ATP敏感性钾通道在帕金森病模型小鼠神经损伤中的作用
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摘要
帕金森病（Parkinson’s disease, PD）是严重危害中老年人健康的的神经退行性疾病，在65岁以上人群中的发病率约为2%，预计未来十年我国罹患PD的人数将占世界PD患者的60%1。PD特征性的病理标志为中脑黑质致密部（Substantia nigra pars compacta, SNpc）多巴胺能（DAergic）神经元进行性丢失，临床症状包括静止性震颤、肌肉僵直、随意运动迟缓和姿态不稳等。研究发现遗传因素和环境因素的相互作用在调节PD易感性中发挥重要作用。目前认为老化与PD发生最密切相关2。家族性PD中发现α-synuclein突变，开启了PD研究的基因时代。近期研究发现反应性胶质细胞是PD中一个关键因素。星形胶质细胞和小胶质细胞通过分泌一系列促炎或抑炎细胞因子、抗氧化分子和神经营养因子在PD病理过程中发挥重要作用。这些调质成为双刃剑，产生神经损伤和神经保护效应。目前，通过抑制神经炎症、恢复线粒体功能和调节能量代谢从而调节胶质细胞功能已成为治疗干预PD富有前景的策略。剖析神经元-胶质间相互作用的决定性调节因素将为发展治疗PD的多潜能神经保护剂提供有益的靶标和思路。
ATP敏感性钾通道（ATP-sensitive potassium channel，K-ATP通道）是一类耦联细胞代谢和电活动、非电压依赖性的特殊钾离子通道。K-ATP通道由通道形成亚基内向整流钾通道（inwardly rectified potassium channel，Kir）和调节亚基磺酰脲类受体（sulfonylureas，SUR）按4∶4比例组成的异源性八聚体（SUR/Kir6.X），其开闭由细胞的代谢状态即ATP/ADP的水平决定。其中Kir为孔形成亚基，决定通道的离子选择性，包括Kir6.1（KCNJ8）和Kir6.2（KCNJ11）两种异构体，存在近71%相同氨基酸序列。SUR是调节亚基磺酰脲类受体，包括SUR1（ABCC8）和SUR2（ABCC9）两种异构体。
K-ATP通道广泛表达于中枢神经系统，但在不同细胞类型中亚基组成不尽相同。中脑DA神经元表达由Kir6.2和SUR1构成的K-ATP通道3-4，而星形胶质细胞和小胶质细胞则主要表达Kir6.1亚基，并且本实验室前期研究发现神经干细胞表达Kir6.1亚基5。本实验室前期研究及Liss B等均发现Kir6.2构成的K-ATP通道与PD的发生、发展相关6-7，应用Kir6.2敲除鼠的研究证实K-ATP通道选择性调节黑质DA神经元的存活和死亡3，开放Kir6.1构成的K-ATP通道可通过调节胶质细胞功能，抑制其过度活化，促进神经再生与修复，发挥神经保护作用4。因此，不同细胞类型K-ATP通道亚基表达的不同提示，Kir6.1与Kir6.2构成的K-ATP通道的作用存在差异，那么表达于胶质细胞上Kir6.1构成的K-ATP通道如何参与调节MPTP/p PD模型小鼠的DA能神经元损伤及其机制，目前尚未见报道。
本文工作第一部分应用野生型（wild-type，WT）和Kir6.2敲除（Kir6.2knockout，Kir6.2-/-）小鼠，建立MPTP/p PD小鼠模型，在前期研究的基础上进一步阐明Kir6.2缺失通过抑制内质网应激、增强自噬参与对PD进程中DA神经元的保护作用；并且离体培养WT及Kir6.2-/-原代星形胶质细胞，研究、阐明Kir6.2构成的K-ATP通道通过抑制神经元释放α-synuclein调节星形胶质细胞功能；第二部分建立WT及Kir6.1+/-小鼠MPTP/p PD模型，研究、阐明Kir6.1构成的K-ATP与PD发生的相关性，并初步探讨其可能的机制。本文工作的研究结果为K-ATP参与PD病程的发生、发展提供直接的支持证据，并阐明其在PD神经损伤中发挥多靶点的保护作用，为PD的临床治疗学提供新的思路和策略。
