in SN and TH fibers in striatum in Kir6.2-/-mice. 2) Chronic MPTP treatment induced increased activation of astrocytes andmicroglia, with remarkable elevations of Cx43 expression and TNF-αrelease in SN,and significant inhibition of cell proliferation in the SVZ and SGZ of Kir6.2+/+ mice,but not in Kir6.2-/- mice. Administration of IPT inhibited MPTP-induced astroglialand microglial activation and the elevations of Cx43 and TNF-αin Kir6.2+/+ mice andreversed MPTP-induced inhibition of cell proliferation in Kir6.2+/+ mice. 3) Chronic MPTP treatment-induced increases of D-Ser and SR were only observed in astrocytesbut not in TH-positive neuron in SN of Kir6.2+/+ mice. IPT treatment couldsignificantly down-regulate the expressions of D-Ser and SR in Kir6.2+/+ mice.CONCLUSION:1. The results from this part provide direct evidence that K-ATP channelsparticipate in the pathophysiological mechanisms of PD.2. Opening of K-ATP channels exerted neuroprotective effects on MPTP-induced neurodegeneration, which may be related to the improvement ofneurogenesis and suppression of glial activation.Part II The regulatory effects of K-ATP channels onastrocyte functionAIM: To investigate the role and the mechanism of K-ATP channels onastrocyte function using the neuron-glia co-culture systems.METHODS: Mesencephalic primary neuron or astrocyte cultures were preparedfrom the ventral mesencephalic tissues of embryonic day 14/15 or postnatal (P1-P2)Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. Tyrosine Hydroxylase immunocyto-chemistry quantification of Tyrosine Hydroxylase immunoreactive (THir) neuronalcounts and processes. MTT assay was used to determine the viability of astrocytes.RT-PCR and Western blotting were used to analysis the expression of Kir6.1 andKir6.2 in astrocytes. Western blotting was used to analysis the expression ofneurotrophic factors in astrocytes. GFAP and D-Ser, GFAP and SR doubleimmunofluorescence were used to investigate the expression of D-Ser and SR inastrocyte. The co-culture system was achieved by inverting the coverslips bearingastrocytes and all cells were exposed to MPP+ (10μM). TH and GFAPimmunocytochemistry was used to quantify THir neuronal counts and processes and astrocyte activation. The level of D-Ser and SR in astrocyte were determined byGFAP and D-Ser, GFAP and SR double immunofluorescence, respectively.RESULTS: 1) MPP+ induces cytotoxicity in Kir6.2+/+ and Kir6.2-/- primarymesencephalic neurons especially dopaminergic neurons in aconcentration-dependent manner, but Kir6.2 subunit deficiency reducesMPP+-induced cytotoxicity in mesencephalic neurons. Pretreatment with KCOsprotects primary mesencephalic neurons against MPP+-induced cytotoxicity inKir6.2+/+ neurons, but not in Kir6.2-/- neurons. 2) MPP+ induced stronger activationof Kir6.2+/+ astrocytes than that of Kir6.2./. astrocytes. Moreover, when co-culturedwith mesencephalic neurons from either Kir6.2+/+ mice or Kir6.2-/- mice, Kir6.2-/-astrocytes could also shown less activation. Knockout of Kir6.2 in astrocytessignificantly protected the MPP+-induced lesion on both Kir6.2+/+ and Kir6.2-/-neurons. 3) Astrocytes expressed both Kir6.1 and Kir6.2 subunits, and knockout ofKir6.2 decreased the expression of Kir6.1 in astrocytes, whereas MPP+ treatmentup-regulated the expression of Kir6.1 in Kir6.2-/- astrocytes. MPP+ inducedincreased expression of D-Ser and SR in Kir6.2+/+ astrocytes, but not in Kir6.2-/-astrocytes. Knockout of the Kir6.2 gene in astrocytes increased the expressions ofBDNF and FGF-2, and MPP+ (10μM) treatment for 48h significantly increasedBDNF expression in Kir6.2-/- astrocytes, but not in Kir6.2+/+ astrocytes.CONCLUSION:Knockout of Kir6.2 in astrocytes protected the MPP+-induced lesion on bothKir6.2+/+ and Kir6.2-/- neurons, which was related to the increased expression ofKir6.1 and BDNF and inhibition of MPP+ induced increased expression of D-Ser andSR. Part III The regulatory effects and mechanisms of K-ATPchannels on neurogenesisAIM: To investigate the effects and mechanisms of K-ATP channels onneurogenesisMETHODS: Kir6.2+/+ and Kir6.2-/- mice were subjected to daily i.p. injection ofFlx (10 mg·kg-1) or IPT (10 mg·kg-1) for 4 weeks under normal condition or followedby chronic mild stress (CMS). The antidepressive effects of Flx and IPT understress were evaluated by Tail suspension test (TST), Sucrose preference test andForced swimming test (FST). BrdU was administrated to both genotypic micefollowed 4-week Flx or IPT treatment. Neurogenesis was evalutated by countingthe BrdU-positive cells and NeuN/BrdU or GFAP/BrdU-positive cells