激活mTORC2/Akt信号通路对6-羟基多巴胺模型小鼠多巴胺能神经元和行为学的影响

边维; 李梦一; 周鹏; 李军伟; 张庭; 吴安婷; 戚双双; 崔怀瑞; 孙臣友

doi:10.16098/j.issn.0529-1356.2023.01.002

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解剖学报 ›› 2023, Vol. 54 ›› Issue (1) : 13-22. DOI: 10.16098/j.issn.0529-1356.2023.01.002

神经生物学

激活mTORC2/Akt信号通路对6-羟基多巴胺模型小鼠多巴胺能神经元和行为学的影响

边维^1,2 李梦一^1,2周鹏^1,2 李军伟^1,2张庭³吴安婷^1,2 戚双双⁴ 崔怀瑞¹ 孙臣友^1,2*

作者信息 +

Effects of activating mTORC2/Akt signaling pathway on dopaminergic neurons and behaviors in 6-hydroxydopamin model mice

BIAN Wei^1,2 LI Meng-yi^1,2 ZHOU Peng^1,2 LI Jun-wei^1,2 ZHANG Ting³ WU An-ting^1,2 QI Shuang-shuang⁴ CUI Huai-rui¹ SUN Chen-you^1,2*

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文章历史 +

摘要

目的探讨激活哺乳动物雷帕霉素靶蛋白复合物2（mTORC2）/Akt信号通路对6-羟基多巴胺（6-OHDA）模型小鼠多巴胺能神经元和行为学的影响及可能的机制。方法将36只体重20~25 g 3月龄Nestin-CreERTM ::ROSA26-LacZ雄性C57BL/6J小鼠分为NS+玉米油组、6-OHDA+玉米油组、6-OHDA+PP242组、6-OHDA+A-443654组，并在小鼠右侧纹状体注射6-OHDA制备帕金森病（PD）小鼠模型以及每日腹腔注射mTORC2/Akt信号通路激动剂A-443654或抑制剂PP242。通过ELISA测定血清肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）的水平；免疫组织化学和免疫荧光染色考察黑质（SN）-纹状体小胶质细胞、脑室周围神经前体细胞（NPCs）和多巴胺能神经元数目，Western blotting检测中脑水管mTORC2/Akt信号通路各相关蛋白Rictor，p-Akt和DNA损伤反应调节1（REDD1）的表达并通过免疫共沉淀验证它们之间的相互作用，最后观察各组小鼠行为学的变化。结果 6-OHDA模型小鼠伴随小胶质细胞的激活和炎症因子的增加，黑质多巴胺能阳性神经元数目明显下降，小鼠的运动功能发生障碍，但NPCs数目较对照组小鼠明显增加，mTORC2/Akt信号通路相关蛋白表达也明显上调，在激动剂A-443654处理后随着相关蛋白的表达进一步上调，上述各指标均有明显改善，而抑制剂PP242处理组则呈现与激动剂A-443654完全相反的情况。结论 A-443654通过上调mTORC2/Akt信号通路关键蛋白促进NPCs的增殖，增加新生多巴胺能神经元的数目并减少小胶质细胞的激活和炎症反应最终导致PD模型小鼠黑质纹状体多巴胺能神经元和小鼠行为学的改善。

Abstract

Objective To explore the effect of activation of mammalian target of rapmycin complex 2(mTORC2)/Akt signaling pathway on dopaminergic neurons and behavior in 6-hydroxydopamine (6-OHDA) model mice and its possible mechanism. Methods Selecting 36 mice which The Nestin-CreERTM and ROSA26-LacZ reporter genes were detected at the same time in 3-month-old male C57BL/6J mice weighing 20-25 g divideng them into 4 gruops, NS+ corn oil group, 6-OHDA+corn oil group, 6-OHDA+PP242 group and 6-OHDA+A-443654 group, and 6-OHDA was injected into the right striatum of the brain to replicate the Parkinson’s disease (PD) model of mice, and then daily intraperitoneal injection of mTORC2/Akt signaling pathway agonist A-443654 or inhibitor PP242. Serum interleukin-1β (IL-1β) and tumor necrosis factor-α(TNF-α)levels were measured by enzymelinked immunosorbent assay. Immunohistochemistry and immunofluorescence staining were performed to investigate the change of microglia, dopaminergic neurons as well as neural progenitor cells (NPCs). Western blotting was used to detect the expression of related protein of mTORC2/Akt signaling pathway including rictor, p-Akt and regulated in development and DNA dgmage responses 1(REDD1) and the interaction between them were verified by immunoprecipitation. Finally, the behavioral performance of each group of mice was observed. Results With the activation of microglia and the increase of inflammatory factors in PD model mice, the number of dopaminergic neurons in the substantia nigra(SN) decreased significantly, and the motor function of the mice was impaired, but the number of NPCs increased significantly compared with the control mice, mTORC2/Akt signaling pathway related protein expression was also significantly up-regulated. A-443654 treatment further up-regulated the expression of these proteins, meanwhile the indicators mentioned above were ameliorated. However, the inhibitor PP242 treatment group showed completely opposite result with the agonist group. Conclusion A-443654 can promote the proliferation of NPCs and the number of new-born dopaminergic neurons by up-regulating related proteins of mTORC2/Akt signaling pathway, and reducing the activation of microglia and the level of inflammation factors, which ultimately lead to the amelioration of SN-striatal dopaminergic neurons and behavioral performance in PD model mice.

关键词

哺乳动物雷帕霉素靶蛋白复合物2 / 帕金森病 / 多巴胺能神经元 / 小胶质细胞 / 神经再生 / 免疫印迹法 / 小鼠

Key words

Mammalian target of rapamycin complex 2

/ Parkinson’s disease / Dopaminergic neuron / Microglial cell / Nerve regeneration / Western blotting / Mouse

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边维李梦一周鹏李军伟张庭吴安婷戚双双崔怀瑞孙臣友. 激活mTORC2/Akt信号通路对6-羟基多巴胺模型小鼠多巴胺能神经元和行为学的影响[J]. 解剖学报. 2023, 54(1): 13-22 https://doi.org/10.16098/j.issn.0529-1356.2023.01.002

BIAN Wei LI Meng-yi ZHOU Peng LI Jun-wei ZHANG Ting WU An-ting QI Shuang-shuang CUI Huai-rui SUN Chen-you. Effects of activating mTORC2/Akt signaling pathway on dopaminergic neurons and behaviors in 6-hydroxydopamin model mice[J]. Acta Anatomica Sinica. 2023, 54(1): 13-22 https://doi.org/10.16098/j.issn.0529-1356.2023.01.002

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