中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (4): 712-723.doi: 10.4103/1673-5374.266916

• 原著:退行性病与再生 • 上一篇    下一篇

腺苷A1受体长期激活诱导分拣蛋白能促进多巴胺能神经元中α-突触核蛋白的表达

  


  • 出版日期:2020-04-15 发布日期:2020-05-29
  • 基金资助:

    中国国家自然科学基金项目（81770460），加拿大萨斯喀彻温省卫生研究基金会博士后研究奖学金项目（SHRF，4144），华南大学船山人才项目（2017CST20），衡阳市科学技术局助学计划（2017KJ268），衡阳市科学技术局临床解剖与生殖医学重点实验室项目（2017KJ182），湖南省研究生科研创新项目（CX2018B62）

Long-term adenosine A1 receptor activation-induced sortilin expression promotes α-synuclein upregulation in dopaminergic neurons

Yun-Cheng Lv1, 2, An-Bo Gao1, 3, Jing Yang4, Li-Yuan Zhong1, Bo Jia1, Shu-Hui Ouyang1, Le Gui2, Tian-Hong Peng1, Sha Sun1,Francisco S. Cayabyab2#br#   

  1. 1 Clinical Anatomy & Reproductive Medicine Application Institute, Hengyang Medical College, University of South China, Hengyang, Hunan Province, China
    2 Department of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, Canada
    3 Institute of Pharmacy and Pharmacology, School of Pharmaceutical Science, University of South China, Hengyang, Hunan Province, China
    4 Department of Metabolism & Endocrinology, the First Affiliated Hospital, Hengyang Medical College, University of South China, Hengyang, Hunan Province, China
  • Online:2020-04-15 Published:2020-05-29
  • Contact: Tian-Hong Peng, PhD,thpeng67@163.com; Sha Sun, BS,1060565208@qq.com; Francisco S. Cayabyab, PhD,frank.cayabyab@usash.ca.
  • Supported by:
    This study was supported by the National Natural Sciences Foundation of China, No. 81770460 (to YCL); the Postdoctoral Research Fellowship of the Saskatchewan Health Research Foundation, No. SHRF, 4144 (to YCL); the third level of the Chuanshan Talent project of the University of South China, No. 2017CST20 (to YCL); the Aid Program, No. 2017KJ268 and the Key Lab for Clinical Anatomy & Reproductive Medicine, No. 2017KJ182 from the Science and Technology Bureau of Hengyang City, China (to YCL and XC); the Postgraduate Student Research Innovation Projects of Hunan Province, China, No. CX2018B62 (to ABG).

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摘要：

腺苷A1受体长期激活可能导致多巴胺能神经元的损伤和神经退行性疾病的发展，但是腺苷A1受体长期激活诱导的神经变性的发病机制目前尚不清楚。(1)此次实验给大鼠连续5周腹腔注射5 mg/kg腺苷A1受体激动剂N6-环戊基腺苷希望揭示此问题。以强迫游泳实验评估大鼠运动能力，以Y迷宫实验评价记忆能力，以Western blot分析检测黑质分拣蛋白、α-突触核蛋白、p-JUN和c-JUN表达，以免疫荧光染色检测大脑黑基中分拣蛋白及α-突触核蛋白的免疫阳性反应。结果显示，与同时给予A1拮抗剂5 mg/kg DPCPX的大鼠相比，只给予腺苷A1受体激动剂的大鼠运动和记忆能力降低，多巴胺能神经元表面分拣蛋白和表面α-突触核蛋白减少，而总分拣蛋白和总α-突触核蛋白增加；(2)以500 nM N6-环戊基腺苷单独或联合10 μM JNK抑制剂SP600125孵育MN9D细胞48h，以实时定量PCR分析MN9D细胞中的分拣蛋白和α-突触核蛋白mRNA水平，发现MN9D细胞与CPA单独培养时，sortilin mRNA表达上调，而联合SP600125干预则抑制sortilin mRNA表达。以在线数据库Jasper，PROMO和Alibaba进行预测，结果发现在sortilin启动子中存在一个高度保守的序列，可与人和啮齿动物中的JUN结合。进一步使用荧光素酶报告分析分拣蛋白启动子的质粒转染的HEK293T细胞证明了上述预测。以sh-SORT1抑制分拣蛋白表达后，以Western blot分析检测细胞p-JUN和c-JUN表达，发现长期的腺苷A1受体激活可在转录后水平通过影响分拣蛋白表达上调α-突触核蛋白的表达；（3）使用在线工具Raptor-X-Binding和Discovery Studio 4.5预测软件来预测sortilin是否可以与α-突触核蛋白结合，结果显示分拣蛋白具有与α-突触核蛋白的结合能力；共沉淀实验显示MN9D细胞中分拣蛋白和α-突触核蛋白存在相互作用；（4）实验结果提示，抑制腺苷A1受体长期激活可有效抑制分拣蛋白表达和α-突触核蛋白积累，并改善宿主认知和运动能力。实验于2007年3月经University Committee of Animal Care and Supply at the University of Saskatchewan批准（批准号AUP#20070090），2017年6月经南华大学动物伦理委员会批准（批准号：LL0387-USC）。

orcid: 0000-0002-5058-7087 (Francisco S. Cayabyab)

关键词: 腺苷A1受体长期激活, 分拣蛋白, JNK/c-JUN信号通路, α-突触核蛋白, 多巴胺能神经元, 运动障碍, 认知功能障碍, 神经退行性疾病, 神经再生

Abstract:

Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases. However, the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear. In this study, rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) for five weeks. The mobility of rats was evaluated by forced swimming test, while their cognitive capabilities were evaluated by Y-maze test. Expression of sortilin, α-synuclein, p-JUN, and c-JUN proteins in the substantia nigra were detected by western blot analysis. In addition, immunofluorescence staining of sortilin and α-synuclein was performed to detect expression in the substantia nigra. The results showed that, compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (5 mg/kg) + CPA co-treated rats, motor and memory abilities were reduced, surface expression of sortin and α-synuclein in dopaminergic neurons was reduced, and total sortilin and total α-synuclein were increased in CPA-treated rats. MN9D cells were incubated with 500 nM CPA alone or in combination with 10 μM SP600125 (JNK inhibitor) for 48 hours. Quantitative real-time polymerase chain reaction analysis of sortilin and α-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone, but the combination of CPA and SP600125 could inhibit this expression. Predictions made using Jasper, PROMO, and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents. A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction. After sortilin expression was inhibited by sh-SORT1, expression of p-JUN and c-JUN was detected by western blot analysis. Long-term adenosine A1 receptor activation levels upregulated α-synuclein expression at the post-transcriptional level by affecting sortilin expression. The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind to α-synuclein. Co-immunoprecipitation revealed an interaction between sortilin and α-synuclein in MN9D cells. Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression and α-synuclein accumulation, and dramatically improved host cognition and kineticism. This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan (approval No. AUP#20070090) in March 2007, and the Animals Ethics Committee of University of South China (approval No. LL0387-USC) in June 2017.

Key words: cognitive dysfunction, dopaminergic neuron, dyskinesia, JNK/c-JUN pathway, long-term adenosine A1 receptor activation, neural regeneration, neurodegenerative diseases, sortilin, α-synuclein