地塞米松对体外培养胎鼠大脑皮质神经元胞浆Dynein重链和Dynactin表达的影响

程琳; 谢紫云; 里健; 薄涛

doi:10.7499/j.issn.1008-8830.2103151

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中国当代儿科杂志 ›› 2021, Vol. 23 ›› Issue (6) : 639-644. DOI: 10.7499/j.issn.1008-8830.2103151

论著·实验研究

地塞米松对体外培养胎鼠大脑皮质神经元胞浆Dynein重链和Dynactin表达的影响

程琳¹, 谢紫云¹, 里健², 薄涛¹

作者信息 +

Effect of dexamethasone on the expression of Dynein heavy chain and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured in vitro

CHENG Lin¹, XIE Zi-Yun¹, LI Jian², BO Tao¹

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文章历史 +

摘要

目的研究地塞米松（dexamethasone，DEX）对体外培养胎鼠大脑皮质神经元胞浆动力蛋白Dynein重链（Dynein heavy chain，DHC）和Dynactin表达的影响。方法体外培养原代胎鼠大脑皮质神经元细胞，制作DEX干预细胞模型。依据DEX终浓度不同，分为对照组（未施加DEX）、0.1 μmol/L组和1.0 μmol/L组。分别于干预1 d、3 d及7 d时，应用实时荧光定量PCR研究DEX对DHC和Dynactin mRNA表达的影响；应用Western blot法研究DEX对DHC和Dynactin蛋白表达的影响。结果各时间点各组之间DHC mRNA和Dynactin mRNA比较差异均无统计学意义（P > 0.05）。DEX干预后7 d，1.0 μmol/L组胎鼠大脑皮质神经元DHC蛋白随时间推移表达至高峰，且明显高于对照组和0.1 μmol/L组（P < 0.05）。DEX干预后，对照组、0.1 μmol/L组在3 d和7 d时体外培养神经元Dynactin蛋白表达均高于1 d时（P < 0.05）；对照组7 d时神经元Dynactin蛋白表达高于3 d时（P < 0.05）；0.1 μmol/L组7 d时神经元Dynactin蛋白表达低于3 d时（P < 0.05）。在DEX干预后3 d及7 d时，0.1 μmol/L组及1.0 μmol/L组胎鼠大脑皮质神经元Dynactin蛋白表达均明显低于对照组（P < 0.05）；且7 d时，1.0 μmol/L组Dynactin蛋白表达低于0.1 μmol/L组（P < 0.05）。结论 DEX影响体外培养发育中胎鼠大脑皮质神经元DHC和Dynactin蛋白表达，并可能存在浓度依赖性及时间依赖性。

Abstract

Objective To study the effect of dexamethasone (DEX) on the expression of Dynein heavy chain (DHC) and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured in vitro. Methods Primary cerebral cortical neurons of fetal rats were cultured in vitro and were used to establish a cellular model of DEX intervention. According to the final concentration of DEX, the neurons were divided into three groups:control (without DEX), 0.1 μmol/L DEX, and 1.0 μmol/L DEX. On days 1, 3, and 7 after intervention, the quantitative PCR was used to observe the effect of DEX on the mRNA expression of DHC and Dynactin. The Western blot was used to observe the effect of DEX on the protein expression of DHC and Dynactin. Results There was no significant difference in the mRNA expression levels of DHC and Dynactin among the three groups at all time points (P > 0.05). On day 7 after DEX intervention, the protein expression of DHC in the 1.0 μmol/L DEX group gradually increased and reached the peak over time, which was significantly higher than that in the control and 0.1 μmol/L DEX groups (P < 0.05). The control and 0.1 μmol/L DEX groups had a significant increase in the protein expression of Dynactin from day 1 to days 3 and 7 after DEX intervention (P < 0.05). The control group had a significant increase in the protein expression of Dynactin from day 3 to day 7 after intervention (P < 0.05), while the 0.1 μmol/L DEX group had a significant reduction in the protein expression of Dynactin from day 3 to day 7 after intervention (P < 0.05). On days 3 and 7 after DEX intervention, the 0.1 μmol/L DEX and 1.0 μmol/L DEX groups had a significantly lower protein expression level of Dynactin in the cerebral cortical neurons than the control group (P < 0.05). On day 7 after DEX intervention, the 1.0 μmol/L DEX group had a significantly lower protein expression level of Dynactin than the 0.1 μmol/L DEX group (P < 0.05). Conclusions DEX affects the protein expression of DHC and Dynactin in the fetal rat cerebral cortical neurons cultured in vitro, possibly in a concentration- and time-dependent manner.

