K-ATP通道与帕金森病的相关性研究
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摘要
ATP敏感性钾通道（ATP-sensitive potassium channel，K-ATP通道）是一类偶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通道由调节亚基磺酰脲类受体（sulfonylureas，SUR）和通道形成亚基内向整流钾通道（inwardly rectified potassium channel，Kir）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的水平所决定。K-ATP广泛表达于中枢神经系统，但其亚基组成不尽相同，在中脑和VTA区的多巴胺（Dopamine，DA）能神经元主要表达Kir6.2亚基，而在星形胶质细胞和小胶质细胞则主要表达Kir6.1亚基，本实验室发现神经干细胞表达Kir6.1亚基。近年来，包括本实验室在内的研究表明K-ATP通道不仅在急性缺血缺氧、代谢抑制等病理因素导致的细胞损伤中发挥重要的作用，而且与帕金森病（Parkinson’sdisease，PD）的发生、发展密切相关。
PD是一类由于黑质致密部DA能神经元的渐进性丢失导致的以运动障碍为特征的进行性神经退行性疾病。PD发病率逐年增加，预计在未来的25年内，在包括中国在内的10个人口最稠密的国家罹患PD的人数将至少翻一番。针对导致DA能神经元缺失的病理机制，尽管已提出兴奋性毒性、线粒体功能障碍、氧化应激和炎症等诸多假说，但是，根据这些假说而研发的NMDA受体拮抗剂、抗氧化剂及凋亡抑制剂等大多仅在实验研究中显现显著神经保护作用，而在临床研究中均未获得理想的神经保护效果。因此，深入研究、探索PD的病因学，对于研发新型治疗药物和临床治疗学的突破具有重要意义。
本实验室的前期研究发现，K-ATP通道开放剂（K-ATP channel openers，KCOs）在6-羟基多巴胺（6-hydroxydopamine，6-OHDA）、鱼藤酮、1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium，MPP+）导致的多种PD动物模型中发挥显著的神经保护作用，其机制涉及抗兴奋性毒性、抗凋亡、抑制神经炎症和保护线粒体功能等。然而，本实验室近期应用Kir6.2敲除鼠的研究却发现：Kir6.2敲除或Kir6.1/Kir6.2混合型开放剂均能取消1-甲基-4-苯基-1，2，3，6-四氢吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydoyndine，MPTP）联合丙磺舒（probenecid）所致PD模型（MPTP/p PD模型）小鼠黑质DA能神经元的损伤作用，该发现不仅有悖于开放Kir6.2构成的K-ATP通道可发挥保护作用的传统观点，也提示表达于胶质细胞和神经干细胞上Kir6.1构成的K-ATP通道在MPTP诱导的DA能神经元损伤中发挥了重要的调节作用。近年认识到胶质细胞，尤其是星形胶质细胞在维持神经电活动正常环境、调节神经递质代谢、保护神经元及促神经再生和修复过程中均发挥重要的作用。本实验室的前期研究发现，开放星形胶质细胞上K-ATP通道可以通过抑制线粒体凋亡通路以及MAPK信号通路保护MPP+诱导的星形胶质细胞的损伤；开放小胶质细胞上的K-ATP通道，可抑制鱼藤酮所致PD模型鼠小胶质细胞的活化，从而保护中脑多巴胺能神经元。但是，表达于胶质细胞的K-ATP通道是否参与调节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Kir6.2野生型（wild-type，Kir6.2+/+）和敲除型（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胶质细胞以及神经干细胞上K-ATP通道对PD的调节作用；第二部分离体培养Kir6.2+/+及Kir6.2-/-原代中脑神经元以及星形胶质细胞，并建立神经元与星形胶质细胞共培养体系，研究、阐明K-ATP通道对星形胶质细胞功能的调节；第三部分建立Kir6.2+/+及Kir6.2-/-小鼠慢性温和刺激（chronic mild stress，CMS）模型，研究K-ATP通道对成年鼠脑内神经再生的调节作用；第四部分应用蛋白质组学技术研究Kir6.2+/+及Kir6.2-/-小鼠中脑组织、中脑神经元以及星形胶质细胞基础状态以及在PD模型中相关蛋白表达的改变，在蛋白水平阐明K-ATP通道调节PD发生与发展的机制。
第一部分K-ATP通道在MPTP/p PD模型小鼠神经损伤中的作用
目的：应用Kir6.2敲除小鼠，研究、阐明K-ATP通道对MPTP/p PD模型小鼠神经损伤的调节作用及机制。
方法：应用MPTP皮下注射，继而丙磺舒腹腔注射（MPTP 20 mg·kg-1，s.c.，丙磺舒250 mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。IPT（10 mg·kg-1·day-1，p.o.）在MPTP首次给药前1h开始灌胃，连续给药6周。此外，应用MPTP皮下注射，继而丙磺舒腹腔注射单次给药模式建立MPTP急性损伤模型。埃他卡林（Iptakalim，IPT）（10 mg·kg-1·day-1，p.o.）在MPTP给药前1 h灌胃，连续给药3天。应用酪氨酸羟化酶（tyrosine hydroxylase，TH）免疫组织化学结合体视学计数、ImageJ软件分析黑质致密部（substantia nigra parscompacta，SNpc）DA能神经元及纹状体DA投射纤维的损伤，同时检测黑质（substantia nigra，SN）区星形胶质细胞的活化（glial fibrillary acidic protein，GFAP染色）、小胶质细胞的活化（ionized calcium binding adaptor molecule1，Iba1染色）以及室管膜下层（subventricular zone，SVZ）和颗粒细胞下层（subgranularzone，SGZ）神经再生的变化（5-bromo-2-deoxyuridine，BrdU免疫组化）；免疫双标观察SN区神经元和星形胶质细胞D-丝氨酸（D-Serine，D-Ser）及其合成酶丝氨酸消旋酶（serine racemase，SR）的表达水平；Western-blotting法检测SN区缝隙连接蛋白Connexin 43（Cx43）表达的变化；ELISA法检测SN中TNF-α蛋白水平的变化。