第一部分Kir6.2构成的K-ATP通道缺失对PD模型小鼠DA神经元保护作用的机制研究
目的：应用Kir6.2敲除小鼠，研究、阐明Kir6.2对MPTP/p PD模型小鼠神经损伤的调节作用及机制。
方法：应用3月龄野生型（wildtype，WT）及Kir6.2敲除（Kir6.2knockout，Kir6.2-/-）雄性C57BL/6J小鼠，MPTP皮下注射，继而丙磺舒腹腔注射（MPTP20mg·kg-1，s.c.，丙磺舒250mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/pPD小鼠模型。应用酪氨酸羟化酶（tyrosine hydroxylase，TH）免疫组织化学结合体视学计数分析黑质致密部（substantia nigra pars compacta，SNpc）和腹侧被盖区（ventral tegmental area，VTA）DA能神经元的损伤，同时应用尼氏染色（Nissl staining）结合体视学计数分析黑质区（substantia nigra，SN）神经元的损伤；检测黑质区星形胶质细胞的活化（glial fibrillary acidic protein，GFAP染色）。应用免疫组织化学和免疫荧光方法检测SN区α-synuclein的沉积。Western-blotting法检测SN区内质网应激标记物GRP78、CHOP和Caspase12，溶酶体自噬标志物LC3、P62，核转录因子NF-κB p65亚基的表达及能量感受器AMPK（AMP-activated protein kinase）的磷酸化水平；酶联免疫吸附测定（ELISA）法检测血清TNF-α、IL-1β及SN区TNF-α蛋白水平的变化。分离、培养WT及Kir6.2-/-小鼠中脑星形胶质细胞。给予AMPK激动剂AICAR和抑制剂Compoud C及MPP+药物处理，MTT法和LDH测定观察MPP+对星形胶质细胞活力的影响。Western-blotting法观察MPP+对WT、Kir6.2-/-星形胶质细胞AMPK磷酸化水平及核转录因子NF-κB p65亚基的表达。应用RT-PCR及Western-blotting法观察中脑星形胶质细胞炎症因子TNF-α、IL-1β及营养因子GDNF、FGF-2和BDNF的表达。
结果：1）基础状态下，两种基因型小鼠SNpc区和VTA区TH神经元数量无显著差异。MPTP模型制备成功后，WT小鼠SNpc区Nissl染色阳性神经元减少为正常对照的50%；SNpc区TH阳性神经元数量减少为正常对照的59%，VTA区为正常对照的21%。Kir6.2敲除逆转了MPTP诱导的SNpc区DA神经元减少。2）Kir6.2敲除抑制MPTP/p诱导的SNc区星形胶质细胞增殖活化。3）MPTP引起WT小鼠SN区内质网应激增强，内质网应激中蛋白伴侣分子GRP78、转录因子CHOP、效应分子Caspase12表达上调，自噬受阻，溶酶体自噬标志物LC3、自噬底物P62表达水平增加，下游NF-κB信号通路激活。Kir6.2敲除抑制SN区内质网应激，增强自噬，并抑制下游NF-κB信号通路，抑制炎症因子分泌。4）基础状态下，Kir6.2-/-小鼠中脑AMPK磷酸化水平显著升高。MPTP诱导WT小鼠中脑AMPK磷酸化水平上调，Kir6.2-/-小鼠AMPK磷酸水平较WT小鼠升高更为显著。5）Kir6.2敲除抑制基础状态和MPTP/p诱导的中脑SNc区α-synuclein蓄积。6）MPP+（50μM）作用48h可降低WT星形胶质细胞的活力，对Kir6.2-/-星形胶质细胞的活力无显著影响。AICAR（10μM）抑制MPP+对中脑星形胶质细胞的损伤，Compound C（10μM）取消Kir6.2敲除对MPP+诱导中脑星形胶质细胞损伤的保护作用。7）Kir6.2敲除取消MPP+对中脑星形胶质细胞NF-κB的激活作用，抑制炎症因子TNF-α和IL-1β的释放；Kir6.2敲除上调营养因子FGF-2、BDNF及GDNF的蛋白水平。
