A53T转基因小鼠黑质Kv4.3 A型钾通道的表达改变

    王怡云 石丽敏 谢俊霞

    [摘要]目的 探讨不同月龄α-突触核蛋白A53T转基因小鼠黑质区Kv4.3 A型钾通道的表达变化。方法选取不同月龄A53T转基因小鼠和同窝野生型(WT)对照小鼠,采用蛋白免疫印迹(Western blot)方法检测小鼠黑质区Kv4.3以及酪氨酸羟化酶(TH)蛋白的表达。结果 3月龄的A53T转基因小鼠Kv4.3及TH蛋白的表达与WT小鼠比较差异无显著性(P>0.05),15月龄的A53T转基因小鼠Kv4.3蛋白表达较WT小鼠升高(t=3.202,P<0.01),TH蛋白表达较WT小鼠降低(t=2.475,P<0.05)。结论 黑质区Kv4.3 A型钾通道随着帕金森病(PD)病情的进展发生改变,可能参与了PD的发病过程。

    [关键词]钾通道,电压门控;黑质;α突触核蛋白;小鼠,转基因

    [中图分类号]R338.2

    [文献标志码]A

    [文章编号]2096-5532(2021)02-0163-04

    [ABSTRACT]Objective To investigate the change in the expression of Kv4.3 A-type potassium channels in transgenic mice with different ages in months expressing A53T human α-synuclein.?Methods A53T transgenic mice with different ages in months and wild-type (WT) littermates were selected, and Western blot was used to measure the protein expression of Kv4.3 and tyrosine hydroxylase (TH) in the substantia nigra of mice.?Results There were no significant differences in the protein expression of Kv4.3 and TH between the A53T transgenic mice aged 3 months and WT mice (P>0.05). Compared with WT mice, the A53T transgenic mice aged 15 months had significantly higher protein expression of Kv4.3 (t=3.202,P<0.01) and significantly lower protein expression of TH (t=2.475,P<0.05).?Conclusion Kv4.3 A-type potassium channels in the substantia nigra change with the progression of Parkinsons disease (PD) and may be involved in the pathogenesis of PD.

    [KEY WORDS]potassium channels, voltage-gated; substantia nigra; alpha-synuclein; mice, transgenic

    帕金森病(PD)是全球第二大神經退行性疾病,其病理特征为中脑黑质(SN)区多巴胺(DA)能神经元选择性死亡,残存的DA能神经元出现以α-突触核蛋白(α-syn)为主要成分的路易小体[1-3]。PD主要的临床症状有静止性震颤、运动迟缓、肌强直、姿势反射障碍等[4-6]。但到目前为止,PD病因及发病机制尚不明确,可能与遗传、环境、铁沉积、氧化应激、线粒体功能障碍等原因有关[7-11]。

    钾通道是目前发现的亚型最多、功能最复杂、分布最广的一类离子通道,几乎存在于所有生物体中[12]。

    近年来有文献报道,钾通道的异常表达可能对PD有一定的调控作用[13]。A型钾通道是电压依赖型钾通道的一个重要分支,在调控DA能神经元的动作电位、放电模式和放电频率上具有重要的作用[14]。A型钾通道共有5种亚型,有研究表明,Kv4.3 A型钾通道在SN区DA能神经元上广泛表达[15]。有研究对PD病人残存的DA能神经元进行PCR分析,发现Kv4.3 mRNA表达明显升高[16],而阻断A型钾通道可以改善PD病人[17]以及PD大鼠模型[18]的运动功能障碍。然而,在PD疾病进展过程中A型钾通道的表达变化目前尚不清楚。本实验在携带人A53T突变型α-syn的转基因小鼠(α-Syn A53T+/+小鼠)上,应用蛋白免疫印迹方法检测了不同月龄的α-Syn A53T+/+小鼠以及同窝野生型(WT)小鼠SN区Kv4.3和酪氨酸羟化酶(TH)蛋白的表达水平,探究在PD的进展过程中A型钾通道的变化,为PD提供潜在的治疗靶点。

