肖雪 毕明霞 焦倩 陈曦 杜希恂 姜宏
[摘要]目的 探讨生长激素促分泌素受体1a(GHSR1a)基因敲除对小鼠黑质区γ-氨基丁酸(GABA)信号传递的影响。方法 取3只GHSR1a敲除(Ghsr-/-)小鼠和3只同窝野生型(WT)小鼠的黑质区,应用转录组学测序(RNA-seq)技术筛选出差异表达基因(DEGs),并通过KEGG富集分析神经活性配体受体信号通路的变化,以及对GABA信号传递的影响。结果 与WT组相比,Ghsr-/-组神经活性配体受体相互作用信号通路上的12个基因表达发生了显著性变化,其中γ-氨基丁酸受体α2(Gabra2)和γ-氨基丁酸受体α4(Gabra4)分别是GABA-A受体α2亚基和α4亚基的编码基因。在Ghsr-/-小鼠中,Gabra2和Gabra4表达出现明显上调。除此之外,与GABA转运相关的转运蛋白溶质载体家族6成员1(Slc6a1)基因表达出现明显下调。结论 GHSR1a基因敲除通过增加GABA受体亚基表达,抑制GABA重摄取,从而调控GABA信號传递。
[关键词]受体,胃促生长素;黑质;γ氨基丁酸;信号传导;转录组测序;小鼠
[中图分类号]R338.2
[文献标志码]A
[文章编号]2096-5532(2021)02-0171-03
[ABSTRACT]Objective To investigate the effect of growth hormone secretagogue receptor 1a (GHSR1a) gene knockout on γ-aminobutyric acid (GABA) signal transduction in the substantia nigra of mice. Methods Three GHSR1a-knockout (Ghsr-/-) mice and three wild-type (WT) littermates were selected, and RNA-seq technology was used to screen out differently expressed genes (DEGs) in the substantia nigra. KEGG enrichment analysis was used to investigate the change in the neuroactive ligand-receptor interaction signaling pathway and the impact of GABA signal transduction. Results Compared with the WT group, the Ghsr-/- group had significant changes in the expression of 12 genes in the neuroactive ligand-receptor interaction signaling pathway, among which γ-aminobutyric acid A receptor α2 (Gabra2) and γ-aminobutyric acid A receptor α4 (Gabra4) were the coding genes of the α2 and α4 subunits of GABA-A receptor, respectively. Gabra2 and Gabra4 were significantly upregulated in Ghsr-/- mice. In addition, solute carrier family 6 member 1 (Slc6a1) related to GABA transport was significantly downregulated.Conclusion GHSR1a knockout regulates GABA signal transduction by increasing the expression of GABA receptor subunits and inhibiting GABA reuptake.
[KEY WORDS]receptors, ghrelin; substantia nigra; gamma-aminobutyric acid; signal transduction; RNA-seq; mice
生长激素促分泌素受体1a(GHSR1a)是G蛋白偶联受体(GPCR)家族成员,同时也是ghrelin的功能型受体[1-2]。GHSR1a由366个氨基酸残基和7个跨膜结构域(TMD)组成,不仅在中枢神经系统广泛表达,在外周器官中也有较低水平表达[2]。本实验室前期研究结果也证实了ghrelin-GHSR1a系统具有神经保护作用[3-6]。除了介导ghrelin的功能外,GHSR1a还具有本构型活性,在无ghrelin刺激时可参与多种生物学活动,如影响学习记忆、生长发育、癫痫发作、血压和癌症等,还可以通过Gi/o信号通路影响γ-氨基丁酸(GABA)释放,产生抑制性突触后电流(IPSCs)并最终导致神经元兴奋性增强[7]。为了更加全面地了解GHSR1a影响GABA释放的分子机制,本研究选用3月龄GHSR1a基因敲除小鼠(Ghsr-/-小鼠)和同窝野生型(WT)小鼠,对其黑质区进行转录组学测序(RNA-seq),筛选差异表达基因(DEGs)并进行KEGG信号通路富集分析,以期为阐明脑内GHSR1a参与GABA信号传递及影响神经元兴奋性提供新的思路。
