Effect and molecular mechanism of mammalian target of rapamycin complex 2/Akt signaling pathway on 6-hydroxydopamine-treated SH-SY5Y cell model

LI Meng-Yi; WU An-Ting; ZHANG Ting; LI Jun-Wei; ZHOU Peng; CUI Huai-Rui; SUN Chen-You

doi:10.16098/j.issn.0529-1356.2023.05.004

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Acta Anatomica Sinica ›› 2023, Vol. 54 ›› Issue (5) : 521-530. DOI: 10.16098/j.issn.0529-1356.2023.05.004

Effect and molecular mechanism of mammalian target of rapamycin complex 2/Akt signaling pathway on 6-hydroxydopamine-treated SH-SY5Y cell model

LI Meng-yi^1,2 WU An-ting^1,2 XU Ze-ting^1,2 ZHANG Ting^1,3 LI Jun-wei^1,2 ZHOU Peng^1,2 CUI Huai-rui^1,2 SUN Chen-you^1,2*

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Abstract

Objective To study whether the regulation of mammalian target of rapamycin complex 2(mTORC2)/Akt signaling pathway has a protective effect on SH-SY5Y cell line damaged by 6-hydroxydopamine (6-OHDA), and to clarify its molecular mechanism. Methods SH-SY5Y cells treated with retinoic acid (RA) were given 6-OHDA, mTORC2 signaling pathway inhibitor PP242 and agonist A-443654 respectively. The changes of cell number in each group were investigated by immunofluorescent staining; The total protein was extracted and the expression level and interaction of key proteins in mTORC2 signaling pathway were determined by Western blotting and co-immunoprecipitation (CoIP); The apoptosis rate of cells in each group was detected by flow cytometry. At the same time, the co-culture Parkinson’s disease(PD) model was made using SH-SY5Y cell line and Bv-2 cell line; MTT colorimetric method was used to detect the cell viability of each group; ELISA was used to detect the content of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell culture supernatant. Results The number of tyrosine hydroxylase(TH)/proliferating cell nuclear antigen(PCNA)/hochest-, TH/5-bronmo-2’-deoxyuridine(BrdU)-labeled positive cells in 6-OHDA-lesioned PD cell model group was significantly lower than that in the normal group; The apoptosis rate was higher; The expression of Rictor, p-Akt and regulated in DNA damage and development 1(REDD1) was increased; There was an interaction between Rictor and p-Akt or REDD1; The cell viability was significantly reduced in the co-culture model; the content of TNF-α and IL-β increased in the cell culture supernatant. With further up-regulation of the abovementioned protein expressions, the cell survival, apoptosis and pro-inflammatory cytokine levels in A-443654 group were significantly ameliorated, while PP242 group showed the opposite changes. Conclusion A-443654 activates mTORC2 signaling pathway by p-Akt, which increases the expression of Rictor and REDD1 protein. These changes contribute to the amelioration in cell survival rate, apoptosis rate, and the proliferation and differentiation and decreasion of apoptosis rate of SH-SY5Y cells. These result improved 6-OHDA-induced cell damage and inhibited the release of pro-inflammatory cytokines.

Key words

Parkinson’s disease

/ Mammalian rapamycin target protein 2 / PP242 / A-443654 / Immunofluorescence / SH-SY5Y cell line

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LI Meng-yi WU An-ting XU Ze-ting ZHANG Ting LI Jun-wei ZHOU Peng CUI Huai-rui SUN Chen-you.

Effect and molecular mechanism of mammalian target of rapamycin complex 2/Akt signaling pathway on 6-hydroxydopamine-treated SH-SY5Y cell model

[J]. Acta Anatomica Sinica. 2023, 54(5): 521-530 https://doi.org/10.16098/j.issn.0529-1356.2023.05.004

References

［1］Malar DS, Prasanth MI, Brimson JM, et al. Neuroprotective properties of green tea (Camellia sinensis) in Parkinson’s disease: a review［J］. Molecules, 2020, 25(17):3926.

［2］Rizek P, Kumar N, Jog MS. An update on the diagnosis and treatment of Parkinson disease［J］. CMAJ, 2016, 188(16):1157-1165.

［3］Nutt JG, Wooten GF. Clinical practice. Diagnosis and initial management of Parkinson’s disease［J］. N Engl J Med, 2005, 353(10):1021-1027.

