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

doi:10.16098/j.issn.0529-1356.2023.01.002

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

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*

1.温州医科大学基础医学院解剖学教研室; 2.神经科学研究所，浙江温州 325035; 3.温州医科大学基础医学院，浙江温州325035; 4.温州医科大学附属第二医院（育英儿童医院）药剂科，浙江温州 325035

收稿日期:2021-05-14 修回日期:2022-09-22 出版日期:2023-02-06 发布日期:2023-02-06
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

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*

1.Department of Anatomy, School of Basic Medical Sciences, Wenzhou Medical University, Zhejiang Wenzhou325035,China; 2.Institution of Neuroscience, School of Basic Medical Sciences, Wenzhou Medical University, Zhejiang Wenzhou325035, China;

3.School of Basic Medical Sciences, Wenzhou Medical University,Zhejiang Wenzhou325035, China; 4.Pharmacy Department of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang Wenzhou325035, China

Received:2021-05-14 Revised:2022-09-22 Online:2023-02-06 Published:2023-02-06
Contact: SUN Chen-you E-mail:sunchenyou1972@aliyun.com

摘要/Abstract

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。

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

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.

Key words:

Mammalian target of rapamycin complex 2, Parkinson’s disease, Dopaminergic neuron, Microglial cell, Nerve regeneration, Western blotting, Mouse

中图分类号:

Q189

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.

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.

参考文献

[1] Morrison BE. Discovery of nigral dopaminergic neurogenesis in adult mice［J］.Neural Regen Res,2016, 11(6):878-881.

［2］Sliter DA, Martinez J, Hao L, et al. Parkin and PINK1 mitigate STING-induced inflammation［J］.Nature,2018, 561(7722):258-262.

［3］Chen, LL, Song JX, Lu JH, et al. Corynoxine, a natural autophagy enhancer, promotes the clearance of alpha-synuclein via Akt/mTOR pathway［J］. J Neuroimmune Pharmacol,2014, 9:380-387.

［4］Lu Z, Shi X, Gong F, et al.RICTOR/mTORC2 affects tumorigenesis and therapeuticefficacy of mTOR inhibitors in esophageal squamous cell carcinoma［J］.Acta Pharm Sin B,2020,10(6):1004-1019.

［5］Hemmati-Dinarvand M, Saedi S, Valilo M, et al. Oxidative stress and Parkinson’s disease:conflict of oxidant-antioxidant systems［J］.Neurosci Lett,2019,709:134296.

［6］Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins［J］.Biochim Biophys Acta,2011,1813(11):1938-1945.

[7] Harms AS, Thome AD, Yan Z, et al.Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease［J］.Exp Neurol,2017, 300:179-187.

［8］Wang TT, Chen ZhCh, Ye X, et al. The effect of allopregnenolone on dopaminergic neurons in the substantia nigra of mice with Parkinson’s disease and its possible molecular mechanism［J］.Acta Anatomica Sinica, 2020, 51(4):473-482.(in Chinese)

王彤彤,陈治池,叶鑫,等.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.

［9］Wang TT, Ye X,Bian W, et al. Allopregnanolone modulates GABAAR-dependent CaMKIIδ3 and BDNF to protect SH-SY5Y cells against 6-OHDA-induced damage［J］.Acta Anatomica Sinica, 2021, 52(1):5-13.(in Chinese)

王彤彤,叶鑫,边维,等.别孕烯醇酮对6-羟基多巴胺损伤的细胞系SH-SY5Y的保护作用［J］.解剖学报,2021, 52(1):5-13.

［10］Ota KT, Liu RJ, Voleti B, et al.REDD1 is essential for stress-induced synaptic loss and depressive behavior ［J］.Nat Med,2014, 20(5):531-535.

［11］Huang L, Deng M, He Y, et al.β-asarone increases MEF2D and TH levels and reduces α-synuclein level in 6-OHDA-induced rats via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway:chaperone-mediated autophagy activation, macroautophagy inhibition and HSP70 up-expression［J］.Behav Brain Res,2016, 313:370-379.

［12］Abe K, Saito H.Effect of basic fibroblast growth factor on development of Parkinson syndrome in mice［J］.Biull Eksp Biol Med,1995, 120(9):260-262.

［13］Chen ZC, Wang TT, Bian W, et al. Allopregnanolone restores the tyrosine hydroxylase-positive neurons and motor performance in 6-OHDA-injected mouse model［J］. CNS Neurosci Ther, 2020, 26(10):1069-1082.

［14］Wang T, Ye X, Bian W, er al. Allopregnanolone modulates GABAAR-dependent CaMKIIδ3 and BDNF to protect SH-SY5Y cells against 6-OHDA-induced damage［J］. Front Cell Neurosci,2020, 13:569.

［15］Saxton RA, Sabatini DM, et al.mTOR Signaling in growth, metabolism, and disease［J］.Cell,2017, 168(6):960-976.

［16］JhanwarUniyal M, Amin AG, Cooper JB, et al. Discrete signaling mechanisms of mTORC1 and mTORC2: connected yet apart in cellular and molecular aspect［J］.Adv Biol Regul,2017,64:39-48.

［17］Hresko RC, Murata H, Mueckler M, et al. Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes［J］.J Biol Chem,2003, 278(24):21615-21622.

［18］Zhu Z, Yang C, Iyaswamy A, et al. Balancing mTOR signaling and autophagy in the treatment of Parkinson’s disease ［J］. Int J Mol Sci,2019, 20(3):728-735.

［19］Lee M, Sim H, Ahn H, et al. Direct reprogramming to human induced neuronal progenitors from fibroblasts of familial and sporadic Parkinson’s disease patients［J］.Int J Stem Cells, 2019, 12(3):474-483.

［20］Yin H, Zhang Y, Wang K, et al.The involvement of regulated in development and DNA damage response 1 (REDD1) in the pathogenesis of intervertebral disc degeneration［J］.Exp Cell Res,2018, 372(2):188-197.

［21］Sun J, Yue F. Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy ［J］.J Cell Biochem,2019, 120(9):14771-14779.

［22］Jin HO, Hong SE, Kim JY, et al. Induction of HSP27 and HSP70 by constitutive overexpression of Redd1 confers resistance of lung cancer cells to ionizing radiation［J］.Oncol Rep,2019, 41(5):3119-3126.

［23］Jia W, Chang B, Sun L, et al. REDD1 and p-AKT over-expression may predict poor prognosis in ovarian cancer ［J］.Int J Clin Exp Pathol, 2014, 7(9):5940-5969.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[5]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[6]	吕海燕沈西雅赵富生朱梅. 松茸多糖对 1-甲基-4-苯基-吡啶离子诱导 PC12 细胞损伤的影响 [J]. 解剖学报, 2024, 55(1): 49-54.
[7]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[8]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[9]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[10]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[11]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[12]	陈子璇司丽娜舒薇菡张欣魏晨阳程露阳杨松鹤乔跃兵. 褪黑素调控下丘脑PI3K/Akt/mTOR信号延缓雌性小鼠青春期启动的机制 [J]. 解剖学报, 2023, 54(6): 644-651.
[13]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[14]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.
[15]	刘哲川李坤马帅男孟家琪王艳芹. 利拉鲁肽对百草枯诱导的小鼠帕金森病模型炎症及线粒体融合/分裂的影响 [J]. 解剖学报, 2023, 54(6): 676-681.

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

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价