结果：1）MPTP慢性给药后，Kir6.2+/+小鼠SNpc DA能神经元数量仅为正常对照的49%，同时伴有纹状体DA能纤维投射的缺失。Kir6.2-/-小鼠未出现SNpc DA能神经元数量的减少，尽管Kir6.2-/-小鼠纹状体也存在着TH阳性纤维投射的丧失，但是其程度较Kir6.2+/+小鼠轻。IPT能改善MPTP慢性给药造成的Kir6.2+/+小鼠SNpc DA能神经元的数量减少和纹状体DA能神经纤维投射的丧失。MPTP单次给药显著降低Kir6.2-/-小鼠SNpc TH阳性细胞数，并且导致纹状体TH阳性纤维密度显著减少。IPT显著改善MPTP单次给药对Kir6.2-/-小鼠SNpcTH神经元的损伤，并且可以减轻纹状体TH阳性纤维投射的损伤。2）MPTP慢性给药引起Kir6.2+/+小鼠SN星形胶质细胞和小胶质细胞显著增殖活化，并且黑质区主要表达于星形胶质细胞上的Cx43以及主要由小胶质细胞释放的TNF-α也出现显著的增加；SVZ和SGZ区神经干细胞数量显著减少。而Kir6.2-/-小鼠未出现SN星形胶质细胞和小胶质细胞的活化以及SVZ和SGZ区神经干细胞数量的减少。IPT能显著抑制MPTP导致的Kir6.2+/+小鼠SN星形胶质细胞和小胶质细胞的活化以及SVZ和SGZ区神经干细胞数量的减少。3）免疫双标显示MPTP慢性给药诱导Kir6.2+/+小鼠黑质星形胶质细胞D-Ser和SR表达增加，不影响TH神经元D-Ser和SR的表达。IPT显著抑制MPTP慢性给药导致的Kir6.2+/+小鼠黑质星形胶质细胞D-Ser和SR表达增加。
结论：
1、应用Kir6.2敲除小鼠的研究证实K-ATP通道与PD的发生发展密切相关，获得了K-ATP通道与PD相关性的直接证据。
2、开放Kir6.1构成的K-ATP通道可通过调节胶质细胞功能，抑制其活化，促进神经再生与修复，发挥神经保护作用。
第二部分K-ATP通道对星形胶质细胞功能的调节：神经-胶质细胞共培养研究
目的：应用Kir6.2敲除小鼠，研究、阐明K-ATP通道对离体PD模型中星形胶质细胞功能的调节及机制。
方法：分离、培养Kir6.2+/+及Kir6.2-/-小鼠中脑神经元以及星形胶质细胞。给予三种不同亚基敏感性的KATP通道开放剂及MPP+药物处理，通过TH免疫细胞化学染色，研究K-ATP通道对MPP+诱导的原代培养中脑神经元尤其是DA能神经元损伤作用的调节。MTT法观察MPP+对星形胶质细胞活力的影响。应用RT-PCR及Western-blotting法观察星形胶质细胞Kir6.2的表达以及MPP+对Kir6.2+/+、Kir6.2-/-星形胶质细胞Kir6.1表达的影响。Western-blotting法观察MPP+对Kir6.2+/+、Kir6.2-/-星形胶质细胞神经营养因子表达的影响。免疫双标检测星形胶质细胞D-Ser及其合成酶SR的表达。建立神经元-星形胶质细胞共培养体系，TH免疫组化以及免疫双标观察星形胶质细胞对MPP+诱导的中脑DA能神经元损伤的影响以及星形胶质细胞D-Ser和SR的表达。
结果：1）MPP+呈浓度依赖性地损伤原代培养的Kir6.2+/+、Kir6.2-/-中脑DA能神经元，但对Kir6.2+/+神经元的损伤作用较Kir6.2-/-神经元显著；吡那地尔（pinacidil，Pin）和二氮嗪（diazoxide，Dia）预处理减轻MPP+对Kir6.2+/+神经元的损伤作用，而克罗卡林（cromakalim，Cro）则无此效应；三种KCOs均不能减轻MPP+对Kir6.2-/-中脑DA能神经元的损伤。2）Kir6.2+/+、Kir6.2-/-星形胶质细胞与Kir6.2+/+、Kir6.2-/-中脑神经元共培养后，Kir6.2-/-星形胶质细胞的存在可减轻MPP+对Kir6.2+/+、Kir6.2-/-中脑DA能神经元的损伤。3）星形胶质细胞与神经元混合培养后，MPP+诱导Kir6.2+/+星形胶质细胞活化、D-Ser和SR表达增加，Kir6.2敲除抑制混合培养中MPP+诱导的星形胶质细胞的活化，减少D-Ser和SR的表达，保护神经元。4）MPP+（50μM）作用48可降低Kir6.2+/+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；MPP+（10，50μM）作用72h可降低Kir6.2+/+、Kir6.2-/-星形胶质细胞的活力，对Kir6.2+/+星形胶质细胞的损伤作用较Kir6.2-/-严重。5）中脑星形胶质细胞表达Kir6.2亚基；Kir6.2敲除后，星形胶质细胞Kir6.1的表达降低；MPP+作用48h诱导Kir6.2敲除星形胶质细胞Kir6.1表达增加。6）Kir6.2基因缺失后，中脑星形胶质细胞神经营养因子FGF2（Fibroblast growth factor-2）、BDNF（Brain-derived neurotrophic factor）表达增加；MPP+（10μM）作用48h可诱导Kir6.2-/-星形胶质细胞BDNF表达增加，但不影响Kir6.2+/+星形胶质细胞FGF2和BDNF的表达。
结论：
1、星形胶质细胞参与了Kir6.2敲除后对MPP+诱导的中脑多巴胺能神经元损伤的保护作用。
2、上调并开放星形胶质细胞上Kir6.1构成的K-ATP通道可通过促进营养因子的表达，维持正常的胶质传递，减轻兴奋性毒性损伤，发挥对神经元的保护作用。
第三部分K-ATP通道对成年小鼠脑神经再生的调节
目的：建立CMS模型，研究、阐明K-ATP通道对神经再生的调节作用及机制。
方法：建立Kir6.2+/+及Kir6.2-/-小鼠CMS模型，连续4周给予氟西汀（Fluoxetine，Flx）（10 mg·kg-1·day-1，i.p.）或IPT（10 mg·kg-1·day-1，i.p.）。通过糖水偏爱、悬尾实验等行为学研究Flx以及IPT对动物抑郁样症状的改善情况。腹腔注射BrdU（50 mg/kg，每两小时一次，共四次）24小时及28天后分批处死小鼠，灌注、取脑，行NeuN、GFAP、BrdU免疫组化/免疫荧光双重标记，通过计数海马CA3区NeuN以及SGZ区BrdU阳性细胞数，NeuN/BrdU、GFAP/BrdU阳性细胞数测定海马CA3神经元的数量、海马神经干细胞增殖、存活及分化。取新鲜脑组织，匀浆后分别测定海马BDNF的含量，Kir6.2、Kir6.1的表达，Akt、GSK3β、ERK1/2、JNK1/2、p38、CREB磷酸化水平及5-HT水平，取躯干血测定血浆皮质酮水平。分离培养成年Kir6.2+/+及Kir6.2-/- C57BL/6J雄性小鼠海马及侧脑室神经干细胞，应用RT-PCR、Western blotting法检测原代培养的成年C57BL/6J小鼠神经干细胞上K-ATP通道的表达；[3H]-脱氧胸腺嘧啶摄取实验检测IPT对原代培养的成年鼠神经干细胞增殖的影响。