    1 材料与方法

    1.1 实验材料

    1.1.1 实验动物 所用A53T转基因小鼠购自美国Jackson实验室。α-Syn A53T+/-小鼠与α-Syn A53T+/-小鼠杂交得到下一代小鼠,通过基因组鉴定得到α-Syn A53T+/+小鼠、α-Syn A53T+/-小鼠及相对应的同窝WT小鼠。本实验选用3月龄和15月龄α-Syn A53T+/+小鼠和同窝WT小鼠作为研究对象,每组6只。小鼠饲养条件:室温(21±2)℃,湿度(50±5)%,12 h昼夜循环光照,可自由饮水、取食。

    1.1.2 实验仪器及试剂 Kv4.3抗体购自中国Absin公司,TH抗体购自美国Millipore公司,β-actin抗体购自中国博奥森公司。山羊抗兔二抗购自中国Absin公司。分离胶缓冲液和浓缩胶缓冲液均购于康为公司,ECL发光液、PVDF膜购自美国Millipore公司,APS、TEMED、RIPA裂解液、BCA试剂盒、Loading buffer购自中国碧云天公司。电泳仪、电转仪购自美国BioRad公司,凝胶成像系统购自美国UVP公司。

    1.2 蛋白免疫印迹法检测SN区TH和Kv4.3蛋白的表达

    小鼠用100 g/L水合氯醛麻醉后快速断头取脑,完整地取出包括中脑SN区的脑组织,置于冰盒内,取出双侧SN区域,分别放入预冷的1.5 mL EP管中,准确称质量。加入蛋白裂解液充分研磨后,于冰上静置30 min,在4 ℃下以12 000 r/min离心20 min,将上清转移至新的EP管中,应用BCA法,测定波长562 nm处的吸光度值,根据标准品的吸光度绘制标准曲线,计算待测样品的蛋白浓度。配制分离胶和浓缩胶,按照样品上样量准确上样,蛋白经SDS-PAGE电泳后,湿转到PVDF膜上,转膜完成后,将目的条带完整切下,用50 g/L的脱脂奶粉于室温摇床上封闭2 h;分别加入一抗Kv4.3(1∶1 000)、TH(1∶3 000)以及β-actin(1∶10 000),于4 ℃摇床上低速摇动孵育过夜。用TBST洗3次,每次10 min,加山羊抗兔(1∶10 000)的二抗在室温下孵育1 h,用TBST洗3次,每次10 min。于ECL显色试剂盒中取适量发光液均匀滴在PVDF膜上,室温孵育1 min,用UVP凝胶成像系统拍摄图片。在Image J图像采集与分析软件上对条带进行灰度值分析。用Kv4.3、TH蛋白与β-actin的比值作为目的蛋白相对表达水平。

    1.3 统计学分析

    应用GraphPad Prism 6软件进行统计学分析。计量资料结果以x2±s表示,两组间比较采用t检验。以P<0.05为差异有统计学意义。

    2 结 果

    2.1 不同月龄α-Syn A53T+/+小鼠SN区Kv4.3蛋白表达比较

    α-Syn A53T+/+组和WT组3月龄小鼠Kv4.3蛋白表达水平分别为0.965±0.040和0.844±0.084(n=6),差异无统计学意义(t=1.402,P>0.05)。α-Syn A53T+/+组和WT组15月龄小鼠Kv4.3蛋白表达水平分别为1.855±0.101和1.343±0.124(n=5),α-Syn A53T+/+组Kv4.3蛋白表达明显上调,差异具有统计学意义(t=3.202,P<0.05)。

    2.2 不同月龄α-Syn A53T+/+小鼠SN区TH蛋白表达变化

    α-Syn A53T+/+组和WT组3月龄小鼠TH蛋白表达水平分别为1.030±0.099和0.928±0.135(n=5),差异无统计学意义(t=0.612,P>0.05)。α-Syn A53T+/+組和WT组15月龄小鼠TH蛋白表达水平分别为1.586±0.1205和1.975±0.1008(n=6),α-Syn A53T+/+组TH表达明显下调,差异具有统计学意义(t=2.475,P<0.05)。