结论：
1、阐明Kir6.2敲除抑制胶质细胞增殖活化，抑制内质网应激和神经炎症，增强自噬，在小鼠MPTP/p PD模型中发挥神经保护作用。
2、阐明Kir6.2敲除通过抑制神经元分泌并释放α-synuclein，致星形胶质细胞摄取减少，引起星形胶质细胞炎症因子分泌减少，营养因子分泌增加，参与对MPP+诱导的中脑多巴胺能神经元损伤的保护作用。
第二部分Kir6.1构成的K-ATP通道在MPTP/p PD模型小鼠神经损伤中的作用
目的：应用Kir6.1杂合子小鼠，研究、阐明Kir6.1构成的K-ATP通道与PD发生的相关性。
方法：应用3月龄野生型（wildtype，WT）及Kir6.1杂合子（Kir6.1heterozygote，Kir6.1+/-）雄性C57BL/6J小鼠，应用Western-blotting法检测Kir6.1+/-小鼠中脑及纹状体Kir6.1蛋白的表达变化。应用MPTP皮下注射，继而丙磺舒腹腔注射（MPTP20mg·kg-1，s.c.，丙磺舒250mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。应用TH免疫组织化学结合体视学计数分析SNpc和VTA DA能神经元的损伤；同时检测SNpc和VTA GFAP染色、小胶质细胞的活化（Macrophage antigen complex1，MAC-1染色）以及室管膜下层（subventricular zone，SVZ）和颗粒细胞下层（subgranular zone，SGZ）神经再生的变化（5-bromo-2-deoxyuridine，BrdU免疫组化）。应用高效液相色谱法（HPLC）检测纹状体脑区单胺类及氨基酸类神经递质及其代谢产物水平的变化。应用Western-blotting法检测SN区内质网应激标记物CHOP和Caspase12，溶酶体自噬标志物LC3、P62，核转录因子NF-κB p65亚基的表达及营养因子GDNF、FGF-2和BDNF的表达变化。应用ELISA法检测血清TNF-α、IL-1β蛋白水平的变化。应用Realtime PCR法检测SN区促炎因子TNF-α、IL-1β和IL-6及抑炎因子IL-10、TGF-β的mRNA变化。
结果：1）基础状态下，两种基因型小鼠SNpc区和VTA区TH神经元数量无显著差异，纹状体DA及其代谢产物水平无显著差异（p>0.05）。MPTP模型制备成功后，两种基因型小鼠均发生SN区神经元损伤，Kir6.1+/-小鼠神经元损伤更为严重（p<0.05）。WT小鼠SNpc区TH阳性神经元存活率为59%，Kir6.1+/-小鼠TH阳性神经元存活率为46%。WT和Kir6.1+/-小鼠VTA区TH神经元存活率分别为78%和71%。MPTP模型成功后，WT和Kir6.1+/-小鼠纹状体DA及其代谢产物水平均显著降低（p<0.05），但两种基因型之间无显著差异（p>0.05）。2）基础状态下，两种基因型小鼠SN区星形胶质细胞和小胶质细胞数量无显著差异，仅出现少数GFAP、MAC-1阳性染色。MPTP引起WT小鼠SN区星形胶质细胞和小胶质细胞显著增殖活化，Kir6.1+/-小鼠SN区星形胶质细胞和小胶质细胞增殖活化更为显著（p<0.05）。3）基础状态下Kir6.1+/-小鼠SVZ区BrdU阳性细胞数显著少于WT小鼠（p<0.05）。MPTP模型成功后，SVZ和SGZ区神经干细胞数量减少，两种基因型小鼠无显著差异（p>0.05）。