结果：1）悬尾实验及强迫游泳实验结果显示，Kir6.2敲除鼠具有抗抑郁样行为；CMS造模对Kir6.2-/-小鼠行为学影响较Kir6.2+/+小鼠显著，IPT及Flx对Kir6.2-/-小鼠治疗作用弱于Kir6.2+/+小鼠。CMS造模及IPT，Flx药物治疗对Kir6.2+/+小鼠海马CA3区神经元数量无显著影响；CMS造模显著减少Kir6.2-/-小鼠海马CA区神经元数量；4周IPT及Flx治疗能阻断CMS对Kir6.2-/-小鼠海马CA3神经元的损伤。2）正常情况下，两种基因型小鼠SGZ区神经再生无显著差异；CMS造模能显著减少两种基因型小鼠海马SGZ区BrdU标记阳性细胞数；4周IPT与Flx药物治疗均能逆转CMS对两种基因型小鼠SGZ区神经再生的抑制。CMS造模及IPT，Flx药物治疗对两种基因型小鼠BrdU阳性细胞的分化均无显著影响。3）Kir6.2-/-小鼠基础状态下海马BDNF含量显著低于Kir6.2+/+小鼠；CMS造模后两种基因型小鼠海马BDNF水平均显著降低，4周IPT及Flx治疗后均能逆转两种基因型小鼠海马BDNF水平的降低。CMS造模后，Kir6.2+/+小鼠海马中Kir6.2亚基表达上调；4周IPT及Flx治疗后未逆转CMS所致的海马Kir6.2亚基的上调。CMS造模后两种小鼠海马Kir6.1表达均上调；4周IPT及Flx治疗后不改变CMS所致海马Kir6.1表达的上调。4）CMS造模不影响海马中Akt的磷酸化水平，抑制两种基因型小鼠海马GSK3β、ERK1/2以及CREB的磷酸化，对Kir6.2+/+小鼠海马JNK1/2的磷酸化无显著影响，但能显著促进Kir6.2-/-小鼠海马JNK1/2的磷酸化，对两种基因型小鼠海马p38磷酸化水平无显著影响。4周IPT及Flx治疗后能逆转CMS造模所致两种基因型小鼠海马GSK3β、ERK1/2磷酸化的抑制；逆转CMS对Kir6.2+/+小鼠海马CREB磷酸化的抑制；Flx能逆转CMS对Kir6.2-/-小鼠CREB磷酸化水平的抑制，IPT能增加Kir6.2-/-小鼠海马CREB磷酸化水平；4周IPT及Flx治疗不影响Kir6.2+/+小鼠海马JNK1/2磷酸化，能逆转CMS所致Kir6.2-/-小鼠海马JNK1/2磷酸化水平的升高；显著降低两种基因型小鼠海马p38磷酸化水平。5）CMS造模使两种基因型小鼠海马5-HT水平降低，对其代谢产物5-HIAA水平无显著影响；4周IPT给药能逆转CMS造模所致两种基因型小鼠海马5-HT水平的降低，对其代谢产物5-HIAA水平无显著影响；4周Flx给药不改变CMS造模所致海马5-HT水平的降低，显著降低海马5-HIAA的水平。CMS造模能显著升高两种基因型小鼠血浆皮质酮水
平；4周IPT治疗后能逆转CMS造模引起的Kir6.2+/+小鼠血浆皮质酮水平，Flx显著降低CMS造模引起的Kir6.2+/+小鼠血浆皮质酮水平；IPT与Flx均不能显著抑制CMS造模所致的Kir6.2-/-小鼠血浆皮质酮水平的升高。6）原代培养的C57BL/6J小鼠神经干细胞表达Kir6.1、SUR1亚基组成的K-ATP通道，而不表达Kir6.2、SUR2亚基。IPT（1μM）可显著增强两种基因型小鼠海马神经干细胞的增殖。
结论：
1、原代培养的成年小鼠神经干细胞表达Kir6.1和SUR1亚基构成的K-ATP通道，其参与调节神经干细胞的增殖。
2、IPT通过开放Kir6.1亚基构成的K-ATP通道促进神经再生，其作用机制与活化ERK-CREB信号通路、促进BDNF的产生相关。
3、Kir6.2亚基构成的K-ATP通道在CMS过程中发挥对CA3区神经元的保护作用。
第四部分K-ATP通道与PD的相关性：蛋白质组学研究
目的：应用整体与离体蛋白质组学，研究、阐明K-ATP通道对帕金森病的调节作用及机制。
方法：应用Kir6.2+/+、Kir6.2-/- C57BL/6J小鼠，MPTP皮下注射，继而丙磺舒腹腔注射（MPTP 20 mg·kg-1，s.c.，丙磺舒250 mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。最后一次给药后7 d时，断头处死小鼠，迅速取出大脑，预冷超纯水洗去血渍，在冰面上分离中脑，-70°C保存备用。部分小鼠灌注、取脑，行黑质区TH免疫组化。分离、培养Kir6.2+/+、Kir6.2-/- C57BL/6J小鼠中脑神经元以及星形胶质细胞。MPP+孵育48 h后，D-hank’s漂洗，经胰酶消化后，1500 rpm离心5 min，收集于5 ml EP管中。在冰浴条件下，向小鼠中脑样品或神经元样品加入裂解液，超声2 min，4°C冰箱作用1 h，4°C 40000 g离心1 h，吸取上清液，分装-70°C保存备用。Bradford比色法检测蛋白浓度。取180μg蛋白/样品，行双向电泳与图像分析，选取差异2倍及以上的蛋白点，经胰蛋白酶消化，行MALDI-TOF质谱分析，Mascot软件（http://www.matrixscience.com）检索Swiss-Prot数据库（http://www.expasy.org）鉴定蛋白质。
结果：1）基础状态下，Kir6.2敲除导致小鼠中脑39个蛋白表达发生显著变化，其中NADH-ubiquinone oxidoreductase 75 kDa subunit、ATP synthase subunitbeta蛋白在Kir6.2-/-小鼠表达显著上调，而Serotransferrin蛋白表达则显著下调；MPTP慢性造模引起Kir6.2+/+小鼠23个蛋白表达发生显著变化，Fascin、Heatshock 70 kDa protein 4等多个蛋白表达下调，而Aldehyde dehydrogenase等蛋白表达显著上调；MPTP慢性造模引起Kir6.2-/-小鼠23个蛋白表达发生显著变化，NADH dehydrogenase flavoprotein 1、Isoform 1 of 4-aminobutyrateaminotransferase、Glutathione synthetase等蛋白表达显著下调，而Aldose reductase等蛋白表达则显著上调。