1 材料与方法
1.1 实验材料
生理盐水,水合氯醛,干冰,液氮,PBS缓冲液,手术剪,眼科镊,EP管,玻璃培养皿,注射器,液氮罐,-80 ℃冰箱,制冰机。
1.2 实验方法
1.2.1 实验动物与分组 3月龄Ghsr-/-雄性小鼠3只(Ghsr-/-组)和同窝WT雄性小鼠3只(WT组),体质量(20±2)g,均购自上海南方模式生物科技发展有限公司。小鼠在室温(23±1)℃、12 h昼夜循环光照的环境下进行饲养,可自由饮水与进食。所有动物实验操作均遵循医学伦理学原则。
1.2.2 小鼠黑质区样本的采集及测序 使用水合氯醛对小鼠进行麻醉,脱臼法处死小鼠,解剖取脑后立即置于生理盐水中冲洗脑组织表面血污,使用眼科镊于冰上进行黑质组织块的取材,取材完毕立即置于脱酶EP管中,并保存于液氮中。测序样本从液氮中取出后干冰运输至北京诺禾致源生物科技有限公司进行RNA-seq。对Ghsr-/-组和WT组小鼠测序所得结果进行分析。
2 结 果
2.1 质量控制
测序错误率随着测序长度的增加而降低,本研究6个样本的测序错误率均小于1%,GC含量均小于50,Q30约为90%,6个样本间的Pearson相关系数均大于0.95,说明数据均质化程度高,可用于后续分析。
2.2 DEGs筛选
共有533个基因在Ghsr-/-组和WT组之间呈现出差异性表达,其中258个基因在WT组高表达,275个基因在Ghsr-/-组高表达。
2.3 KEGG富集通路分析
有12个DEGs富集到神经活性配体受体相互作用通路上,与WT组相比,Ghsr-/-组中表达上调基因有6个,分别为γ-氨基丁酸受体α2(Gabra2)、γ-氨基丁酸受体α4(Gabra4)、胆碱能受体烟碱α4(Chrnb4)、AMPA离子型谷氨酸受体3(Gria3)、阿片受体1(Oprd1)和甘氨酸受体(Glrb);表达下调基因6个,分别为胆碱能受体烟碱α5(Chrna5)、N-甲基-D-天冬氨酸离子能谷氨酸受体2D(Grin2d)、离子型谷氨酸受体海藻酸盐5(Grik5)、神经降压素受体2(Ntsr2)和黑色素皮质素受体3(Mc3r)。其中,Gabra2和Gabra4是编码GABA受体亚基的基因,其上调提示GABA-A受体表达增多。除此之外,与GABA转运有关的转运蛋白溶质载体家族6成员1(Slc6a1)基因表达出现明显下调,其下调会导致突触间隙中GABA不能被重吸收,从而增强其神经抑制作用。以上结果表明,GHSR1a基因敲除对GABA信号传递具有显著性影响。
3 讨 论
GHSR1a是由7个TMD组成的GPCR家族中的一员,主要分布于下丘脑和垂体,是ghrelin的功能型受体[8-9]。当GHSR1a被ghrelin激活时,能够发挥抗炎、抗凋亡、增强学习记忆、促进生长发育等作用[7,10]。本实验室前期研究结果表明,PD病人的血浆ghrelin水平显著降低[4]。而且ghrelin激活GHSR1a之后能够拮抗1-甲基-4-苯基-1,2,3,6-四氢吡啶对小鼠黑质多巴胺能神经元的神经毒性作用[6]。以上结果表明,ghrelin-GHSR1a系统具有神经保护作用。另外,GHSR1a不依赖于ghrelin刺激的本构型活性对脑内功能、生长发育等多种生物学过程也产生影响[11-13]。缺乏ghrelin时,GHSR1a可以通過Gi/o信号通路影响突触前Ca2+电流,减少抑制性神经递质GABA的释放和IPSCs的形成并最终导致神经元兴奋性增强[14-15]。
GABA是重要的抑制性神经递质,通过与其受体结合能够调节神经元兴奋性[16-17]。GABA受体属于氯离子通道受体,分布于整个中枢神经系统,介导中枢神经系统大部分抑制性神经传递,Gabra2和Gabra4是编码该受体亚基的基因[18-19]。本研究结果显示,在Ghsr-/-组小鼠中Gabra2和Gabra4的表达明显升高,这可能会增加GABA受体组装进而增强GABA与受体结合,通过增强IPSCs导致神经元兴奋性降低[20-21]。GABA发挥抑制性突触传递作用除了与受体有关,还受到GABA转运体的影响[22]。在本研究中,Ghsr-/-小鼠GABA转运蛋白Slc6a1基因表达出现明显下调。Slc6a1为溶质载体家族6的成员之一,是大脑中主要的GABA转运蛋白,负责从突触间隙重新摄取GABA[19,23]。突触间隙中的GABA主要来源于神经胶质细胞细胞膜、突触前膜或囊泡膜上GABA转运体(CAT)的摄取。Slc6a1可使GABA在突触间隙中的浓度降低,从而减弱GABA的突触传递作用[24-25]。当Slc6a1蛋白表达降低时,从突触间隙重新摄取GABA可能会受到抑制,从而增强GABA的抑制作用。因此,研究GHSR1a敲除对GABA信号传递的影响能够更加深入地了解神经元兴奋性的影响因素,从而为神经系统疾病的治疗提供更多思路。
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(本文編辑 马伟平)