in the SGZ.Fresh hippocampal homogenates were used to determine the levels of BDNF,Serotonin, the phosphoralations of Akt,GSK3β,ERK1/2,JNK1/2,p38,CREB, andthe expression of Kir6.2 and Kir6.1. Trunk blood was collected for thedetermination of corticosterone levels. Western blotting and RT-PCR were used toidentify the existence and subunit composition of K-ATP channels in the primarycultured adult neural stem cells (ANSCs). [3H]-thymidine incorporation was used toassess the cell proliferation rate of the primary cultured adult NSCs.RESULTS: 1) Knockout of Kir6.2 resulted in antidepressant-like behaviors, butaggravated depressive behaviors in mice followed by CMS. Four-week treatment ofIPT or Flx prevented CMS-induced depressive behaviors in Kir6.2+/+ mice butpartially alleviated these behavioral symptoms in Kir6.2-/- mice. CMS significantlydecreased the hippocampal CA3 neuronal number in Kir6.2-/- mice, IPT and Flxprevent hippocampal CA3 neuron from losing in Kir6.2-/- mice. 2) Kir6.2 knockoutfailed to influence hippocampal ANSC proliferation in normal and stressed mice,while IPT increased proliferation of ANSCs in normal mice and reversed CMS-induced inhibition of ANSCs in both genotypic mice. Kir6.2 knockout, CMSprocedure, IPT or Flx treatment had no effect on the differentiation of BrdU-positivecells in SGZ. Knockout of Kir6.2 inhibits new-born cell survival in SGZ. 3)Knockout of Kir6.2 decreased the level of BDNF. IPT reversed CMS-inducedattenuation of 5-HT and BDNF to the normal levels in both genotypic mice. IPTonly reversed the CMS-induced increment of serous corticosterone in Kir6.2+/+ micebut not in Kir6.2-/- mice. 4) In both genotypic mice, CMS procedure significantlyinhibited the phosphorylation of CREB, ERK1/2 and GSK3β, increased thephosphorylation of JNK in Kir6.2-/- mice, IPT and Flx markedly extenuated theCMS-induced decrement of phosphorylated CREB, ERK1/2 and GSK3βin bothgenotypic mice and the CMS-induced increased phosphorylation of JNK in Kir6.2-/-mice. 5) Western blotting and RT-PCR analysis showed that ANSC expressedKir6.1/SUR1-composed K-ATP channels. [3H]-thymidine incorporation assayrevealed the promotive effects of IPT on the proliferation in both genotypes ANSC.CONCLUSION:1. Neural stem cells express Kir6.1/SUR1-composed K-ATP channels.2. IPT promoted neurogenesis through Kir6.1-composed K-ATP channels viaphosphorating ERK and CREB.3. Kir6.2-containing K-ATP channels play a protective role in neuronal injury.Part IV Proteomic analysis of the effects of K-ATPchannels on Parkinson's diseaseAIM: To gain insight into the role of K-ATP channels in the pathomechanism ofPD at the molecular level.METHODS: Kir6.2+/+ and Kir6.2-/- mice were treated with chronic MPTPintoxication protocol: 20 mg·kg-1 MPTP in saline was injected subcutaneously, and 250 mg·kg-1 probenecid in DMSO was injected intraperitoneally every 3.5 d over aperiod of 5 weeks. Mice were sacrificed by cervical dislocation 7 days after the lastMPTP or saline injection. Midbrains were dissected rapidly, frozen in liquidnitrogen, and stored at -70°C until used. Primary cultured mesencephalic neuronsand astrocytes were treated with 10μM MPP+ for 48 h, digested with 0.25% trypsinand collected. The tissue was homogenized twenty times, with sonication at 70%intensity in 10 s bursts, with 10 parts v/w of buffer that consisted of 7 M urea, 2 Mthiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, 1% protease inhibitor cocktail (v/v), and2% (v/v) IPG buffer (pH 3-10). Samples were then lysised for 1 h at 4°C andcentrifuged at 13000 rpm for 1 hour at 4°C. The supernatant was collected anddispensed, then stored at -80°C. Protein concentrations were determined using theBradford assay. Samples containing 180μg of protein were profiled by comparative2-DE and image analysis. Protein spots with significant differences between the twogroups were excised. Gel pieces were denatured, alkylated, trypsin digested andanalyzed by an Ultraflex II MALDI-TOF-TOF mass spectrometer. Proteinidentification was confirmed by sequence information obtained from MS/MS analysisin"LIFT"mode. Acquired MS/MS spectra were also processed using the softwareFlexAnalysisTM 2.4 using a SNAP method set at a signal-to-noise ratio threshold of3.0. In order to avoid quantitative deviations, for the key proteins, Western blottingwas used to verify their expressional alterations.RESULTS: 1) 45 protein spots were significantly altered in response to Kir6.2deletion in midbrains. 