2）基础状态下，Kir6.2敲除导致小鼠中脑神经元20个蛋白表达发生显著变化，其中Isoform 3 of Septin-11、Proteasome subunit betatype-3等与细胞传递、蛋白降解相关的蛋白表达则显著下调，而Tubulin beta-5chain、Collapsin response mediator protein 4A、Dihydropyrimidinase-related protein2、78 kDa glucose-regulated protein和Elongation factor 2等分别与细胞骨架、突触发育、应激分子伴侣、蛋白合成等功能相关的蛋白在Kir6.2-/-小鼠中脑神经元表达显著上调；MPP+处理引起Kir6.2+/+中脑神经元48个蛋白表达发生显著变化，Vimentin、Heat shock cognate 71 kDa protein等多个蛋白表达下调，而Tubulinbeta-3 chain、78 kDa glucose-regulated protein、Enolase等蛋白表达显著上调；MPP+处理引起Kir6.2-/-小鼠中脑神经元44个蛋白表达发生显著变化，Alpha-enolase、Vinculin、Heat shock cognate 71 kDa protein等蛋白表达则显著下调，而Tubulin alpha-1A chain、Protein disulfide-isomerase A3、centromere proteinF等蛋白表达显著上调。3）基础状态下，Kir6.2敲除导致小鼠中脑星形胶质细胞30个蛋白表达发生显著变化，其中Isoform 3 of Glial fibrillary acidic protein、Nuclear pore complex protein Nup93、Peroxiredoxin-4、Heat shock protein HSP90-beta和Zinc finger protein 497等分别与细胞骨架、细胞运输、抗氧化、应激分子伴侣、蛋白合成等功能相关的蛋白在Kir6.2-/-小鼠中脑星形胶质细胞表达显著上调，而Tubulin alpha-1A chain、Phosphoglycerate kinase 1等与细胞骨架、能量代谢相关的蛋白表达则显著下调；MPP+处理引起Kir6.2+/+中脑星形胶质细胞44个蛋白表达发生显著变化，Isoform 3 of Glial fibrillary acidic protein、ATPsynthase subunit beta、HSPA5 protein、26S proteasome non-ATPase regulatorysubunit 14等多个蛋白表达下调，而Vimentin、Transaldolase等蛋白表达显著上调；MPP+处理引起Kir6.2-/-小鼠中脑星形胶质细胞44个蛋白表达发生显著变化，Tubulin、Thioredoxin-like protein 1、Heat shock 70kDa protein 8 isoform 2 variant（Fragment）等蛋白表达则显著下调，而Actin、Glutamate dehydrogenase 1,mitochondrial precursor、Protein DJ-1、Vascular endothelial growth factor C precursor等蛋白表达显著上调。
结论：
1、Kir6.2敲除导致小鼠中脑能量代谢、应激分子伴侣、细胞骨架、蛋白转录等多种蛋白的表达发生改变，提示K-ATP通道可通过调节能量代谢、细胞应激、细胞骨架、蛋白转录和降解等过程参与PD的发生与发展过程。
2、Kir6.2敲除可通过维持细胞骨架、神经元突触功能及线粒体功能抵抗神经毒素MPTP或MPP+导致的神经损伤。
综上所述，本文工作的主要创新之处在于：
1、K-ATP通道与PD的发生与发展密切相关揭示Kir6.2基因缺失以及K-ATP通道开放剂在MPTP/p慢性PD模型中发挥相似的神经保护作用，其机制与抑制星形胶质细胞活化，减少D-Ser和SR的表达，抑制神经炎性反应，逆转MPTP诱导的神经再生的抑制相关，为深化对PD发生发展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、K-ATP通道调节离体PD模型中星形胶质细胞的功能Kir6.2基因缺失能抑制MPP+对TH神经元的损伤，Kir6.2基因缺失的星形胶质细胞与神经元共培养可进一步减轻MPP+对TH神经元的损伤，其机制与Kir6.2敲除后，促进星形胶质细胞基础状态以及MPP+应激时营养因子表达，抑制MPP+诱导的星形胶质细胞活力降低、D-Ser和SR表达增加，促进MPP+应激时星形胶质细胞Kir6.1表达相关。研究结果为PD的治疗提供了新的思路，也为研发靶向于K-ATP通道的治疗PD的保护剂提供了新的靶标。
3、K-ATP通道调节成年小鼠脑神经再生发现神经干细胞表达Kir6.1和SUR1亚基构成的K-ATP通道。应用Kir6.2敲除小鼠建立CMS模型，发现Kir6.2在CMS过程中发挥对CA3区神经元损伤的保护作用；K-ATP通道开放剂IPT通过作用于Kir6.1亚基组成的K-ATP通道促进神经再生，其机制与激活ERK-CREB信号通路相关。研究结果为发展靶向于调制神经再生的药物和神经干细胞移植用于PD的临床治疗提供了理论依据。
4、K-ATP通道调节PD模型小鼠脑内蛋白的表达发现Kir6.2敲除引起小鼠中脑与能量代谢、应激分子伴侣、细胞骨架、蛋白转录和降解等功能相关的多种蛋白的表达发生改变。Kir6.2敲除增强中脑神经元蛋白合成以及维持细胞骨架和突触完整性的功能，减轻MPP+对中脑神经元细胞骨架的损伤。Kir6.2敲除增强中脑星形胶质细胞抵抗氧化应激、降解错误折叠蛋白、合成蛋白、激活转录的功能，减轻MPP+对中脑星形胶质细胞线粒体功能的损伤。研究结果从蛋白水平进一步证实K-ATP通道与PD相关，拓展了对K-ATP通道调节PD发生与发展的分子机制的认识。
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 disease
AIM: 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 function
AIM: 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 neurogenesis
AIM: To investigate the effects and mechanisms of K-ATP channels onneurogenesis
METHODS: 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 disease
AIM: 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.
