| ATP-sensitive potassium channels (K-ATP 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 K-ATP channels in different cells in the brain. Kir6.2 subunit constitutes the neuronal K-ATP channels, while Kir6.1-containing K-ATP channels are expressed in the neuroglia and neural stem cells. K-ATP channels are highly expressed in substantia nigra and striatum, and participate in the regulation of release of neurotransmitters which are involved in the pathophysiology of PD, including DA, glutamate and GABA. These evidences suggest the relationship between K-ATP channels and PD.Parkinson's disease (PD), a common progressive neurodegenerative disorder, is characterized by selective degeneration of dopamine neurons in the substantia nigra and neuronal proteinaceous aggregates called Lewy bodies (LBs). Its prevalence and incidence rates increase with age, and more than 2% of the population aged over 65 years are affected by the 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.Our previous studies have demonstrated that K-ATP channel opener (KCO) 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. KCOs could also modulate the adult neurogenesis in mice hippocampal dentatus gyrus under normal conditions. Recent reseach reveavled K-ATP channels promoted the differential degeneration of dopaminergic midbrain neurons. However, there are no direct evidences to support the contribution of K-ATP channels to PD. Whether KCOs exert any effects on PD model in Kir6.2 KO mice remains unclear.The aims of the present studies are:1) to elucidate that Kir6.2-containing K-ATP channels participate in the regulation of striatal neurotransmission; 2) to support the direct evidence that K-ATP channels participate in the initation and progress of PD employing Kir6.2 knockout (KO) mice and classical PD animal model; 3) to suggest the involvement of neurogenesis in neuroprotection exerted by opening K-ATP channels.Partâ… Effects of Kir6.2 deficiency on neurotransmitters releasesfrom mouse striatumAIM: To elucidate that Kir6.2-containing K-ATP channels participate in regulating high K+-evoked releases of striatal neurotransmitter, providing the direct evidence that K-ATP channels regulate neurotransmission.METHODS: 1) Twelve-week-old wildtype (WT) and Kir6.2 KO mice were used for the experiments. The mice were placed in a stereotaxic apparatus. The concentric microdialysis probe was implanted into the right ventral striatum. The whole assembly was secured with the dental cement. Neurotransmitter release was measured in the striatum of mice at 7 d after surgery. On day of the experiment, the probes were perfused with an artificial cerebrospinal fluid (aCSF) for 100 min. After collection of five high-K+ (100mM) samples for 100 min, the mice were continually injected by aCSF for 160 min to normalize the levels of neurotransmitters. Each collected sample was stored for analysis of monoamines and amino acids levels. The levels of monoamines and amino acids in dialysate samples were measured by high-performance liquid chromatography (HPLC).RESULTS: The extracellular levels of monoamine and amino acid neurotransmitters in KO mice striatum were similar to those in WT mice under basal conditions. After high K+ (100 mM) perfusion, the extracellular levels of dopamine (DA) and amino acids were increased in both genotypes. These increases, however, were significantly lower in KO mice than those in WT mice. Extracellular levels of 3, 4-dihydroxyphenylacetic acid (DOPAC), a major metabolite of DA, were increased in KO mice whereas decreased in WT mice in response to high K+ stimulus. The releases of 4-hydroxy-3-methoxy-phenylacetic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were attenuated to similar extents in the two genotype mice.CONCLUSION: These findings provide direct in vivo evidence for that Kir6.2-containing K-ATP channels participate in the regulation of K+-stimulated releases of striatal neurotransmitters. Kir6.2 deficiency decreased high K+ perfusion-induced releases of DA and amino acids.Partâ…¡Effects of Kir6.2 deficiency on MPTP-induced mouse modelof Parkinson's diseaseAIM: To investigate the effects of K-ATP channels on MPTP-induced chronic PD model using Kir6.2 deficiency mice, and to obtain the direct evidence that K-ATP channels participate in pathophysiological mechanism of PD. METHODS: Kir6.2 KO and WT mice were treated with chronic MPTP intoxication 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 a period of 5 weeks. Iptakalim (Ipt, 10 mg·kg-1·day-1, p.o.), diazoxide (Dia, 10 mg·kg-1·day-1, p.o.) or cromakalim (Cro, 150 or 300μg·kg-1·day-1, p.o.) was administered to mice one hour before the first injection with MPTP on a daily basis for 6 weeks. Mice were killed 1 week after the final injection of MPTP. Locomotor activity test and a pole test were performed under baseline conditions and at 3 days after the final injection of MPTP or saline. Immunohistochemistry was taken