    3 讨 论

    近年来的研究显示,PD的发病可能与钾离子通道功能异常有关,以钾离子通道为靶点来治疗PD也成为一个重要的研究方向[19]。钾离子通道广泛存在于神经元、心肌细胞、骨骼肌细胞、红细胞、平滑肌细胞和淋巴细胞等多种细胞中[20-21]。A型钾通道是一种电压依赖型钾通道,又称瞬时外向型钾通道,在SN区DA能神经元上有广泛的表达,可影响神经元的自发放电[22-24]。该通道介导的电流具有瞬时出现、快速激活、快速失活等特点[25-26]。A型钾通道由Kv4基因家族形成的α亚基和KChip基因家族形成的辅助β亚基共同组成,其中在SN区DA能神经元上主要表达的是Kv4.3/KChip3.1[27-29]。然而,在PD的SN区DA能神经元退行性变过程中,A型钾通道究竟发挥了何种作用尚不清楚。

    本实验选取α-Syn A53T+/+小鼠作为动物模型,该模型可用来研究PD发病过程中运动及非运动行为的改变[30]。本文研究结果显示,3月龄的A53T转基因小鼠SN区Kv4.3及TH蛋白的表达水平尚未发生明显变化,提示3月龄的A53T转基因小鼠SN区并无损伤,A型钾通道也尚未发生变化。有文献报道,3月龄A53T转基因小鼠运动协调能力无明显障碍,但认知功能出现一定程度的下降[31]。另有研究表明,A53T转基因小鼠在8月龄时才出现明显的总体运动功能障碍,表现为毛发梳理减少、移动减少,在12月龄时表现出步幅的缩短和速度的减慢[32-33]。本研究在15月龄的A53T转基因小鼠SN区检测到,Kv4.3蛋白的表达显著升高,TH蛋白的表达显著降低,说明SN区的DA能神经元出现了一定程度的损伤,A型钾通道的表达也出现了异常,该结果与以往研究PD病人存活的SN区DA能神经元中Kv4.3 mRNA的表达显著增加相吻合[16]。

    钾离子通道的表达及功能异常通过多种机制影响SN、纹状体的功能,从而在PD的发病中发挥重要作用。例如,近期已有研究结果显示,钙激活型钾通道KCa3.1在1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的PD小鼠模型SN区表达显著增加,基因敲除或用通道阻断剂药理干预能有效拮抗MPTP诱导的SN区DA能神经元损伤及纹状体DA含量下降,减轻小胶质细胞增生所导致的炎症反应[34]。ATP敏感性钾通道(KATP)的SUR1亚单位在PD小鼠模型SN区的表达也显著增高[35],激活KATP可以导致DA能细胞内铁含量增加,损伤线粒体功能并增加细胞氧化应激[36]。本实验首次证明,在15月龄的A53T转基因小鼠SN区A型钾通道Kv4.3蛋白表达增加。A型钾通道是调节SN区DA能神经元兴奋性的关键因素,可影响突触传递和神经递质释放。我们的前期研究已经观察到,A型钾通道阻断剂4-氨基吡啶可以抑制A型钾通道电流,增加SN区DA能神经元自发放电频率[37];腹腔注射4-氨基吡啶能显著缩短MPTP诱导的PD小鼠模型爬杆实验中转头和爬杆的时间,改善PD小鼠的运动障碍[38]。因此,我们推测,在PD的进展过程中,A型钾通道的表达及功能发生改变,影响SN区神经元的兴奋性,进而改变了纹状体DA的释放,从而影响机体的运动功能。本实验结果为阐明A型钾通道参与PD发病提供了初步的实验证据。

    [參考文献]

    [1]GOEDERT M. NEURODEGENERATION. Alzheimers and Parkinsons diseases: the prion concept in relation to assembled Aβ, tau, and α-synuclein[J]. Science (New York, N Y), 2015,349(6248):1255555.

