Role Of K-atp Channels In Neural Injury In Mptp/p Mouse Model Of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease,which charactered by the gradual, irreversible loss of dopaminergic (DA) neurons inthe substantia nigra compacta (SNc). However, the detailed mechanisms underlyingPD remain unclear8-10, which have been the bottleneck of the PD clinical therapeuticsand the ideal treatment. The crucial cause results in the onset of PD is considered tobe age or the aging process. α-synuclein is a major component of Lewy bodies inidiopathic, apparently sporadic PD and point mutations or multiplications in theα-synuclein gene are a cause of autosomal recessive PD. In recent decades thefunctional relevance and versatility of neuroglia have started to be fullyappreciated11-13. Impairments in astrocytic function are increasingly beingrecognized as an important contributor to neuronal dysfunction and, in particular,neurodegenerative processes. Meanwhile, morphologic changes of microglia andaccumulation of proinflammatory factors are associated with degenerating DAneurons in the SNc of PD patients and various PD animal models14-16.ATP-sensitive potassium (K-ATP) channels provide a unique link betweencellular energetics and electrical excitability17. Metabolic stresses such as hypoxia,ischemia, or hypoglycemia lead to activation of the K-ATP channel. K-ATPchannels are known to be present in many tissue types with a variety of functions18.It has been documented that neurons mainly express K-ATP channel-forming Kir6.2subunit whereas Kir6.1is the principal pore-forming subunit of astrocytes19.Activation of the neuronal K-ATP channels during depletion of ATP hyperpolarizes the neuron membrane, thereby reducing neuronal excitability and ATP consumption.Therefore, pharmacological agents that target this channel may potentially haveclinical utility for therapy. Emerging evidence indicates that K-ATP channel may bea promising target for protecting DA neurons in PD. Birgit Liss et. al had reportedthat genetic inactivation of Kir6.2resulted in a selective rescue of SNc DA neurons inthe neurotoxicological MPTP model. We have also demonstrated previously thatboth genetic inactivation of Kir6.2and activation of K-ATP channels withnon-selective K-ATP channel openers, contributed to a selective rescue of SNc DAneurons in the MPTP model of PD. We have found that not only astrocytes but alsomicroglia expressed Kir6.1-containing K-ATP channels7. In glia, Kir6.1-containingK-ATP channels have been implicated in the regulation of neurodegeneration. Theymay thereby harbor the potential to translate signals of demand, as occur undersyndromes of systemic metabolic stress such as in PD. Although theelectrophysiological and pharmacological properties of Kir6.2-containing K-ATPchannels have been well characterized in reconstituted systems20-22and nativetissues23-27, those of Kir6.1-containing K-ATP channels are not fully understood.Given this background, the aim of present studies is to investigate the regulationmechanism of K-ATP channels in PD. We first explore the roles and the involvedmechanisms of Kir6.2containing K-ATP channels in MPTP/p PD mouse model.Then, the primary cultured astrocytes were used to study the regulatory effects ofα-synuclein and Kir6.2containing K-ATP channels on astrocytic function. Finally,we probed the outcome of genetic disruption of Kir6.1in a chronic MPTP/p model, todemonstrate the potential impact of Kir6.1-containing K-ATP channel in PD.Part I The protective effects of Kir6.2deficiency inMPTP/p mouse model of Parkinson’s diseaseAIM: To investigate the protective mechanism of Kir6.2deficiency on thedegeneration of dopaminergic neurons in MPTP/p PD model using Kir6.2deficiencymice.METHODS: WT and Kir6.2-/-mice were treated with chronic MPTP intoxication protocol:20mg·kg-1MPTP in saline was injected subcutaneously, and250mg·kg-1probenecid in DMSO was injected intraperitoneally every3.5d over aperiod of5weeks. Mice were killed1week after the final injection of MPTP.Nissl-stained nigral neurons represented survival neurons. Immunohistochemistrywas taken for tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP)expression. The total numbers of TH-positive neurons and GFAP-positive cells inthe substantia nigra pars compacts (SNpc) and ventral tegmental area (VTA) wereobtained stereologically using the optical fractionator method.Immunohistochemistry and immunofluorescence were taken for α-synuclein in SNc.The levels of endoplasmic reticulum stress (ERS), autophagy and NF-κB p65subunitin SN were determined by western blotting, respectively. The levels of TNF-α andIL-1β were determined by Realtime PCR and ELISA, respectively. Mesencephalicprimary astrocyte culture was prepared from the ventral mesencephalic tissues ofpostnatal (P1-P2) WT and Kir6.2-/-C57BL/6J mice. Preincubate astrocytes withAMPK agonist AICAR (10μM) and antagonist Compound C (10μM) after MPP+(50μM) treatment for48hours. MTT assay was employed to examine astrocyteviability and LDH measurement was applied to detect cellular injury. Westernblotting was used to analysis the phosphorylation of AMPK and the expression ofNF-κB p65subunit. Realtime PCR and Western blotting were used to analysis theexpression of neurotrophic factors in astrocytes, including FGF-2, BDNF and GDNF.Realtime PCR and ELISA were used to analysis the expression of