别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

doi:10.16098/j.issn.0529-1356.2020.04.001

解剖学报 ›› 2020, Vol. 51 ›› Issue (4): 473-482.doi: 10.16098/j.issn.0529-1356.2020.04.001

• 神经生物学 • 下一篇

别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

王彤彤^1,2,3陈治池^1,3叶鑫^1,3边维^1,3 杜娟娟^3,4 傅维达⁵ 陈梦娇⁵ 李俊楠⁵孙臣友^1,3*

1.温州医科大学基础医学院人体解剖学教研室，浙江温州 325035； 2.台州职业技术学院医学与制药工程学院，浙江台州 318000； 3.温州医科大学基础医学院神经科学研究所，浙江温州 325035；4.温州医科大学基础医学院组织学胚胎学教研室，浙江温州 325035；5.温州医科大学第一临床医学院2015、2016级监床医学、医学影像学本科，浙江温州 325035

收稿日期:2019-04-02 修回日期:2019-05-23 出版日期:2020-08-06 发布日期:2020-08-06
通讯作者: 孙臣友 E-mail:sunchenyou1972@aliyun.com
基金资助:
别孕烯醇酮促进PD小鼠多巴胺能神经元新生的作用及机制研究;CD133追踪APα诱导的6-OHDA PD小鼠新生多巴胺能神经元起源的应用研究

Allopregnanolone restores dopaminergic neurons and motor performance in Parkinson’s disease mice and its molecular mechanisms

WANG Tong-tong^1,2,3CHEN Zhi-chi^1,3 YE Xin^1,3 BIAN wei^1,3 DU Juan-juan^3,4 FU Wei-da⁵ CHEN Meng-jiao⁵ LI Jun-nan⁵ SUN Chen-you^1,3*

1.Department of Anatomy，School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 35035，China; 2.School of Medicine and Pharmaceutical Engineering, Taizhou Vocational and Technical College, Zhejiang Taizhou 318000; 3.Institution of Neuroscience, School of Basic Medical Sciences of Wenzhou Medical University，Zhejiang Wenzhou 325035, China; 4.Department of Histology and Embryology, School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 35035, China; 5.Bachelor of Clinical Medicine and Medical Imaging, Grade 2015 and 2016, the First Clinical Medical College， Wenzhou Medical University，Zhejiang Wenzhou 325035,China

Received:2019-04-02 Revised:2019-05-23 Online:2020-08-06 Published:2020-08-06
Contact: SUN Chen-you E-mail:sunchenyou1972@aliyun.com

摘要/Abstract

摘要：

目的探讨别孕烯醇酮（APα）对帕金森病（PD）小鼠黑质-纹状体多巴胺能系统及行为学的影响及可能的分子机制。方法 90只3月龄体重为20~25 g雄性C57BL/6小鼠1侧纹状体被注射6-羟多巴胺（6-OHDA）复制小鼠PD模型，再给予APα及γ-氨基丁酸A受体（GABAAR）拮抗剂荷包牡丹碱（Bic）。采用ELISA检测血清及大脑皮质APα含量和纹状体多巴胺水平，免疫组织化学法检测黑质多巴胺能神经元及其纹状体纤维投射数量的变化，Western blotting检测中脑胞膜GABAAR、胞质及胞核各蛋白水平的变化，并通过免疫共沉淀验证它们之间的相互作用，观察小鼠行为学的变化。结果 PD小鼠大脑皮质内源性APα水平显著降低，黑质多巴胺能阳性神经元及其纹状体纤维投射含量下降，胞膜、胞质和胞核各蛋白的水平也明显下降，小鼠的运动功能发生障碍。在给予外源性APα处理后，上述情况均有所改善。但在应用Bic后，上述调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号呈现相反的变化，免疫共沉淀结果显示，蛋白之间存在相互作用。结论外源性的APα通过增加其内源性水平调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号通路促进PD模型小鼠黑质-纹状体多巴胺能神经系统和小鼠行为学的改善。