PD是一类由于黑质致密部DA能神经元的渐进性丢失导致的以运动障碍为特征的进行性神经退行性疾病。PD发病率逐年增加，预计在未来的25年内，在包括中国在内的10个人口最稠密的国家罹患PD的人数将至少翻一番。针对导致DA能神经元缺失的病理机制，尽管已提出兴奋性毒性、线粒体功能障碍、氧化应激和炎症等诸多假说，但是，根据这些假说而研发的NMDA受体拮抗剂、抗氧化剂及凋亡抑制剂等大多仅在实验研究中显现显著神经保护作用，而在临床研究中均未获得理想的神经保护效果。因此，深入研究、探索PD的病因学，对于研发新型治疗药物和临床治疗学的突破具有重要意义。
本实验室的前期研究发现，K-ATP通道开放剂（K-ATP channel openers，KCOs）在6-羟基多巴胺（6-hydroxydopamine，6-OHDA）、鱼藤酮、1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium，MPP+）导致的多种PD动物模型中发挥显著的神经保护作用，其机制涉及抗兴奋性毒性、抗凋亡、抑制神经炎症和保护线粒体功能等。然而，本实验室近期应用Kir6.2敲除鼠的研究却发现：Kir6.2敲除或Kir6.1/Kir6.2混合型开放剂均能取消1-甲基-4-苯基-1，2，3，6-四氢吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydoyndine，MPTP）联合丙磺舒（probenecid）所致PD模型（MPTP/p PD模型）小鼠黑质DA能神经元的损伤作用，该发现不仅有悖于开放Kir6.2构成的K-ATP通道可发挥保护作用的传统观点，也提示表达于胶质细胞和神经干细胞上Kir6.1构成的K-ATP通道在MPTP诱导的DA能神经元损伤中发挥了重要的调节作用。近年认识到胶质细胞，尤其是星形胶质细胞在维持神经电活动正常环境、调节神经递质代谢、保护神经元及促神经再生和修复过程中均发挥重要的作用。本实验室的前期研究发现，开放星形胶质细胞上K-ATP通道可以通过抑制线粒体凋亡通路以及MAPK信号通路保护MPP+诱导的星形胶质细胞的损伤；开放小胶质细胞上的K-ATP通道，可抑制鱼藤酮所致PD模型鼠小胶质细胞的活化，从而保护中脑多巴胺能神经元。但是，表达于胶质细胞的K-ATP通道是否参与调节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Kir6.2野生型（wild-type，Kir6.2+/+）和敲除型（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胶质细胞以及神经干细胞上K-ATP通道对PD的调节作用；第二部分离体培养Kir6.2+/+及Kir6.2-/-原代中脑神经元以及星形胶质细胞，并建立神经元与星形胶质细胞共培养体系，研究、阐明K-ATP通道对星形胶质细胞功能的调节；第三部分建立Kir6.2+/+及Kir6.2-/-小鼠慢性温和刺激（chronic mild stress，CMS）模型，研究K-ATP通道对成年鼠脑内神经再生的调节作用；第四部分应用蛋白质组学技术研究Kir6.2+/+及Kir6.2-/-小鼠中脑组织、中脑神经元以及星形胶质细胞基础状态以及在PD模型中相关蛋白表达的改变，在蛋白水平阐明K-ATP通道调节PD发生与发展的机制。
第一部分K-ATP通道在MPTP/p PD模型小鼠神经损伤中的作用
目的：应用Kir6.2敲除小鼠，研究、阐明K-ATP通道对MPTP/p PD模型小鼠神经损伤的调节作用及机制。
方法：应用MPTP皮下注射，继而丙磺舒腹腔注射（MPTP 20 mg·kg-1，s.c.，丙磺舒250 mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。IPT（10 mg·kg-1·day-1，p.o.）在MPTP首次给药前1h开始灌胃，连续给药6周。此外，应用MPTP皮下注射，继而丙磺舒腹腔注射单次给药模式建立MPTP急性损伤模型。埃他卡林（Iptakalim，IPT）（10 mg·kg-1·day-1，p.o.）在MPTP给药前1 h灌胃，连续给药3天。应用酪氨酸羟化酶（tyrosine hydroxylase，TH）免疫组织化学结合体视学计数、ImageJ软件分析黑质致密部（substantia nigra parscompacta，SNpc）DA能神经元及纹状体DA投射纤维的损伤，同时检测黑质（substantia nigra，SN）区星形胶质细胞的活化（glial fibrillary acidic protein，GFAP染色）、小胶质细胞的活化（ionized calcium binding adaptor molecule1，Iba1染色）以及室管膜下层（subventricular zone，SVZ）和颗粒细胞下层（subgranularzone，SGZ）神经再生的变化（5-bromo-2-deoxyuridine，BrdU免疫组化）；免疫双标观察SN区神经元和星形胶质细胞D-丝氨酸（D-Serine，D-Ser）及其合成酶丝氨酸消旋酶（serine racemase，SR）的表达水平；Western-blotting法检测SN区缝隙连接蛋白Connexin 43（Cx43）表达的变化；ELISA法检测SN中TNF-α蛋白水平的变化。
结果：1）MPTP慢性给药后，Kir6.2+/+小鼠SNpc DA能神经元数量仅为正常对照的49%，同时伴有纹状体DA能纤维投射的缺失。Kir6.2-/-小鼠未出现SNpc DA能神经元数量的减少，尽管Kir6.2-/-小鼠纹状体也存在着TH阳性纤维投射的丧失，但是其程度较Kir6.2+/+小鼠轻。IPT能改善MPTP慢性给药造成的Kir6.2+/+小鼠SNpc DA能神经元的数量减少和纹状体DA能神经纤维投射的丧失。MPTP单次给药显著降低Kir6.2-/-小鼠SNpc TH阳性细胞数，并且导致纹状体TH阳性纤维密度显著减少。IPT显著改善MPTP单次给药对Kir6.2-/-小鼠SNpcTH神经元的损伤，并且可以减轻纹状体TH阳性纤维投射的损伤。2）MPTP慢性给药引起Kir6.2+/+小鼠SN星形胶质细胞和小胶质细胞显著增殖活化，并且黑质区主要表达于星形胶质细胞上的Cx43以及主要由小胶质细胞释放的TNF-α也出现显著的增加；SVZ和SGZ区神经干细胞数量显著减少。而Kir6.2-/-小鼠未出现SN星形胶质细胞和小胶质细胞的活化以及SVZ和SGZ区神经干细胞数量的减少。IPT能显著抑制MPTP导致的Kir6.2+/+小鼠SN星形胶质细胞和小胶质细胞的活化以及SVZ和SGZ区神经干细胞数量的减少。3）免疫双标显示MPTP慢性给药诱导Kir6.2+/+小鼠黑质星形胶质细胞D-Ser和SR表达增加，不影响TH神经元D-Ser和SR的表达。IPT显著抑制MPTP慢性给药导致的Kir6.2+/+小鼠黑质星形胶质细胞D-Ser和SR表达增加。