4）MPTP诱导WT小鼠产生神经炎症反应，增加SN区NF-κB p65亚基核转位，引起促炎因子TNF-α、IL-1β和IL-6mRNA水平增加，抑炎因子IL-10、TGF-β mRNA水平降低，并伴有外周血清TNF-α、IL-1β产生增加；同时，MPTP诱导内质网应激增强，内质网特异性转录因子CHOP、效应分子Caspase12表达上调，自噬功能受到抑制，溶酶体自噬标志物LC3、自噬底物P62表达均上调。而MPTP引起Kir6.1+/-小鼠炎症反应加剧，内质网应激增强，自噬功能减弱，协同加重DA神经元损伤。5）基础状态下，两种基因型小鼠中脑营养因子GDNF、FGF-2及BDNF表达水平无显著差异。MPTP诱导WT小鼠营养因子GDNF、FGF-2表达增加，BDNF表达降低；但MPTP不影响Kir6.1+/-小鼠GDNF、FGF-2的表达，却进一步加重BDNF表达降低的现象。
结论：
1、应用Kir6.1杂合子小鼠，发现Kir6.1构成的K-ATP通道与PD的发生密切相关。
2、阐明Kir6.1构成的K-ATP通道通过调节胶质细胞功能（加增强营养支持功能，抑制神经炎症），促进神经再生，在PD中发挥神经保护作用。
综上所述，本文工作的主要创新之处在于：
1、应用Kir6.2-/-小鼠，阐明Kir6.2敲除逆转MPTP/p诱导的SNc区DA神经元损伤，其机制与抑制内质网应激和神经炎性反应，增强自噬水平相关；为深化对PD发生发展的认识积累了重要的学术基础，也为PD临床治疗学的突破提供了新的思路。
2、揭示Kir6.2构成的K-ATP通道通过调节神经元α-synuclein的释放影响星形胶质细胞功能。进一步证实星形胶质细胞在PD发生中的重要作用，为靶向于星形胶质细胞功能调节药物应用于PD等神经系统疾病的临床治疗提供了理论依据。
3、应用Kir6.1+/-小鼠，发现Kir6.1构成的K-ATP通道与PD的发生密切相关。Kir6.1基因缺失加重MPTP诱导的SNc区DA神经元损伤，其机制与诱导胶质细胞活化，损伤胶质细胞功能，加重MPTP引起的神经再生抑制相关。研究结果丰富了K-ATP通道在PD神经损伤中的作用机制，为PD的临床治疗提供了有益的靶标和策略。
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 disease
AIM: 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 disease
AIM: 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.
ATP敏感性钾通道（ATP-sensitive potassium channel，K-ATP通道）是一类耦联细胞代谢和电活动、非电压依赖性的特殊钾离子通道。K-ATP通道由通道形成亚基内向整流钾通道（inwardly rectified potassium channel，Kir）和调节亚基磺酰脲类受体（sulfonylureas，SUR）按4∶4比例组成的异源性八聚体（SUR/Kir6.X），其开闭由细胞的代谢状态即ATP/ADP的水平决定。其中Kir为孔形成亚基，决定通道的离子选择性，包括Kir6.1（KCNJ8）和Kir6.2（KCNJ11）两种异构体，存在近71%相同氨基酸序列。SUR是调节亚基磺酰脲类受体，包括SUR1（ABCC8）和SUR2（ABCC9）两种异构体。
K-ATP通道广泛表达于中枢神经系统，但在不同细胞类型中亚基组成不尽相同。中脑DA神经元表达由Kir6.2和SUR1构成的K-ATP通道3-4，而星形胶质细胞和小胶质细胞则主要表达Kir6.1亚基，并且本实验室前期研究发现神经干细胞表达Kir6.1亚基5。本实验室前期研究及Liss B等均发现Kir6.2构成的K-ATP通道与PD的发生、发展相关6-7，应用Kir6.2敲除鼠的研究证实K-ATP通道选择性调节黑质DA神经元的存活和死亡3，开放Kir6.1构成的K-ATP通道可通过调节胶质细胞功能，抑制其过度活化，促进神经再生与修复，发挥神经保护作用4。因此，不同细胞类型K-ATP通道亚基表达的不同提示，Kir6.1与Kir6.2构成的K-ATP通道的作用存在差异，那么表达于胶质细胞上Kir6.1构成的K-ATP通道如何参与调节MPTP/p PD模型小鼠的DA能神经元损伤及其机制，目前尚未见报道。