关键词: 别孕烯醇酮, 帕金森病, 多巴胺能神经元, 钙离子-钙调素依赖性蛋白激酶Ⅱδ3, 脑源性神经营养因子, 免疫印迹法, 小鼠

Abstract:

Objective To investigate the effects of allopregnanolone(APα) on the dopaminergic neurons in substantia nigra, striatal dopaminergic neural fibers and behavioral performance in Parkinson’s disease (PD) model mice, as well as its possible molecular mechanisms. Methods C57BL/6 adult male mice with 20-25 g at 3-month old (n=90) were successively injected with 6-hydroxydopamine (6-OHDA) to generate a PD animal model. APα and its receptor γ-aminobutyric acid A receptor (GABAAR) antagonist—bicuculline (Bic) were successively injected. ELISA was used to detect the APα or dopamine concentration in the serum, cerebral cortex and striatum. The number of tyrosine hydroxylase (TH) in the substantia nigra (SN) and striatal dopaminergic neural projections were examined by immunohistochemical staining. The expression levels of GABAAR in membrane fractions and Ca²⁺/calmodulin-dependent protein kinase Ⅱ δ3 (CaMKⅡδ3), phosphorylated CaMKⅡδ3 (p-CaMKⅡδ3), brain derived neurotrophic factor (BDNF), and cyclin-dependent kinases 1 (CDK1) were detected by Western blotting in the cytoplasmic or nuclear fractions. The interaction of CaMK Ⅱδ3/p-CaMK Ⅱδ3 with BDNF and CDK1 was verified by immunoprecipitation. In addition, the behavioral changes of animals were recorded. Results There was a significant decrease in the endogenous APα levels of the cerebral cortex, or the dopaminergic neurons of the SN, or the striatal dopaminergic neural fibers, or various protein levels of the membrane, cytoplasmic and nuclear fractions, as well as motor function in PD mice. As compared with PD mice, the abovementioned results were ameliorated in the mice administrated with APα, which were pulled down in mice pre-treated with Bic. The immuno-precipitated result indicated that CaMKⅡδ3 or p-CaMKⅡδ3 interacted with BDNF or CDK1. Conclusion By increasing the endogenous APα levels, exogenous APα treatment can improve SN-striatum dopaminergic neurons in PD model mice by regulating GABAAR/CaMKⅡδ3/BDNF/CDK1 signaling pathway, which in turn promotes behavioral performance in 6-OHDA-lesioned mice.

Key words: Allopregnanolone, Parkinson’s disease, Dopaminergic neurons, Ca²⁺/calmodulin-dependent protein kinase Ⅱδ3, Brain-derived neurotrophic factor, Western blotting, Mouse

中图分类号:

Q189

王彤彤陈治池叶鑫边维杜娟娟傅维达陈梦娇李俊楠孙臣友. 别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制[J]. 解剖学报, 2020, 51(4): 473-482.

WANG Tong-tong CHEN Zhi-chi YE Xin BIAN wei DU Juan-juan FU Wei-da CHEN Meng-jiao LI Jun-nan SUN Chen-you. Allopregnanolone restores dopaminergic neurons and motor performance in Parkinson’s disease mice and its molecular mechanisms [J]. Acta Anatomica Sinica, 2020, 51(4): 473-482.

参考文献

［1］ Xie MQ, Chen ZhCh, Qi ShSh, et al. Effects of 6-hydroxydopamine on dopaminergic neurons in nigrostriatal system and on behavior of adult mice［J］. Acta Anatomica Sinica, 2017, 48(4); 361-374.(in Chinese)

谢明琦，陈治池，戚双双,等.六羟多巴胺对成年小鼠黑质-纹状体系统多巴胺能神经元及其行为学的影响［J］. 解剖学报, 2017, 48(4);361-374.

［2］ Zhang P, Qi ShSh, Xie MQ, et al. Effect of allopregnanolone on the dopaminergic neurons in the substania nigra of APPswe/PSEN1 mice［J］. Acta Anatomica Sinica, 2015,46(3):317-323. (in Chinese)

张鹏，戚双双，谢明琦，等. 别孕烯醇酮对APPswe/PSEN1小鼠黑质多巴胺能神经元的影响［J］. 解剖学报, 2015,46(3):317-323.