    [2]ZARRANZ J J, ALEGRE J, GMEZ-ESTEBAN J C, et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia[J]. Annals of Neurology, 2004,55(2):164-173.

    [3]MULLIN S, SCHAPIRA A H. Pathogenic mechanisms of neurodegeneration in Parkinson disease[J]. Neurologic Clinics, 2015,33(1):1-17.

    [4]ASCHERIO A, SCHWARZSCHILD M A. The epidemiology of Parkinsons disease: risk factors and prevention[J]. The Lancet Neurology, 2016,15(12):1257-1272.

    [5]SUMMA S, TOSI J, TAFFONI F, et al. Assessing bradykinesia in Parkinsons disease using gyroscope signals[C]//2017 International Conference on Rehabilitation Robotics (ICORR). London, UK: IEEE, 2017:1556-1561.

    [6]HELMICH R C, HALLETT M, DEUSCHL G, et al. Cerebral causes and consequences of parkinsonian resting tremor: a tale of two circuits[J]? Brain: a Journal of Neurology, 2012,135(Pt 11):3206-3226.

    [7]PRZEDBORSKI S. The two-century journey of Parkinson di-sease research[J]. Nature Reviews Neuroscience, 2017,18(4):251-259.

    [8]ISOBE C, ABE T, TERAYAMA Y. Levels of reduced and oxidized coenzyme Q-10 and 8-hydroxy-2-deoxyguanosine in the cerebrospinal fluid of patients with living Parkinsons di-sease demonstrate that mitochondrial oxidative damage and/or oxidative DNA damage contributes to the neurodegenerative process[J]. Neuroscience Letters, 2010,469(1):159-163.

    [9]MAITI P, MANNA J, DUNBAR G L. Current understanding of the molecular mechanisms in Parkinsons disease: targets for potential treatments[J]. Translational Neurodegeneration, 2017,6:28.

    [10]ZENG X S, GENG W S, JIA J J, et al. Cellular and molecular basis of neurodegeneration in Parkinson disease[J]. Frontiers in Aging Neuroscience, 2018,10:109.

    [11]CHARTIER-HARLIN M C, KACHERGUS J, ROUMIER C, et al. Alpha-synuclein locus duplication as a cause of fami-lial Parkinsons disease[J]. Lancet (London, England), 2004,364(9440):1167-1169.

    [12]SHIEH C C, COGHLAN M, SULLIVAN J P, et al. Potas-sium channels: molecular defects, diseases, and therapeutic opportunities[J]. Pharmacological reviews, 2000,52(4):557-594.

    [13]KUMAR P, KUMAR D, JHA S K, et al. Ion channels inneurological disorders[J]. Advances in Protein Chemistry and Structural Biology, 2016,103:97-136.

    [14]SEGEV D, KORNGREEN A. Kinetics of two voltage-gated K+ conductances in substantia nigra dopaminergic neurons[J]. Brain Research, 2007,1173:27-35.

    [15]CHEN X Y, XUE B, WANG J, et al. Potassium channels: a potential therapeutic target for Parkinsons disease[J]. Neuroscience Bulletin, 2018,34(2):341-348.

    [16]DRAGICEVIC E, SCHIEMANN J, LISS B. Dopamine midbrain neurons in health and Parkinsons disease: emerging roles of voltage-gated calcium channels and ATP-sensitive potassium channels[J]. Neuroscience, 2015,284:798-814.

    [17]LUCA C C, SINGER C. 4-aminopyridine improves freezing of gait in Parkinsons disease[J]. Journal of Neurology, 2013,260(10):2662-2664.

    [18]TAHERIAN R, ARAB AHMADI M. 4-aminopyridine decreases MPTP-induced behavioral disturbances in animal model of Parkinsons disease[J]. International Clinical Neuroscience Journal, 2016,2(4):142-146.