本文工作第一部分应用野生型（wild-type，WT）和Kir6.2敲除（Kir6.2knockout，Kir6.2-/-）小鼠，建立MPTP/p PD小鼠模型，在前期研究的基础上进一步阐明Kir6.2缺失通过抑制内质网应激、增强自噬参与对PD进程中DA神经元的保护作用；并且离体培养WT及Kir6.2-/-原代星形胶质细胞，研究、阐明Kir6.2构成的K-ATP通道通过抑制神经元释放α-synuclein调节星形胶质细胞功能；第二部分建立WT及Kir6.1+/-小鼠MPTP/p PD模型，研究、阐明Kir6.1构成的K-ATP与PD发生的相关性，并初步探讨其可能的机制。本文工作的研究结果为K-ATP参与PD病程的发生、发展提供直接的支持证据，并阐明其在PD神经损伤中发挥多靶点的保护作用，为PD的临床治疗学提供新的思路和策略。
第一部分Kir6.2构成的K-ATP通道缺失对PD模型小鼠DA神经元保护作用的机制研究
目的：应用Kir6.2敲除小鼠，研究、阐明Kir6.2对MPTP/p PD模型小鼠神经损伤的调节作用及机制。
方法：应用3月龄野生型（wildtype，WT）及Kir6.2敲除（Kir6.2knockout，Kir6.2-/-）雄性C57BL/6J小鼠，MPTP皮下注射，继而丙磺舒腹腔注射（MPTP20mg·kg-1，s.c.，丙磺舒250mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/pPD小鼠模型。应用酪氨酸羟化酶（tyrosine hydroxylase，TH）免疫组织化学结合体视学计数分析黑质致密部（substantia nigra pars compacta，SNpc）和腹侧被盖区（ventral tegmental area，VTA）DA能神经元的损伤，同时应用尼氏染色（Nissl staining）结合体视学计数分析黑质区（substantia nigra，SN）神经元的损伤；检测黑质区星形胶质细胞的活化（glial fibrillary acidic protein，GFAP染色）。应用免疫组织化学和免疫荧光方法检测SN区α-synuclein的沉积。Western-blotting法检测SN区内质网应激标记物GRP78、CHOP和Caspase12，溶酶体自噬标志物LC3、P62，核转录因子NF-κB p65亚基的表达及能量感受器AMPK（AMP-activated protein kinase）的磷酸化水平；酶联免疫吸附测定（ELISA）法检测血清TNF-α、IL-1β及SN区TNF-α蛋白水平的变化。分离、培养WT及Kir6.2-/-小鼠中脑星形胶质细胞。给予AMPK激动剂AICAR和抑制剂Compoud C及MPP+药物处理，MTT法和LDH测定观察MPP+对星形胶质细胞活力的影响。Western-blotting法观察MPP+对WT、Kir6.2-/-星形胶质细胞AMPK磷酸化水平及核转录因子NF-κB p65亚基的表达。应用RT-PCR及Western-blotting法观察中脑星形胶质细胞炎症因子TNF-α、IL-1β及营养因子GDNF、FGF-2和BDNF的表达。