结论：
1、应用Kir6.2敲除小鼠的研究证实K-ATP通道与PD的发生发展密切相关，获得了K-ATP通道与PD相关性的直接证据。
2、开放Kir6.1构成的K-ATP通道可通过调节胶质细胞功能，抑制其活化，促进神经再生与修复，发挥神经保护作用。
第二部分K-ATP通道对星形胶质细胞功能的调节：神经-胶质细胞共培养研究
目的：应用Kir6.2敲除小鼠，研究、阐明K-ATP通道对离体PD模型中星形胶质细胞功能的调节及机制。
方法：分离、培养Kir6.2+/+及Kir6.2-/-小鼠中脑神经元以及星形胶质细胞。给予三种不同亚基敏感性的KATP通道开放剂及MPP+药物处理，通过TH免疫细胞化学染色，研究K-ATP通道对MPP+诱导的原代培养中脑神经元尤其是DA能神经元损伤作用的调节。MTT法观察MPP+对星形胶质细胞活力的影响。应用RT-PCR及Western-blotting法观察星形胶质细胞Kir6.2的表达以及MPP+对Kir6.2+/+、Kir6.2-/-星形胶质细胞Kir6.1表达的影响。Western-blotting法观察MPP+对Kir6.2+/+、Kir6.2-/-星形胶质细胞神经营养因子表达的影响。免疫双标检测星形胶质细胞D-Ser及其合成酶SR的表达。建立神经元-星形胶质细胞共培养体系，TH免疫组化以及免疫双标观察星形胶质细胞对MPP+诱导的中脑DA能神经元损伤的影响以及星形胶质细胞D-Ser和SR的表达。
结果：1）MPP+呈浓度依赖性地损伤原代培养的Kir6.2+/+、Kir6.2-/-中脑DA能神经元，但对Kir6.2+/+神经元的损伤作用较Kir6.2-/-神经元显著；吡那地尔（pinacidil，Pin）和二氮嗪（diazoxide，Dia）预处理减轻MPP+对Kir6.2+/+神经元的损伤作用，而克罗卡林（cromakalim，Cro）则无此效应；三种KCOs均不能减轻MPP+对Kir6.2-/-中脑DA能神经元的损伤。2）Kir6.2+/+、Kir6.2-/-星形胶质细胞与Kir6.2+/+、Kir6.2-/-中脑神经元共培养后，Kir6.2-/-星形胶质细胞的存在可减轻MPP+对Kir6.2+/+、Kir6.2-/-中脑DA能神经元的损伤。3）星形胶质细胞与神经元混合培养后，MPP+诱导Kir6.2+/+星形胶质细胞活化、D-Ser和SR表达增加，Kir6.2敲除抑制混合培养中MPP+诱导的星形胶质细胞的活化，减少D-Ser和SR的表达，保护神经元。4）MPP+（50μM）作用48可降低Kir6.2+/+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；MPP+（10，50μM）作用72h可降低Kir6.2+/+、Kir6.2-/-星形胶质细胞的活力，对Kir6.2+/+星形胶质细胞的损伤作用较Kir6.2-/-严重。5）中脑星形胶质细胞表达Kir6.2亚基；Kir6.2敲除后，星形胶质细胞Kir6.1的表达降低；MPP+作用48h诱导Kir6.2敲除星形胶质细胞Kir6.1表达增加。6）Kir6.2基因缺失后，中脑星形胶质细胞神经营养因子FGF2（Fibroblast growth factor-2）、BDNF（Brain-derived neurotrophic factor）表达增加；MPP+（10μM）作用48h可诱导Kir6.2-/-星形胶质细胞BDNF表达增加，但不影响Kir6.2+/+星形胶质细胞FGF2和BDNF的表达。
结论：
1、星形胶质细胞参与了Kir6.2敲除后对MPP+诱导的中脑多巴胺能神经元损伤的保护作用。
2、上调并开放星形胶质细胞上Kir6.1构成的K-ATP通道可通过促进营养因子的表达，维持正常的胶质传递，减轻兴奋性毒性损伤，发挥对神经元的保护作用。
第三部分K-ATP通道对成年小鼠脑神经再生的调节
目的：建立CMS模型，研究、阐明K-ATP通道对神经再生的调节作用及机制。
方法：建立Kir6.2+/+及Kir6.2-/-小鼠CMS模型，连续4周给予氟西汀（Fluoxetine，Flx）（10 mg·kg-1·day-1，i.p.）或IPT（10 mg·kg-1·day-1，i.p.）。通过糖水偏爱、悬尾实验等行为学研究Flx以及IPT对动物抑郁样症状的改善情况。腹腔注射BrdU（50 mg/kg，每两小时一次，共四次）24小时及28天后分批处死小鼠，灌注、取脑，行NeuN、GFAP、BrdU免疫组化/免疫荧光双重标记，通过计数海马CA3区NeuN以及SGZ区BrdU阳性细胞数，NeuN/BrdU、GFAP/BrdU阳性细胞数测定海马CA3神经元的数量、海马神经干细胞增殖、存活及分化。取新鲜脑组织，匀浆后分别测定海马BDNF的含量，Kir6.2、Kir6.1的表达，Akt、GSK3β、ERK1/2、JNK1/2、p38、CREB磷酸化水平及5-HT水平，取躯干血测定血浆皮质酮水平。分离培养成年Kir6.2+/+及Kir6.2-/- C57BL/6J雄性小鼠海马及侧脑室神经干细胞，应用RT-PCR、Western blotting法检测原代培养的成年C57BL/6J小鼠神经干细胞上K-ATP通道的表达；[3H]-脱氧胸腺嘧啶摄取实验检测IPT对原代培养的成年鼠神经干细胞增殖的影响。