    [19]LAWSON K, MCKAY N G. Modulation of potassium channels as a therapeutic approach[J]. Current Pharmaceutical Design, 2006,12(4):459-470.

    [20]MATHIE A, WOOLTORTON J R, WATKINS C S.Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents[J]. General pharmacology, 1998,30(1):13-24.

    [21]SARKAR S, NGUYEN H M, MALOVIC E, et al. Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinsons disease[J]. The Journal of Clinical Investigation, 2020,130(8):4195-212.

    [22]SMIRNOV S V, AARONSON P I. Ca(2+)-activated and voltage-gated K+ currents in smooth muscle cells isolated from human mesenteric arteries[J]. The Journal of Physiology, 1992,457:431-454.

    [23]DUDA J, PTSCHKE C, LISS B. Converging roles of ion channels, calcium, metabolic stress, and activity pattern of Substantia nigra dopaminergic neurons in health and Parkinsons disease[J]. Journal of Neurochemistry, 2016,139(Suppl 1):156-178.

    [24]HUANG H Y, LIAO C W, CHEN P H, et al. Transient expression of A-type K channel alpha subunits Kv4.2 and Kv4.3 in rat spinal neurons during development[J]. The European Journal of Neuroscience, 2006,23(5):1142-1150.

    [25]SHAH N H, AIZENMAN E. Voltage-gated potassium channels at the crossroads of neuronal function, ischemic tole-rance, and neurodegeneration[J]. Translational Stroke Research, 2014,5(1):38-58.

    [26]LISS B, FRANZ O, SEWING S, et al. Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription[J]. The EMBO Journal, 2001,20(20):5715-5724.

    [27]DUFOUR M A, WOODHOUSE A, GOAILLARD J M. So-matodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development[J]. Journal of Neuroscience Research, 2014,92(8):981-999.

    [28]ZEMEL B M, RITTER D M, COVARRUBIAS M, et al. A-type KV channels in dorsal root ganglion neurons: diversity, function, and dysfunction[J]. Frontiers in Molecular Neuroscience, 2018,11:253.

    [29]GIASSON B I, DUDA J E, QUINN S M, et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[J]. Neuron, 2002,34(4):521-533.

    [30]徐玉钰,马泽刚. 3月龄α-突触核蛋白A53T转基因小鼠认知功能的改变[J]. 青岛大学学报(医学版), 2018,54(2):202-205.

    [31]PAUMIER K L, SUKOFF RIZZO S J, BERGER Z, et al. Behavioral characterization of A53T mice reveals early and late stage deficits related to Parkinsons disease[J]. PLoS One, 2013,8(8):e70274.

    [32]STOLZE H, KUHTZ-BUSCHBECK J P, DRCKE H, et al. Comparative analysis of the gait disorder of normal pressure hydrocephalus and Parkinsons disease[J]. Journal of Neurology, Neurosurgery, and Psychiatry, 2001,70(3):289-297.

    [33]LU J, DOU F F, YU Z H. The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinsons disease[J]. Journal of Neuroinflammation, 2019,16(1):273.

    [34]LISS B, HAECKEL O, WILDMANN J, et al. K-ATP channels promote the differential degeneration of dopaminergic midbrain neurons[J]. Nature Neuroscience, 2005,8(12):1742-1751.

    [35]DU X X, XU H M, SHI L M, et al. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro[J]. Scientific Reports, 2016,6:33674.

    [36]WHICHER J R, MACKINNON R. Structure of the voltage-gated K(+) channel Eag1 reveals an alternative voltage sen-sing mechanism[J]. Science, 2016,353(6300):664-669.

    [37]XUE B, LI C, CHANG X L, et al. Ghrelin reduces A-type potassium currents in dopaminergic nigral neurons via the PLC/PKCδ pathway[J]. Neuroscience Bulletin, 2020,36(8):947-950.

    [38]賈璐,石丽敏,谢俊霞. 4-AP对MPTP诱导PD模型小鼠运动行为影响[J]. 青岛大学学报(医学版), 2019,55(1):44-46.

    (本文编辑 马伟平)