结果：1）基础状态下，两种基因型小鼠SNpc区和VTA区TH神经元数量无显著差异。MPTP模型制备成功后，WT小鼠SNpc区Nissl染色阳性神经元减少为正常对照的50%；SNpc区TH阳性神经元数量减少为正常对照的59%，VTA区为正常对照的21%。Kir6.2敲除逆转了MPTP诱导的SNpc区DA神经元减少。2）Kir6.2敲除抑制MPTP/p诱导的SNc区星形胶质细胞增殖活化。3）MPTP引起WT小鼠SN区内质网应激增强，内质网应激中蛋白伴侣分子GRP78、转录因子CHOP、效应分子Caspase12表达上调，自噬受阻，溶酶体自噬标志物LC3、自噬底物P62表达水平增加，下游NF-κB信号通路激活。Kir6.2敲除抑制SN区内质网应激，增强自噬，并抑制下游NF-κB信号通路，抑制炎症因子分泌。4）基础状态下，Kir6.2-/-小鼠中脑AMPK磷酸化水平显著升高。MPTP诱导WT小鼠中脑AMPK磷酸化水平上调，Kir6.2-/-小鼠AMPK磷酸水平较WT小鼠升高更为显著。5）Kir6.2敲除抑制基础状态和MPTP/p诱导的中脑SNc区α-synuclein蓄积。6）MPP+（50μM）作用48h可降低WT星形胶质细胞的活力，对Kir6.2-/-星形胶质细胞的活力无显著影响。AICAR（10μM）抑制MPP+对中脑星形胶质细胞的损伤，Compound C（10μM）取消Kir6.2敲除对MPP+诱导中脑星形胶质细胞损伤的保护作用。7）Kir6.2敲除取消MPP+对中脑星形胶质细胞NF-κB的激活作用，抑制炎症因子TNF-α和IL-1β的释放；Kir6.2敲除上调营养因子FGF-2、BDNF及GDNF的蛋白水平。
结论：
1、阐明Kir6.2敲除抑制胶质细胞增殖活化，抑制内质网应激和神经炎症，增强自噬，在小鼠MPTP/p PD模型中发挥神经保护作用。
2、阐明Kir6.2敲除通过抑制神经元分泌并释放α-synuclein，致星形胶质细胞摄取减少，引起星形胶质细胞炎症因子分泌减少，营养因子分泌增加，参与对MPP+诱导的中脑多巴胺能神经元损伤的保护作用。
第二部分Kir6.1构成的K-ATP通道在MPTP/p PD模型小鼠神经损伤中的作用
目的：应用Kir6.1杂合子小鼠，研究、阐明Kir6.1构成的K-ATP通道与PD发生的相关性。
方法：应用3月龄野生型（wildtype，WT）及Kir6.1杂合子（Kir6.1heterozygote，Kir6.1+/-）雄性C57BL/6J小鼠，应用Western-blotting法检测Kir6.1+/-小鼠中脑及纹状体Kir6.1蛋白的表达变化。应用MPTP皮下注射，继而丙磺舒腹腔注射（MPTP20mg·kg-1，s.c.，丙磺舒250mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。应用TH免疫组织化学结合体视学计数分析SNpc和VTA DA能神经元的损伤；同时检测SNpc和VTA GFAP染色、小胶质细胞的活化（Macrophage antigen complex1，MAC-1染色）以及室管膜下层（subventricular zone，SVZ）和颗粒细胞下层（subgranular zone，SGZ）神经再生的变化（5-bromo-2-deoxyuridine，BrdU免疫组化）。应用高效液相色谱法（HPLC）检测纹状体脑区单胺类及氨基酸类神经递质及其代谢产物水平的变化。应用Western-blotting法检测SN区内质网应激标记物CHOP和Caspase12，溶酶体自噬标志物LC3、P62，核转录因子NF-κB p65亚基的表达及营养因子GDNF、FGF-2和BDNF的表达变化。应用ELISA法检测血清TNF-α、IL-1β蛋白水平的变化。应用Realtime PCR法检测SN区促炎因子TNF-α、IL-1β和IL-6及抑炎因子IL-10、TGF-β的mRNA变化。