［4］Sarkar S, Nguyen HM, Malovic E, et al. Kv13 modulates neuroinflammation and neurodegeneration in Parkinson’s disease［J］. J Clin Invest, 2020, 130(8):4195-4212.

［5］Hare SH, Harvey AJ. mTOR function and therapeutic targeting in breast cancer［J］. Am J Cancer Res. 2017;7(3):383-404.

［6］Switon K, Kotulska K, Janusz-Kaminska A, et al. Molecular neurobiology of mTOR［J］. Neuroscience, 2017, 341:112-153.

［7］Chen CJ, Sgritta M, Mays J, et al. Therapeutic inhibition of mTORC2 rescues the behavioral and neurophysiological abnormalities associated with Pten-deficiency［J］. Nat Med, 2019, 25(11):1684-1690.

［8］Pérez-Sisqués L, Solana-Balaguer J, Campoy-Campos G, et al. RTP801/REDD1 is involved in neuroinflammation and modulates cognitive dysfunction in Huntington’s disease［J］. Biomolecules, 2021, 12(1):34.

［9］Jin HO, Hong SE, Kim JH, et al. Sustained overexpression of Redd1 leads to Akt activation involved in cell survival［J］. Cancer Lett, 2013, 336(2):319-324. ［10］Britto FA, Dumas K, Giorgetti-Peraldi S,et al. Is REDD1 a metabolic double agent? Lessons from physiology and pathology［J］. Am J Physiol Cell Physiol, 2020, 319(5):C807-C824.

［11］Lu Z, Zhang Y, Xu Y, et al. mTOR inhibitor PP242 increases antitumor activity of sulforaphane by blocking Akt/mTOR pathway in esophageal squamous cell carcinoma［J］. Mol Biol Rep, 2022, 49(1):451-461.

［12］Han EK, Leverson JD, McGonigal T, et al. Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition［J］. Oncogene, 2007,26(38):5655-5661.

［13］Ponzoni M, Bachetti T, Corrias MV, et al. Recent advances in the developmental origin of neuroblastoma: an overview［J］. J Exp Clin Cancer Res, 2022, 41(1):92.

［14］Xicoy H, Wieringa B, Martens GJ. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review［J］. Mol Neurodegener, 2017, 12(1):10.

［15］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.

［16］Kilo L, Stürner T, Tavosanis G, et al. Drosophila dendritic arborisation neurons: fantastic actin dynamics and where to find them［J］. Cells, 2021, 10(10):2777.

［17］Siuta MA, Robertson SD, Kocalis H, et al. Dysregulation of the norepinephrine transporter sustains cortical hypodopaminergia and schizophrenia-like behaviors in neuronal rictor null mice［J］. PLoS Biol, 2010, 8(6):e1000393.

［18］Urbanska M, Gozdz A, Swiech LJ, et al. Mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) control the dendritic arbor morphology of hippocampal neurons［J］. J Biol Chem, 2012, 287(36):30240-30256.

［19］Laguesse S, Morisot N, PhamLuong K,et al. mTORC2 in the dorsomedial striatum of mice contributes to alcohol-dependent -Actin polymerization, structural modifications, and consumption［J］. Neuropsychopharmacology, 2018, 43(7):1539-1547.

［20］Johnson JL, Huang W, Roman G, et al. TORC2: a novel target for treating age-associated memory impairment［J］. Sci Rep, 2015, 5: 15193.

［21］Brugarolas J, Lei K, Hurley RL, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex［J］. Genes Dev, 2004, 18(23):2893-2904.

［22］Nagatsu T, Sawada M. Inflammatory process in Parkinson’s disease: role for cytokines［J］. Curr Pharm Des, 2005,11(8):999-1016.

［23］Doorn KJ, Moors T, Drukarch B, et al. Microglial phenotypes and toll-like receptor 2 in the substantia nigra and hippocampus of incidental Lewy body disease cases and Parkinson’s disease patients［J］. Acta Neuropathol Commun, 2014, 2:90.

［24］Fan W, Cheng K, Qin X, et al. mTORC1 and mTORC2 play different roles in the functional survival of transplanted adipose-derived stromal cells in hind limb ischemic mice via regulating inflammation in vivo［J］. Stem Cells, 2013, 31(1):203-214.