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Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study
Environmental Health Perspectives ( IF 10.4 ) Pub Date : 2022-5-5 , DOI: 10.1289/ehp9505
Sida Han 1 , Yiwei Feng 1 , Min Guo 1 , Yining Hao 1 , Jian Sun 1 , Yichen Zhao 2 , Qiang Dong 3, 4, 5 , Yanxin Zhao 2 , Mei Cui 1, 5
Affiliation  

Abstract

Background:

Paraquat (PQ) is a pesticide, exposure to which has been associated with an increased risk of Parkinson’s disease; however, PQ transport mechanisms in the brain are still unclear. Our previous studies indicated that the organic cation transporter 3 (OCT3) expressed on astrocytes could uptake PQ and protect the dopaminergic (DA) neurons from a higher level of extracellular PQ. At present, it is unknown how OCT3 levels are altered during chronic PQ exposure or aging, nor is it clear how the compensatory mechanisms are triggered by OCT3 deficiency. Dynamic related protein 1 (DRP1) was previously reported to ameliorate the loss of neurons during Parkinson’s disease. Nowadays, mounting studies have revealed the functions of astrocyte DRP1, prompting us to hypothesize that DRP1 could regulate the PQ transport capacity of astrocytes.

Objectives:

The present study aimed to further explore PQ transport mechanisms in the nigrostriatal system and identify pathways involved in extracellular PQ clearance.

Methods:

Models of PQ-induced neurodegeneration were established by intraperitoneal (i.p.) injection of PQ in wild-type (WT) and organic cation transporter-3–deficient (Oct3/) mice. DRP1 knockdown was achieved by viral tools in vivo and small interfering RNA (siRNA) in vitro. Extracellular PQ was detected by in vivo microdialysis. In vitro transport assays were used to directly observe the functions of different transporters. PQ-induced neurotoxicity was evaluated by tyrosine hydroxylase immunohistochemistry, in vivo microdialysis for striatal DA and behavior tests. Western blotting analysis or immunofluorescence was used to evaluate the expression levels and locations of proteins in vitro or in vivo.

Results:

Older mice and those chronically exposed to PQ had a lower expression of brain OCT3 and, following exposure to a 10-mg/kg i.p. PQ2+ loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and Oct3/ mice after chronic exposure to PQ; this was supported by finding higher levels of DRP1 after PQ treatment of dopamine transporter-expressing neurons with and without OCT3 inhibition and in primary astrocytes of WT and Oct3/ mice. Selective astrocyte DRP1 knockdown ameliorated the PQ2+-induced neurotoxicity in Oct3/ mice but not in WT mice. GL261 astrocytes with siRNA-mediated DRP1 knockdown had a higher expression of alanine–serine–cysteine transporter 2 (ASCT2), and transport studies suggest that extracellular PQ was transported into astrocytes by ASCT2 when OCT3 was absent.

Discussion:

The present study mainly focused on the transport mechanisms of PQ between the dopaminergic neurons and astrocytes. Lower OCT3 levels were found in the older or chronically PQ-treated mice. Astrocytes with DRP1 inhibition (by viral tools or mitochondrial division inhibitor-1) had higher levels of ASCT2, which we hypothesize served as an alternative transporter to remove extracellular PQ when OCT3 was deficient. In summary, our data suggest that OCT3, ASCT2 located on astrocytes and the dopamine transporter located on DA terminals may function in a concerted manner to mediate striatal DA terminal damage in PQ-induced neurotoxicity. https://doi.org/10.1289/EHP9505



中文翻译:

OCT3 和 DRP1 在星形胶质细胞中百草枯转运中的作用:一项小鼠研究

摘要

背景:

百草枯 (PQ) 是一种杀虫剂,接触它会增加患帕金森病的风险;然而,大脑中的 PQ 转运机制仍不清楚。我们之前的研究表明,星形胶质细胞上表达的有机阳离子转运蛋白 3 (OCT3) 可以摄取 PQ 并保护多巴胺能 (DA) 神经元免受更高水平的细胞外 PQ 的影响。目前,尚不清楚在慢性 PQ 暴露或衰老过程中 OCT3 水平如何改变,也不清楚 OCT3 缺乏如何触发补偿机制。先前报道动态相关蛋白 1 (DRP1) 可改善帕金森病期间神经元的损失。如今,越来越多的研究揭示了星形胶质细胞 DRP1 的功能,促使我们假设 DRP1 可以调节星形胶质细胞的 PQ 转运能力。

目标:

本研究旨在进一步探索黑质纹状体系统中的百草枯转运机制,并确定参与细胞外百草枯清除的途径。

方法:

通过腹膜内 (ip) 注射野生型 (WT) 和有机阳离子转运蛋白 3 缺陷型 PQ (ip) 建立 PQ 诱导的神经变性模型。C3-/-) 老鼠。DRP1 敲低是通过体内病毒工具和体外小干扰 RNA (siRNA)实现的。通过体内微透析检测细胞外百草枯。体外转运试验用于直接观察不同转运蛋白的功能。PQ 诱导的神经毒性通过酪氨酸羟化酶免疫组织化学、纹状体 DA的体内微透析和行为测试来评估。Western印迹分析或免疫荧光用于评估蛋白质在体外体内的表达水平和位置。

结果:

年长小鼠和长期暴露于 PQ 的小鼠大脑 OCT3 表达较低,并且在暴露于10-毫克/公斤ip质量问题2+负荷剂量,较高浓度的细胞外百草枯。WT 的星形胶质细胞和神经元中的 DRP1 水平较高,C3-/-长期暴露于百草枯后的小鼠;这通过在 PQ 处理表达多巴胺转运蛋白的神经元(有和没有 OCT3 抑制)和 WT 的原代星形胶质细胞中发现更高水平的 DRP1 得到支持C3-/-老鼠。选择性星形胶质细胞 DRP1 敲低改善了质量问题2+-诱导神经毒性C3-/-小鼠,但不是 WT 小鼠。具有 siRNA 介导的 DRP1 敲低的 GL261 星形胶质细胞具有更高的丙氨酸-丝氨酸-半胱氨酸转运蛋白 2 (ASCT2) 表达,并且转运研究表明,当 OCT3 不存在时,细胞外 PQ 通过 ASCT2 转运到星形胶质细胞中。

讨论:

目前的研究主要集中在多巴胺能神经元和星形胶质细胞之间 PQ 的转运机制。在老年或长期 PQ 治疗的小鼠中发现了较低的 OCT3 水平。具有 DRP1 抑制作用(通过病毒工具或线粒体分裂抑制剂-1)的星形胶质细胞具有更高水平的 ASCT2,我们假设当 OCT3 缺乏时,ASCT2 可作为去除细胞外 PQ 的替代转运蛋白。总之,我们的数据表明位于星形胶质细胞上的 OCT3、ASCT2 和位于 DA 末端的多巴胺转运蛋白可能以协同方式在 PQ 诱导的神经毒性中介导纹状体 DA 末端损伤。https://doi.org/10.1289/EHP9505

更新日期:2022-05-06
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