Wnt 1 silencing enhances neurotoxicity induced by paraquat and maneb in SH ‐ SY 5 Y cells

Wingless (Wnt) signaling regulates the proliferation and differentiation of midbrain dopamine (DA) neurons. Paraquat (PQ) and maneb (MB) are environmental pollutants that can be used to model Parkinson's disease (PD) in rodents. A previous study demonstrated that developmental exposure to PQ and MB affects the expression of Wnt1, Wnt5a, nuclear receptor-related factor 1 (NURR1) and tyrosine hydroxylase (TH). However, how Wnt signaling regulates these developmental factors in vitro is yet to be determined. To explore this, SH-SY5Y cells were exposed to PQ and MB. The results of the current study indicated that exposure to PQ and MB decreased Wnt1, β-catenin, NURR1 and TH levels and increased Wnt5a levels. Furthermore, Wnt1 silencing has the same effect as exposure to PQ and MB. Additionally, the neurotoxicity induced by PQ and MB is more severe in siWnt1-SH-SY5Y cells compared with normal SH-SY5Y cells. Therefore, Wnt1 may serve an important role in regulating developmental DA factors, and may be a candidate gene for PD diagnosis or gene therapy. Introduction Parkinson's disease (PD) is the second most common neurological disorder worldwide and is caused by the degeneration of midbrain dopamine (DA) neurons (1). The exposure to environmental toxins during development, which impact DA precursor factors, may lead to neurodegeneration and can increase the risk of developing PD (2,3). Wingless 1 (Wnt1) and Wingless 5a (Wnt5a), which are two members of the Wnt family, are developmental factors that regulate the proliferation and differentiation of DA precursors (4). Wnt1 enhances mesencephalic DA neuron differentiation (5), and Wnt5a can regulate midbrain DA axon growth and guidance (6). Additionally, Wnt1 and Wnt5a cooperate to regulate nuclear receptor-related factor 1 (NURR1) and tyrosine hydroxylase (TH) expression (7,8). NURR1 is a brain‐specific transcription factor located in DA neurons (9) and induces the neurogenesis of DA-phenotype neurons (10), while TH is a rate-limiting enzyme located in DA neurons and is associated with synthesizing DA (11). NURR1 can activate the promoter of TH gene in neural progenitor cells increasing TH expression (12,13). A previous study demonstrated that the developmental exposure to the herbicide paraquat (PQ) and the fungicide maneb (MB) in pregnant and lactating rats influenced the expression of Wnt1, Wnt5a, NURR1 and TH in the midbrain DA neurons of offspring (14). However, the mechanism of Wnt signaling in PD is still yet to be determined. PQ and MB are two common agricultural chemicals, and their combined exposure has been used to model Parkinson-like motor deficits in rodents and to investigate the mechanisms of pathogenesis in this disease (15-18). However, the combined effect of PQ and MB in vitro, and their mechanism causing PD, is rarely reported. In the current study, the human SH-SY5Y neuroblastoma cell line, which is used as an in vitro cellular model of DA neurons, was exposed to PQ and MB to explore the mechanisms of Wnt signaling in PD. Materials and methods Chemicals, reagents and antibodies. PQ was purchased from J&K Technology Co. Ltd. and MB was purchased from Sigma-Aldrich (Merck KGaA). Cell counting kit-8 (CCK-8) Wnt1 silencing enhances neurotoxicity induced by paraquat and maneb in SH‐SY5Y cells CUI HUANG1,2, JING MA1, BAI-XIANG LI1* and YAN SUN1* 1Department of Toxicology, School of Public Health, Harbin Medical University, Harbin, Heilongjiang 150081; 2Safety and Quality Institute of Agricultural Products, Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang 150086, P.R. China Received July 30, 2018; Accepted July 23, 2019 DOI: 10.3892/etm.2019. 7963 Correspondence to: Professor Bai-Xiang Li or Professor Yan Sun, Department of Toxicology, School of Public Health, Harbin Medical University, 157 Baojian Road, Nan Gang, Harbin, Heilongjiang 150081, P.R. China E-mail: libaix@ems.hrbmu. edu.cn E-mail: 13946167049@163.com *Contributed equally Abbreviations: PD, Parkinson's disease; DA, dopamine; PQ, paraquat; MB, maneb; Wnt1, Wingless 1; Wnt5a, Wingless 5a; NURR1, nuclear receptor-related factor 1; TH, tyrosine hydroxylase; TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween; PBS, phosphate buffered saline; PBST, phosphate buffered saline with Tween; DAPI, 4',6-diamidino-2-phenylindole