导出引用

程琳, 谢紫云, 里健, 薄涛. 地塞米松对体外培养胎鼠大脑皮质神经元胞浆Dynein重链和Dynactin表达的影响[J]. 中国当代儿科杂志. 2021, 23(6): 639-644 https://doi.org/10.7499/j.issn.1008-8830.2103151

CHENG Lin, XIE Zi-Yun, LI Jian, BO Tao. Effect of dexamethasone on the expression of Dynein heavy chain and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured in vitro[J]. Chinese Journal of Contemporary Pediatrics. 2021, 23(6): 639-644 https://doi.org/10.7499/j.issn.1008-8830.2103151

参考文献

[1] Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants[J]. Pediatrics, 1972, 50(4):515-525. PMID:4561295.
[2] Hallman M. The story of antenatal steroid therapy before preterm birth[J]. Neonatology, 2015, 107(4):352-357. DOI:10.1159/000381130. PMID:26044103.
[3] Doyle LW, Cheong JL, Ehrenkranz RA, et al. Early (< 8 days) systemic postnatal corticosteroids for prevention of bronchopulmonary dysplasia in preterm infants[J]. Cochrane Database Syst Rev, 2017, 10(10):CD001146. DOI:10.1002/14651858.CD001146.pub5. PMID:29063585.
[4] Doyle LW, Cheong JL, Ehrenkranz RA, et al. Late (> 7 days) systemic postnatal corticosteroids for prevention of bronchopulmonary dysplasia in preterm infants[J]. Cochrane Database Syst Rev, 2017, 10(10):CD001145. DOI:10.1002/14651858.CD001145.pub4. PMID:29063594.
[5] Scott SM, Rose SR. Use of glucocorticoids for the fetus and preterm infant[J]. Clin Perinatol, 2018, 45(1):93-102. DOI:10.1016/j.clp.2017.11.002. PMID:29406009.
[6] Zhang RL, Bo T, Shen L, et al. Effect of dexamethasone on intelligence and hearing in preterm infants:a meta-analysis[J]. Neural Regen Res, 2014, 9(6):637-645. DOI:10.4103/1673-5374.130085. PMID:25206867.
[7] Nürnberg E, Horschitz S, Schloss P, et al. Basal glucocorticoid receptor activation induces proliferation and inhibits neuronal differentiation of human induced pluripotent stem cell-derived neuronal precursor cells[J]. J Steroid Biochem Mol Biol, 2018, 182:119-126. DOI:10.1016/j.jsbmb.2018.04.017. PMID:29751108.
[8] Chen QF, Wang FF, Zhang YC, et al. Neonatal DEX exposure leads to hyperanxious and depressive-like behaviors as well as a persistent reduction of BDNF expression in developmental stages[J]. Biochem Biophys Res Commun, 2020, 527(1):311-316. DOI:10.1016/j.bbrc.2020.04.084. PMID:32446386.
[9] Hippenmeyer S. Molecular pathways controlling the sequential steps of cortical projection neuron migration[J]. Adv Exp Med Biol, 2014, 800:1-24. DOI:10.1007/978-94-007-7687-6_1. PMID:24243097.
[10] Dantas TJ, Carabalona A, Hu DJ, et al. Emerging roles for motor proteins in progenitor cell behavior and neuronal migration during brain development[J]. Cytoskeleton (Hoboken), 2016, 73(10):566-576. DOI:10.1002/cm.21293. PMID:26994401.
[11] Shi L, Muthusamy N, Smith D, et al. Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member, calcyon[J]. Int J Biochem Cell Biol, 2017, 90:93-102. DOI:10.1016/j.biocel.2017.07.005. PMID:28734834.
[12] Bercier V, Hubbard JM, Fidelin K, et al. Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities[J]. Mol Neurodegener, 2019, 14(1):27. DOI:10.1186/s13024-019-0327-3. PMID:31291987.
[13] Carter AP, Diamant AG, Urnavicius L. How dynein and dynactin transport cargos:a structural perspective[J]. Curr Opin Struct Biol, 2016, 37:62-70. DOI:10.1016/j.sbi.2015.12.003. PMID:26773477.
[14] 罗森林, 薄涛, 刘通, 等. 皮质酮对体外培养发育中胎鼠脑皮层神经元迁移蛋白LIS1表达的影响[J]. 中国当代儿科杂志, 2017, 19(9):1008-1013. DOI:10.7499/j.issn.1008-8830.2017.09.015. PMID:28899473.
[15] Chen YL, Xu YF, Li GQ, et al. Exome sequencing identifies de novo DYNC1H1 mutations associated with distal spinal muscular atrophy and malformations of cortical development[J]. J Child Neurol, 2017, 32(4):379-386. DOI:10.1177/0883073816683083. PMID:28193117.