结果：1）悬尾实验及强迫游泳实验结果显示，Kir6.2敲除鼠具有抗抑郁样行为；CMS造模对Kir6.2-/-小鼠行为学影响较Kir6.2+/+小鼠显著，IPT及Flx对Kir6.2-/-小鼠治疗作用弱于Kir6.2+/+小鼠。CMS造模及IPT，Flx药物治疗对Kir6.2+/+小鼠海马CA3区神经元数量无显著影响；CMS造模显著减少Kir6.2-/-小鼠海马CA区神经元数量；4周IPT及Flx治疗能阻断CMS对Kir6.2-/-小鼠海马CA3神经元的损伤。2）正常情况下，两种基因型小鼠SGZ区神经再生无显著差异；CMS造模能显著减少两种基因型小鼠海马SGZ区BrdU标记阳性细胞数；4周IPT与Flx药物治疗均能逆转CMS对两种基因型小鼠SGZ区神经再生的抑制。CMS造模及IPT，Flx药物治疗对两种基因型小鼠BrdU阳性细胞的分化均无显著影响。3）Kir6.2-/-小鼠基础状态下海马BDNF含量显著低于Kir6.2+/+小鼠；CMS造模后两种基因型小鼠海马BDNF水平均显著降低，4周IPT及Flx治疗后均能逆转两种基因型小鼠海马BDNF水平的降低。CMS造模后，Kir6.2+/+小鼠海马中Kir6.2亚基表达上调；4周IPT及Flx治疗后未逆转CMS所致的海马Kir6.2亚基的上调。CMS造模后两种小鼠海马Kir6.1表达均上调；4周IPT及Flx治疗后不改变CMS所致海马Kir6.1表达的上调。4）CMS造模不影响海马中Akt的磷酸化水平，抑制两种基因型小鼠海马GSK3β、ERK1/2以及CREB的磷酸化，对Kir6.2+/+小鼠海马JNK1/2的磷酸化无显著影响，但能显著促进Kir6.2-/-小鼠海马JNK1/2的磷酸化，对两种基因型小鼠海马p38磷酸化水平无显著影响。4周IPT及Flx治疗后能逆转CMS造模所致两种基因型小鼠海马GSK3β、ERK1/2磷酸化的抑制；逆转CMS对Kir6.2+/+小鼠海马CREB磷酸化的抑制；Flx能逆转CMS对Kir6.2-/-小鼠CREB磷酸化水平的抑制，IPT能增加Kir6.2-/-小鼠海马CREB磷酸化水平；4周IPT及Flx治疗不影响Kir6.2+/+小鼠海马JNK1/2磷酸化，能逆转CMS所致Kir6.2-/-小鼠海马JNK1/2磷酸化水平的升高；显著降低两种基因型小鼠海马p38磷酸化水平。5）CMS造模使两种基因型小鼠海马5-HT水平降低，对其代谢产物5-HIAA水平无显著影响；4周IPT给药能逆转CMS造模所致两种基因型小鼠海马5-HT水平的降低，对其代谢产物5-HIAA水平无显著影响；4周Flx给药不改变CMS造模所致海马5-HT水平的降低，显著降低海马5-HIAA的水平。CMS造模能显著升高两种基因型小鼠血浆皮质酮水
平；4周IPT治疗后能逆转CMS造模引起的Kir6.2+/+小鼠血浆皮质酮水平，Flx显著降低CMS造模引起的Kir6.2+/+小鼠血浆皮质酮水平；IPT与Flx均不能显著抑制CMS造模所致的Kir6.2-/-小鼠血浆皮质酮水平的升高。6）原代培养的C57BL/6J小鼠神经干细胞表达Kir6.1、SUR1亚基组成的K-ATP通道，而不表达Kir6.2、SUR2亚基。IPT（1μM）可显著增强两种基因型小鼠海马神经干细胞的增殖。
结论：
1、原代培养的成年小鼠神经干细胞表达Kir6.1和SUR1亚基构成的K-ATP通道，其参与调节神经干细胞的增殖。
2、IPT通过开放Kir6.1亚基构成的K-ATP通道促进神经再生，其作用机制与活化ERK-CREB信号通路、促进BDNF的产生相关。
3、Kir6.2亚基构成的K-ATP通道在CMS过程中发挥对CA3区神经元的保护作用。
第四部分K-ATP通道与PD的相关性：蛋白质组学研究
目的：应用整体与离体蛋白质组学，研究、阐明K-ATP通道对帕金森病的调节作用及机制。
方法：应用Kir6.2+/+、Kir6.2-/- C57BL/6J小鼠，MPTP皮下注射，继而丙磺舒腹腔注射（MPTP 20 mg·kg-1，s.c.，丙磺舒250 mg·kg-1，i.p.，一周2次，连续5周）建立MPTP/p PD小鼠模型。最后一次给药后7 d时，断头处死小鼠，迅速取出大脑，预冷超纯水洗去血渍，在冰面上分离中脑，-70°C保存备用。部分小鼠灌注、取脑，行黑质区TH免疫组化。分离、培养Kir6.2+/+、Kir6.2-/- C57BL/6J小鼠中脑神经元以及星形胶质细胞。MPP+孵育48 h后，D-hank’s漂洗，经胰酶消化后，1500 rpm离心5 min，收集于5 ml EP管中。在冰浴条件下，向小鼠中脑样品或神经元样品加入裂解液，超声2 min，4°C冰箱作用1 h，4°C 40000 g离心1 h，吸取上清液，分装-70°C保存备用。Bradford比色法检测蛋白浓度。取180μg蛋白/样品，行双向电泳与图像分析，选取差异2倍及以上的蛋白点，经胰蛋白酶消化，行MALDI-TOF质谱分析，Mascot软件（http://www.matrixscience.com）检索Swiss-Prot数据库（http://www.expasy.org）鉴定蛋白质。
结果：1）基础状态下，Kir6.2敲除导致小鼠中脑39个蛋白表达发生显著变化，其中NADH-ubiquinone oxidoreductase 75 kDa subunit、ATP synthase subunitbeta蛋白在Kir6.2-/-小鼠表达显著上调，而Serotransferrin蛋白表达则显著下调；MPTP慢性造模引起Kir6.2+/+小鼠23个蛋白表达发生显著变化，Fascin、Heatshock 70 kDa protein 4等多个蛋白表达下调，而Aldehyde dehydrogenase等蛋白表达显著上调；MPTP慢性造模引起Kir6.2-/-小鼠23个蛋白表达发生显著变化，NADH dehydrogenase flavoprotein 1、Isoform 1 of 4-aminobutyrateaminotransferase、Glutathione synthetase等蛋白表达显著下调，而Aldose reductase等蛋白表达则显著上调。2）基础状态下，Kir6.2敲除导致小鼠中脑神经元20个蛋白表达发生显著变化，其中Isoform 3 of Septin-11、Proteasome subunit betatype-3等与细胞传递、蛋白降解相关的蛋白表达则显著下调，而Tubulin beta-5chain、Collapsin response mediator protein 4A、Dihydropyrimidinase-related protein2、78 kDa glucose-regulated protein和Elongation factor 2等分别与细胞骨架、突触发育、应激分子伴侣、蛋白合成等功能相关的蛋白在Kir6.2-/-小鼠中脑神经元表达显著上调；MPP+处理引起Kir6.2+/+中脑神经元48个蛋白表达发生显著变化，Vimentin、Heat shock cognate 71 kDa protein等多个蛋白表达下调，而Tubulinbeta-3 chain、78 kDa glucose-regulated protein、Enolase等蛋白表达显著上调；MPP+处理引起Kir6.2-/-小鼠中脑神经元44个蛋白表达发生显著变化，Alpha-enolase、Vinculin、Heat shock cognate 71 kDa protein等蛋白表达则显著下调，而Tubulin alpha-1A chain、Protein disulfide-isomerase A3、centromere proteinF等蛋白表达显著上调。