结果：1）基础状态下，两种基因型小鼠SNpc区和VTA区TH神经元数量无显著差异，纹状体DA及其代谢产物水平无显著差异（p>0.05）。MPTP模型制备成功后，两种基因型小鼠均发生SN区神经元损伤，Kir6.1+/-小鼠神经元损伤更为严重（p<0.05）。WT小鼠SNpc区TH阳性神经元存活率为59%，Kir6.1+/-小鼠TH阳性神经元存活率为46%。WT和Kir6.1+/-小鼠VTA区TH神经元存活率分别为78%和71%。MPTP模型成功后，WT和Kir6.1+/-小鼠纹状体DA及其代谢产物水平均显著降低（p<0.05），但两种基因型之间无显著差异（p>0.05）。2）基础状态下，两种基因型小鼠SN区星形胶质细胞和小胶质细胞数量无显著差异，仅出现少数GFAP、MAC-1阳性染色。MPTP引起WT小鼠SN区星形胶质细胞和小胶质细胞显著增殖活化，Kir6.1+/-小鼠SN区星形胶质细胞和小胶质细胞增殖活化更为显著（p<0.05）。3）基础状态下Kir6.1+/-小鼠SVZ区BrdU阳性细胞数显著少于WT小鼠（p<0.05）。MPTP模型成功后，SVZ和SGZ区神经干细胞数量减少，两种基因型小鼠无显著差异（p>0.05）。4）MPTP诱导WT小鼠产生神经炎症反应，增加SN区NF-κB p65亚基核转位，引起促炎因子TNF-α、IL-1β和IL-6mRNA水平增加，抑炎因子IL-10、TGF-β mRNA水平降低，并伴有外周血清TNF-α、IL-1β产生增加；同时，MPTP诱导内质网应激增强，内质网特异性转录因子CHOP、效应分子Caspase12表达上调，自噬功能受到抑制，溶酶体自噬标志物LC3、自噬底物P62表达均上调。而MPTP引起Kir6.1+/-小鼠炎症反应加剧，内质网应激增强，自噬功能减弱，协同加重DA神经元损伤。5）基础状态下，两种基因型小鼠中脑营养因子GDNF、FGF-2及BDNF表达水平无显著差异。MPTP诱导WT小鼠营养因子GDNF、FGF-2表达增加，BDNF表达降低；但MPTP不影响Kir6.1+/-小鼠GDNF、FGF-2的表达，却进一步加重BDNF表达降低的现象。
结论：
1、应用Kir6.1杂合子小鼠，发现Kir6.1构成的K-ATP通道与PD的发生密切相关。
2、阐明Kir6.1构成的K-ATP通道通过调节胶质细胞功能（加增强营养支持功能，抑制神经炎症），促进神经再生，在PD中发挥神经保护作用。
综上所述，本文工作的主要创新之处在于：
1、应用Kir6.2-/-小鼠，阐明Kir6.2敲除逆转MPTP/p诱导的SNc区DA神经元损伤，其机制与抑制内质网应激和神经炎性反应，增强自噬水平相关；为深化对PD发生发展的认识积累了重要的学术基础，也为PD临床治疗学的突破提供了新的思路。
2、揭示Kir6.2构成的K-ATP通道通过调节神经元α-synuclein的释放影响星形胶质细胞功能。进一步证实星形胶质细胞在PD发生中的重要作用，为靶向于星形胶质细胞功能调节药物应用于PD等神经系统疾病的临床治疗提供了理论依据。
3、应用Kir6.1+/-小鼠，发现Kir6.1构成的K-ATP通道与PD的发生密切相关。Kir6.1基因缺失加重MPTP诱导的SNc区DA神经元损伤，其机制与诱导胶质细胞活化，损伤胶质细胞功能，加重MPTP引起的神经再生抑制相关。研究结果丰富了K-ATP通道在PD神经损伤中的作用机制，为PD的临床治疗提供了有益的靶标和策略。
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 disease
AIM: 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 disease
AIM: 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.
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