Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis

DING Kang; ZHANG Feng-Ping; QI Gao-Xiu; LIN Meng; CHEN Min; CHEN Yan-Chun; GUO Zhang-Yu; ZHOU Feng-Hua; GUAN Ying-Jun

doi:10.16098/j.issn.0529-1356.2022.03.001

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Acta Anatomica Sinica ›› 2022, Vol. 53 ›› Issue (3) : 273-280. DOI: 10.16098/j.issn.0529-1356.2022.03.001

Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis

DING Kang^1,4 ZHANG Feng-ping^1,4 QI Gao-xiu^1,2 LIN Meng^1,4 CHEN Min^1,2 CHEN Yan-chun^3,4 GUO Zhang-yu^1,4 ZHOU Feng-hua^1,4* GUAN Ying-jun^3,4*

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Abstract

Objective To investigate the role of zinc finger protein 36,C3H type-like 1（ZFP36L1）mediating astrocytes activation in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Methods Superoxide dismutase 1 (SOD1)-G93A transgenic mice were used as animal models, the wild-type littermates as the control (13 mice were taken from mutant and wild-type mice at each time point). The ZFP36L1 mRNA and protein levels of the spinal cord in the early, middle and late stage were detected by Real-time PCR and Western blotting. The expression and distribution of ZFP36L1 in the spinal cord were detected by immunofluorescence. Primary astrocyte model was established from 15 postnatal 1-2 day mice. The ZFP36L1 mRNA and protein levels in astrocytes were detected by Real-time PCR and Western blotting. Si-ZFP36L1 was transfected into SOD1-G93A mutant primary astrocytes. The transfection efficiency was detected by Western blotting. Tumor necrosis factor α (TNF-α) and interleukin-18 (IL-18) secreted from astrocytes after transfection were assessed by Western blotting and ELISA. After silencing ZFP36L1 in SOD1-G93A mutant primary astrocytes, it was cocultured with SOD1-G93A mutant NSC34 cells. 5’-ethyyl-2’deoxyuridine (EdU) test and the level of proliferating cell nuclear antigen (PCNA) were used to determine the effect of ZFP36L1 on NSC34 cell proliferation. TUNEL test and the level of cleaved-Caspase-3 were used to determine the effect of ZFP36L1 on NSC34 cell apoptosis. Blank small interfering RNA(siRNA) was transfected as the control group. Results Compared with the wild-type mice, the mRNA and protein levels of ZFP36L1 were downregulated in the spinal cord of SOD1-G93A transgenic mice. In wild type mice, ZFP36L1 positive cells were mainly β-tubulin Ⅲ positive. In SOD1-G93A mutant mice, ZFP36L1 positive cells were mainly glial fibrillary acidic protein (GFAP) positive. The expression of ZFP36L1 in SOD1-G93A mutant primary astrocytes increased, and si-ZFP36L1 reduced the level of ZFP36L1 in SOD1-G93A mutant primary astrocytes significantly. Inflammatory factors including TNF-α, IL-18 decreased significantly after silencing ZFP36L1. In addition, after silencing ZFP36L1 expression, SOD1-G93A mutant primary astrocytes enhanced the proliferation activity of NSC34 cells and inhibited NSC34 cell apoptosis significantly. Conclusion Astrocytes are activated in the process of ALS. ZFP36L1 promotes the degeneration of motor neurons in ALS through the inflammatory factors secreted by astrocytes.

Key words

Amyotrophic lateral sclerosis / Zinc finger protein 36, C3H type-like 1 / Inflammatory factor / Motor neuron / Astrocyte / Immunofluorescence / Transgenic mouse

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DING Kang ZHANG Feng-ping QI Gao-xiu LIN Meng CHEN Min CHEN Yan-chun GUO Zhang-yu ZHOU Feng-hua GUAN Ying-jun. Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis[J]. Acta Anatomica Sinica. 2022, 53(3): 273-280 https://doi.org/10.16098/j.issn.0529-1356.2022.03.001

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