3 proteins were up-regulated and 37 down-regulated inKir6.2-/- midbrains. NADH-ubiquinone oxidoreductase 75 kDa subunit wasexpressed in Kir6.2-/- and undetectable in Kir6.2+/+ midbrains, 8 proteins includingaspartoacylase, keratin, ornithine aminotransferase, heterogeneous nuclearribonucleoprotein H, isoform 2 of Dihydrolipoyllysine-residue succinyltransferase, visinin-like protein 1, serum albumin, and fascin were only expressed in Kir6.2+/+midbrains. MPTP treatment induced increased expression of 12 and decreasedexpression of 12 proteins in Kir6.2+/+ midbrains, as compared to saline-treatedcontrols. Fascin and Heat shock 70 kDa protein 4 were down-regulated, Aldehydedehydrogenase was up-regulated. Kir6.2 deficient mice showed 35 differentiallyexpressed proteins after chronic MPTP treatment. Among them, 2 were increasedand 33 decreased in expression as compared to untreated controls. NADHdehydrogenase flavoprotein 1, Isoform 1 of 4-aminobutyrate aminotransferase andGlutathione synthetase were down-regulated,Aldose reductase was up-regulated. 2)20 protein spots were significantly altered in response to Kir6.2 deletion inmesencephalic neurons. After MPP+ (10μM) treatment for 48 h, 48 protein spotsshowed significantly altered expresssion in Kir6.2+/+ mesencephalic neurons. AfterMPP+ (10μM) treatment for 48 h, 44 protein spots showed significantly alteredexpresssion in Kir6.2-/- mesencephalic neurons. The proteins in functional complexesresponsible for cytoskeletal integrity, synaptic integrity and protein biosynthesis wereup-regulated in Kir6.2-/- mesencephalic neurons. While, the proteins responsible forstress-related chaperones and ubiquitin proteaosome degradation weredown-regulated in Kir6.2-/- mesencephalic neurons. The proteins responsible forcytoskeletal integrity were down-regulated in Kir6.2+/+ mesencephalic neurons afterMPP+ treatment, but were up-regulated in Kir6.2-/- mesencephalic neurons. MPP+treatment induced up-regulation of the proteins responsible for glycolysis in Kir6.2+/+mesencephalic neurons, but not in Kir6.2-/- mesencephalic neurons. 3) 30 proteinspots were significantly altered in response to Kir6.2 deletion in mesencephalicastrocytes. After MPP+ (10μM) treatment for 48 h, 44 protein spots showedsignificantly altered expresssion in Kir6.2+/+ mesencephalic astrocytes. After MPP+(10μM) treatment for 48 h, 44 protein spots showed significantly altered expresssion in Kir6.2-/- mesencephalic astrocytes. The proteins in functional complexesresponsible for cytoskeletal integrity and stress-related chaperones were up-regulatedin Kir6.2-/- mesencephalic astrocytes. After MPP+ treatment, the proteinsresponsible for protein biosynthesis and mitochondrial function were down-regulatedin Kir6.2+/+ mesencephalic astrocytes, but not in Kir6.2-/- mesencephalic astrocytes.CONCLUSION:1. Knockout of Kir6.2 induced altered expression of the proteins responsible forcytoskeletal structural integrity, synaptic and axonal integrity, stress-relatedchaperones, ubiquitin proteaosome degradation, protein biosynthesis, and energymetabolism.2. Kir6.2 knockout mice respond to MPTP/MPP+ with a less impairment ofcytoskeleton, synaptic and axonal integrity, oxidative phosphorylation, mitochondrialfunction and less stress response, which may be involved in the mechanism of theprotective role of Kir6.2 deficiency on Parkinson's disease.In summary, the major contributions of the present study lie in:1. K-ATP channels participate in the initiation and progress of PDKnockout of Kir6.2 and opening of Kir6.1-containing K-ATP channels exertedneuroprotective effects on MPTP-induced neurodegeneration through improvingneurogenesis and suppressing glial activation, which provides a novel therapeuticapproach for neurodegenerative diseases.2. K-ATP channels regulate astrocyte function Knockout of Kir6.2 inastrocytes protected the MPP+-induced lesion on both Kir6.2+/+ and Kir6.2-/- neuronsthrough increasing the expression of Kir6.1 and BDNF and inhibiting MPP+ inducedincreased expression of D-Ser and SR.3. K-ATP channels regulate adult neurogenesis Opening of Kir6.1-con -taining K-ATP channels in neural stem cells promoted neurogenesis viaphosphorating ERK and CREB. K-ATP channels may be a novel target to regulateadult neurogenesis.4. K-ATP channels regulate protein expression in the brain of PD mice andMPP+ treated neurons and astrocytes In response to equivalent MPP+ stress,Kir6.2 knockout neuron and astrocyte are much more efficient in coping with MPP+toxicity through inhibiting MPP+-induced down-regulation of protein biosynthesisand mitochondrial function. The proteomic results provide more definitive proofs atthe molecular levels of the role of K-ATP channels in PD.