inflammationfactor, including TNF-α and IL-1β, respectively.RESULTS:1) There was no significant difference (p>0.05) of DAergic neuronsin SNc and VTA in saline group between WT and Kir6.2-/-mice. Chronic MPTPtreatment decreased Nissl-stained neurons, and reduced DAergic neurons in SNpc inWT mice, but not in Kir6.2-/-mice.2) Chronic MPTP treatment induced increasedactivation of astrocytes, but not in Kir6.2-/-mice.3) Chronic MPTP treatmentinduced activation of ERS, upregulated GRP78, CHOP, and caspase12expression.Simultaneously, chronic MPTP treatment inhibited autophagy, upregulated LC3andP62, induced downstream inflammation. On the contrary, Kir6.2knockout inhibitedERS and inflammation, enhanced autophagy.4) Kir6.2knockout increased the phosphorylation of AMPK both in normal and MPTP/p treatment conditions.5)Kir6.2deficiency abolished MPTP/p-induced accumulation of α-synuclein in SNc.6) Kir6.2knockout resulted in hyposensitive to MPP+(50μM) induced neurotoxicityin primary cultured astrocytes. AICAR could reverse MPP+-induced loss ofastrocytes, and Compound C remarkably aggravate loss of Kir6.2-/-astrocytes.7)MPP+induced increased expression of inflammatory factors including TNF-α andIL-1β in WT astrocytes, but not in Kir6.2-/-astrocytes. MPP+(50μM) treatment for48h significantly increased GDNF and FGF-2protein expression in Kir6.2-/-astrocytes, but less extent in WT astrocytes.CONCLUSION:1). Kir6.2knockout inhibited proliferation and activation of astrocytes,concomitantly, inhibited ERS and enhanced autophagy, which may exertedneuroprotective effects on MPTP-induced neurodegeneration.2). Kir6.2deficiency in Dopaminergic neurons led to less α-synucleinaccumulation in PD model, consequently influenced astrocytes function less, whichwas related to the increased expression of neurotrophic factors and decreasedexpression of inflammatory factors in astrocytes.Part II Effects of Kir6.1containing K-ATP channels inMPTP/p mouse model of Parkinson’s diseaseAIM: To investigate the effects of Kir6.1on MPTP-induced chronic PD modelusing Kir6.1heterozygote mice, and to obtain the direct evidence that Kir6.1containing K-ATP channels participate in pathophysiological mechanism of PD.Methods: WT and Kir6.1+/-mice were treated with chronic MPTP intoxicationprotocol:20mg kg–1MPTP in saline was injected subcutaneously, and250mg kg–1probenecid in DMSO was injected intraperitoneally every3.5d over a period of5weeks. Mice were killed1week after the final injection of MPTP.Immunohistochemistry was taken for tyrosine hydroxylase (TH), glial fibrillary acidicprotein (GFAP), macrophage-1antigen (Mac-1) and5-bromodeoxyuridine (BrdU) expression. The total numbers of TH-positive neurons, GFAP and MAC-1positivecells in the substantia nigra pars compacts (SNpc) and ventral tegmental area (VTA),and BrdU-positive cells in the subventricular zone (SVZ) and subgranular zone (SGZ)were obtained stereologically using the optical fractionator method. HPLC withelectrochemical detection was used to measure striatal levels of differentneurotransmitters, including DA, DOPAC, HVA,5-HT,5-HIAA, glutamate, GABAand so on. The levels of endoplasmic reticulum stress (ERS), autophagy and NF-κBp65subunit in SN were determined by western blotting, respectively. Realtime PCRand Western blotting were used to analysis the expression of neurotrophic factors inSN, including FGF-2, BDNF and GDNF. The levels of proinflammatory factors andantiinflammatory cytokines were determined by Realtime PCR and ELISA,respectively.Results:1) Kir6.1+/-mice show increased nigrostriatal dopaminergic neurondegeneration in response to chronic MPTP/p treatment. However, there was nosignificant difference (p>0.05) in neurotransmitters in striatum between WT andKir6.1+/-mice.2) MPTP induces SNc glial activation and proliferation in Kir6.1+/-mice, including astrocytes and microglias.3) The BrdU-ir cell number of Kir6.1+/-mice in the SVZ is smaller than WT mice.4) ER stress is aggravated, andautophagy is inhibited more severely in SN after chronic MPTP/p treatment inKir6.1+/-mice compared with WT mice. Furthermore, neuroinflammation isdeteriorated in SN after chronic MPTP/p treatment in Kir6.1+/-mice.5)Neurotrophic factor is downregulated after chronic MPTP/p treatment in Kir6.1+/-mice.CONCLUSION:1) The results from this part provide direct evidence that Kir6.1containingK-ATP channels participate in the pathophysiological mechanisms of PD.2) The neuroprotective effects of Kir6.1containing K-ATP channels may berelated to ameliorating glia functions (increasing expression of neurotrophic factorsand suppression of inflammation) and improvement of neurogenesis. In summary, the major contributions of the present study lie in:1、Knockout of Kir6.2-containing K-ATP channels exerts neuroprotectiveeffects on MPTP-induced neurodegeneration through suppressing ERS andinflammation, enhancing autophagy, which provides a novel therapeutic approachfor neurodegenerative diseases.2、Deficiency of Kir6.2-containing K-ATP channels in dopaminergic neuronregulates astrocyte functions through α-synuclein. The therapeutic strategy targetedto astrocytic modulation may offer a new perspective for the development of noveloptions for PD treatment.3、Kir6.1-containing K-ATP channels are involved in the initiation andprogress of PD and the underlying mechanisms predominantly lie in the modulationof glia functions and neurogenesis.