［3］ Sun C, Ou X, Farley JM, et al. Allopregnanolone increases the number of dopaminergic neurons in substantia nigra of triple transgenic mouse model of Alzheimer’s disease［J］. Curr Alzheimer Res, 2012, 9(4):473-480.

［4］ Wang JM, Singh C, Liu L, et al. Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer’s disease［J］. Proc Natl Acad Sci USA, 2010,107(14):6498-6503.

［5］ Deumens R, Blokland A, Prickaerts J. Modeling Parkinson’s disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway［J］. Exp Neurol, 2002, 175(2):303-317.

［6］ Naylor JC, Kilts JD, Hulette, CM, et al. Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer’s disease compared to cognitively intact control subjects［J］. Biochim Biophys Acta, 2010,1801(8):951-959.

［7］ Locci A , Pinna G. Neurosteroid biosynthesis down-regulation and changes in GABAA receptor subunit composition: a biomarker axis in stress-induced cognitive and emotional impairment［J］. Br J Pharmacol, 2017, 174(19):3226-3241.

［8］ Adeosun SO, Hou X, Jiao Y, et al. Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson’s disease［J］. PLoS One, 2012,7(11):e50040.

［9］ Daadi MM, Weiss S. Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain［J］. J Neurosci, 1999, 19(11):4484-4497.

［10］ Wang JM, Brinton RD. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential［J］. BMC Neurosci, 2008,9(Suppl 2):S11.

［11］ Jagasia R, Steib K, Englberger E, et al. GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus［J］. J Neurosci, 2009,29(25):7966-7977.

［12］ Wang JM, Johnston PB, Ball BG, et al. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression［J］. J Neurosci, 2005,25(19):4706-4718.

［13］ Wang JM, Singh C, Liu L, et al. Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer’s disease［J］.Proc Natl Acad Sci USA, 2010,107(14),6498-6503.

［14］ Moriguchi S, Kita S, Fukaya M, et al. Reduced expression of Na+/Ca2+ exchangers is associated with cognitive deficits seen in Alzheimer’s disease model mice［J］. Neuropharmacology, 2018, 131: 291-303.

［15］ Sobhia ME, Grewal BK, Paul MLS, et al. Protein kinase C inhibitors: a patent review (2010-present)［J］. Expert Opin Ther Pat, 2013, 23(11): 1451-1468.

［16］ Quijada P, Hariharan N, Cubillo JD, et al. Nuclear calcium/calmodulin-dependent protein kinase Ⅱsignaling enhances cardiac progenitor cell survival and cardiac lineage commitment［J］. J Biol Chem, 2015, 290(42):25411-25426.

［17］ Takeuchi Y, Fukunaga K, Miyamoto E. Activation of nuclear Ca2+/calmodulin-dependent protein kinaseⅡ and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells［J］. J Neurochem, 2002,82(2): 316-328.

［18］ Shioda N, Sawai M, Ishizuka Y, et al. Nuclear translocation of calcium/calmodulin-dependent protein kinase Ⅱδ3 promoted by protein phosphatase-1 enhances brain-derived neurotrophic factor expression in dopaminergic neurons［J］. J Biol Chem, 2015, 290(35):21663-21675.

［19］ Kamata A, Takeuchi Y, Fukunaga K. Identification of the isoforms of Ca2+/calmodulin-dependent protein kinaseⅡ and expression of brain-derived neurotrophic factor mRNAs in the substantia nigra［J］. J Neurochem, 2006, 96(1):195-203.

［20］ Li W, Li H, Sanders PN, et al. The multifunctional Ca2+/calmodulin-dependent kinase Ⅱ delta (CaMKⅡdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21［J］. J Bio Chem, 2011, 286(10):7990-7999.

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别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

Allopregnanolone restores dopaminergic neurons and motor performance in Parkinson’s disease mice and its molecular mechanisms

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