3）基础状态下，Kir6.2敲除导致小鼠中脑星形胶质细胞30个蛋白表达发生显著变化，其中Isoform 3 of Glial fibrillary acidic protein、Nuclear pore complex protein Nup93、Peroxiredoxin-4、Heat shock protein HSP90-beta和Zinc finger protein 497等分别与细胞骨架、细胞运输、抗氧化、应激分子伴侣、蛋白合成等功能相关的蛋白在Kir6.2-/-小鼠中脑星形胶质细胞表达显著上调，而Tubulin alpha-1A chain、Phosphoglycerate kinase 1等与细胞骨架、能量代谢相关的蛋白表达则显著下调；MPP+处理引起Kir6.2+/+中脑星形胶质细胞44个蛋白表达发生显著变化，Isoform 3 of Glial fibrillary acidic protein、ATPsynthase subunit beta、HSPA5 protein、26S proteasome non-ATPase regulatorysubunit 14等多个蛋白表达下调，而Vimentin、Transaldolase等蛋白表达显著上调；MPP+处理引起Kir6.2-/-小鼠中脑星形胶质细胞44个蛋白表达发生显著变化，Tubulin、Thioredoxin-like protein 1、Heat shock 70kDa protein 8 isoform 2 variant（Fragment）等蛋白表达则显著下调，而Actin、Glutamate dehydrogenase 1,mitochondrial precursor、Protein DJ-1、Vascular endothelial growth factor C precursor等蛋白表达显著上调。
结论：
1、Kir6.2敲除导致小鼠中脑能量代谢、应激分子伴侣、细胞骨架、蛋白转录等多种蛋白的表达发生改变，提示K-ATP通道可通过调节能量代谢、细胞应激、细胞骨架、蛋白转录和降解等过程参与PD的发生与发展过程。
2、Kir6.2敲除可通过维持细胞骨架、神经元突触功能及线粒体功能抵抗神经毒素MPTP或MPP+导致的神经损伤。
综上所述，本文工作的主要创新之处在于：
1、K-ATP通道与PD的发生与发展密切相关揭示Kir6.2基因缺失以及K-ATP通道开放剂在MPTP/p慢性PD模型中发挥相似的神经保护作用，其机制与抑制星形胶质细胞活化，减少D-Ser和SR的表达，抑制神经炎性反应，逆转MPTP诱导的神经再生的抑制相关，为深化对PD发生发展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、K-ATP通道调节离体PD模型中星形胶质细胞的功能Kir6.2基因缺失能抑制MPP+对TH神经元的损伤，Kir6.2基因缺失的星形胶质细胞与神经元共培养可进一步减轻MPP+对TH神经元的损伤，其机制与Kir6.2敲除后，促进星形胶质细胞基础状态以及MPP+应激时营养因子表达，抑制MPP+诱导的星形胶质细胞活力降低、D-Ser和SR表达增加，促进MPP+应激时星形胶质细胞Kir6.1表达相关。研究结果为PD的治疗提供了新的思路，也为研发靶向于K-ATP通道的治疗PD的保护剂提供了新的靶标。
3、K-ATP通道调节成年小鼠脑神经再生发现神经干细胞表达Kir6.1和SUR1亚基构成的K-ATP通道。应用Kir6.2敲除小鼠建立CMS模型，发现Kir6.2在CMS过程中发挥对CA3区神经元损伤的保护作用；K-ATP通道开放剂IPT通过作用于Kir6.1亚基组成的K-ATP通道促进神经再生，其机制与激活ERK-CREB信号通路相关。研究结果为发展靶向于调制神经再生的药物和神经干细胞移植用于PD的临床治疗提供了理论依据。
4、K-ATP通道调节PD模型小鼠脑内蛋白的表达发现Kir6.2敲除引起小鼠中脑与能量代谢、应激分子伴侣、细胞骨架、蛋白转录和降解等功能相关的多种蛋白的表达发生改变。Kir6.2敲除增强中脑神经元蛋白合成以及维持细胞骨架和突触完整性的功能，减轻MPP+对中脑神经元细胞骨架的损伤。Kir6.2敲除增强中脑星形胶质细胞抵抗氧化应激、降解错误折叠蛋白、合成蛋白、激活转录的功能，减轻MPP+对中脑星形胶质细胞线粒体功能的损伤。研究结果从蛋白水平进一步证实K-ATP通道与PD相关，拓展了对K-ATP通道调节PD发生与发展的分子机制的认识。
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 disease
AIM: 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 function
AIM: 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 neurogenesis
AIM: To investigate the effects and mechanisms of K-ATP channels onneurogenesis
METHODS: 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 disease
AIM: 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.
引文
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