for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), 5-bromodeoxyuridine (BrdU) expression. The total numbers of TH-positive neurons in the substantia nigra pars compacts (SNpc), glial cell line-derived neurotrophic factor (GFAP)-positive cells densities in the substantia nigra (SN) and striatum, and BrdU-positive cells in the subventricular zone (SVZ) and subgranular zone (SGZ) were obtained stereologically using the optical fractionator method. HPLC with electrochemical detectionwas used to measure striatal and hippocampal levels of DA, DOPAC, HVA, 5-HT, and 5-HIAA.RESULTS: 1) Kir6.2 KO mice showed less locomotor activity and retarded behavior in pole test compared with WT mice under physiological conditions; 2) Chronic MPTP-treated WT mice exhibited loss of DAergic neurons in SNpc, suppressive neurogenesis in SVZ and SGZ, increased proliferation of astroglial in SN and striatum, and decreased levels of DA, DOPAC, and HVA in striatum. 3) Chronic treatment with MPTP reduced the levels of DA, DOAPC, and HVA in striatum but failed to injure DA ergic neurons in SNpc of KO mice. Compared with WT mouse, MPTP treatment induced slight suppression of neurogenesis and astroglia proliferation. 4) Administration of Ipt (10 mg·kg-1·day-1, p.o), Dia (10 mg·kg-1·day-1, p.o) or Cro (150 or 300μg·kg-1·day-1, p.o) improved the survival of DAergic neurons and neurogenesis, and inhibited MPTP-induced astroglial proliferation; however, these K-ATP channel openers failed to incease the levels of DA, DOPAC, and HVA in the striatum.CONCLUSION: The results from this part provide direct evidence that K-ATP channels participate in the pathophysiological mechanisms of PD. Opening K-ATP channels exerted neuroprotective effects on MPTP-induced neurodegeneration, which may be related to improvement of neurogenesis and suppression of neuroglial proliferation.Partâ…¢Effects of Kir6.2 deficiency on MPTP-induced acute neurotoxicity in C57BL/6J miceAIM: To investigate the effects of K-ATP channels on acute MPTP neurotoxicity using Kir6.2 deficiency mice.METHODS: Kir6.2 KO and WT 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.), Dia (10 mg·kg-1·day-1, p.o.) or Cro (150 or 300μg·kg-1·day-1, p.o.) was administered to mice one hour before the injection of MPTP on a daily basis for 3 days. Mice were killed 3.5 days after the injection. Locomotor activity test and a pole test were performed under baseline conditions and at 2 days after the final injection of MPTP or saline. Immunohistochemistry was taken for TH, GFAP, BrdU expression. The total numbers of TH-positive neurons in the SNc and striatum, GFAP-positive cells densities in the SNc and striatum, and BrdU-positive cells in SVZ and SGZ were obtained stereologically using the optical fractionator method. HPLC with electrochemical detectionwas used to measure striatal and hippocampal levels of DA, DOPAC, HVA, 5-hydroxytryptamine (5-HT), and 5-HIAA.RESULTS: 1) A single MPTP injection decreased the levels of DA, DOPAC, and HVA in the striatum, induced loss of DAergic neurons and fibers in the SNpc and striatum, and increased astroglial proliferation in Kir6.2 KO mice. MPTP injection also inhibited neurogenesis in SVZ and SGZ of KO mice. However, a single MPTP injection only decreased levels of DA, DOPAC, and HVA, and increased astroglial proliferation in the striatum, and induced suppression of neurogenesis in SGZ in WT mice. 2) In WT mice, Ipt (10 mg·kg-1·day-1, p.o.) or Cro (150 or 300μg·kg-1·day-1, p.o.) significantly inhibited MPTP-induced astroglial proliferation. Ipt (10 mg·kg-1day-1, p.o.), Dia (10 mg·kg-1·day-1, p.o.) or Cro (300μg·kg-1·day-1, p.o.) alleviated suppression of neurogenesis in SGZ. In KO mice, Ipt (10 mg·kg-1·day-1, p.o.) or Cro (300μg·kg-1·day-1, p.o.) prevented MPTP-induced DAergic neurons loss in SNpc, inhibited astroglial proliferation in the SN and striatum, and alleviated suppression of neurogenesis in both SVZ and SGZ. However, K-ATP channel openers failed to increase the levels of striatal DA and its metabolites in both mouse genotypes.CONCLUSION: Kir6.2 knockout exacerbated MPTP-induced acute neurotoxicity, suggesting that K-ATP channels could influence the acute neurotoxicity of MPTP. K-ATP channel openers could reverse the acute nerve injure induced by MPTP, which confirmed the neuroprotective effects of K-ATP channels openers.In summary, the present study firstly confirmed that K-ATP channels could regulate DAergic neurotransmission. Then Kir6.2 knockout mice were used to estabolish MPTP-induced acute and chronic neurotoxicity model, providing direct evidence that K-ATP channels participated in the pathophysiological mechanism of PD. The neuroprotective effects of KCOs against MPTP-induced nerve injure suggested that K-ATP channels participate in the reparative process via improvement of adult neurogenesis and modulation of neuroglial functions in mice. |
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