[16] Olenick MA, Dominguez R, Holzbaur ELF. Dynein activator Hook1 is required for trafficking of BDNF-signaling endosomes in neurons[J]. J Cell Biol, 2019, 218(1):220-233. DOI:10.1083/jcb.201805016. PMID:30373907.
[17] Francis F, Cappello S. Neuronal migration and disorders - an update[J]. Curr Opin Neurobiol, 2021, 66:57-68. DOI:10.1016/j.conb.2020.10.002. PMID:33096394.
[18] Scoto M, Rossor AM, Harms MB, et al. Novel mutations expand the clinical spectrum of DYNC1H1-associated spinal muscular atrophy[J]. Neurology, 2015, 84(7):668-679. DOI:10.1212/WNL.0000000000001269. PMID:25609763.
[19] Tirumala NA, Ananthanarayanan V. Role of dynactin in the intracellular localization and activation of cytoplasmic dynein[J]. Biochemistry, 2020, 59(2):156-162. DOI:10.1021/acs.biochem.9b00772. PMID:31591892.
[20] Htet ZM, Gillies JP, Baker RW, et al. LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes[J]. Nat Cell Biol, 2020, 22(5):518-525. DOI:10.1038/s41556-020-0506-z. PMID:32341549.
[21] Allan VJ. Cytoplasmic dynein[J]. Biochem Soc Trans, 2011, 39(5):1169-1178. DOI:10.1042/BST0391169. PMID:21936784.
[22] Nicholas MP, Höök P, Brenner S, et al. Control of cytoplasmic dynein force production and processivity by its C-terminal domain[J]. Nat Commun, 2015, 6:6206. DOI:10.1038/ncomms7206. PMID:25670086.
[23] Hoogenraad CC, Akhmanova A. Bicaudal D family of motor adaptors:linking dynein motility to cargo binding[J]. Trends Cell Biol, 2016, 26(5):327-340. DOI:10.1016/j.tcb.2016.01.001. PMID:26822037.
[24] Martín-Cófreces NB, Sánchez-Madrid F. Sailing to and docking at the immune synapse:role of tubulin dynamics and molecular motors[J]. Front Immunol, 2018, 9:1174. DOI:10.3389/fimmu.2018.01174. PMID:29910809.
[25] Levy JR, Holzbaur EL. Cytoplasmic dynein/dynactin function and dysfunction in motor neurons[J]. Int J Dev Neurosci, 2006, 24(2-3):103-111. DOI:10.1016/j.ijdevneu.2005.11.013. PMID:16406469.
[26] Egan MJ, Tan K, Reck-Peterson SL. Lis1 is an initiation factor for dynein-driven organelle transport[J]. J Cell Biol, 2012, 197(7):971-982. DOI:10.1083/jcb.201112101. PMID:22711696.
[27] Clark GD. Platelet-activating factor acetylhydrolase and brain development[J]. Enzymes, 2015, 38:37-42. DOI:10.1016/bs.enz.2015.09.009. PMID:26612645.
[28] Aiken J, Moore JK, Bates EA. TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity[J]. Hum Mol Genet, 2019, 28(8):1227-1243. DOI:10.1093/hmg/ddy416. PMID:30517687.
[29] Gonçalves JC, Dantas TJ, Vallee RB. Distinct roles for dynein light intermediate chains in neurogenesis, migration, and terminal somal translocation[J]. J Cell Biol, 2019, 218(3):808-819. DOI:10.1083/jcb.201806112. PMID:30674581.
[30] Heinrich G, Lum T. Fish neurotrophins and Trk receptors[J]. Int J Dev Neurosci, 2000, 18(1):1-27. DOI:10.1016/s0736-5748(99)00071-4. PMID:10708902.
[31] Hoang HT, Schlager MA, Carter AP, et al. DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes[J]. Proc Natl Acad Sci U S A, 2017, 114(9):E1597-E1606. DOI:10.1073/pnas.1620141114. PMID:28196890.
[32] Lanshakov DA, Sukhareva EV, Kalinina TS, et al. Dexamethasone-induced acute excitotoxic cell death in the developing brain[J]. Neurobiol Dis, 2016, 91:1-9. DOI:10.1016/j.nbd.2016.02.009. PMID:26873551.
[33] Sukhareva EV, Dygalo NN, Kalinina TS. Influence of dexamethasone on the expression of immediate early genes c-fos and c-jun in different regions of the neonatal brain[J]. Mol Biol (Mosk), 2016, 50(2):266-271. DOI:10.7868/S0026898416020257. PMID:27239846.