Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study.
Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study.
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DOI：
10.1289/ehp9505
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发表时间：
2022-05
影响因子：
10.4
通讯作者：
中科院分区：
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百草枯（PQ）是一种杀虫剂，暴露于此与帕金森病的风险增加有关;然而，PQ在大脑中的运输机制仍不清楚。我们的前期研究表明，星形胶质细胞上表达的有机阳离子转运蛋白3（OCT 3）可以摄取PQ，保护多巴胺能（DA）神经元免受细胞外高水平PQ的损伤。目前，尚不清楚OCT 3水平在慢性PQ暴露或老化期间如何改变，也不清楚OCT 3缺乏如何触发代偿机制。先前报道动态相关蛋白1（DRP 1）可改善帕金森病期间的神经元损失。近年来，越来越多的研究揭示了星形胶质细胞DRP 1的功能，促使我们推测DRP 1可以调节星形胶质细胞的PQ转运能力。本研究旨在进一步探讨PQ在黑质纹状体系统的转运机制，并确定参与细胞外PQ清除的途径。采用腹腔注射（i. p.）在野生型（WT）和有机阳离子转运蛋白-3-缺陷型（）小鼠中注射PQ。DRP 1基因敲减是通过体内病毒工具和体外小干扰RNA（siRNA）实现的。采用体内微透析法检测细胞外PQ。采用体外转运实验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。采用Western印迹分析或免疫荧光法检测蛋白质在体内外的表达水平和定位。老年小鼠和长期暴露于PQ的小鼠脑OCT 3表达较低，暴露于腹腔负荷剂量后，细胞外PQ浓度较高。长期暴露于PQ后，WT和小鼠的星形胶质细胞和神经元中的DRP 1水平较高;这得到了PQ处理多巴胺转运蛋白表达神经元（有和无OCT 3抑制）后以及WT和小鼠的原代星形胶质细胞中DRP 1水平较高的支持。选择性星形胶质细胞DRP 1敲除可改善小鼠的神经毒性，但在WT小鼠中则不然。siRNA介导的DRP 1敲低的GL 261星形胶质细胞具有较高的丙氨酸-丝氨酸-半胱氨酸转运蛋白2（ASCT 2）表达，并且转运研究表明，当OCT 3不存在时，细胞外PQ通过ASCT 2转运到星形胶质细胞中。本研究主要探讨PQ在多巴胺能神经元和星形胶质细胞之间的转运机制。较低的OCT 3水平被发现在老年人或长期PQ治疗的小鼠。具有DRP 1抑制（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ASCT 2，我们假设当OCT 3缺乏时，ASCT 2作为替代转运蛋白去除细胞外PQ。总之，我们的数据表明，OCT 3，ASCT 2位于星形胶质细胞和多巴胺转运蛋白位于DA终端可能以协调的方式介导的纹状体DA末端损伤PQ诱导的神经毒性。https://doi.org/10.1289/EHP9505
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. The present study aimed to further explore PQ transport mechanisms in the nigrostriatal system and identify pathways involved in extracellular PQ clearance. Models of PQ-induced neurodegeneration were established by intraperitoneal (i.p.) injection of PQ in wild-type (WT) and organic cation transporter-3–deficient () 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. Older mice and those chronically exposed to PQ had a lower expression of brain OCT3 and, following exposure to a i.p. loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and 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 mice. Selective astrocyte DRP1 knockdown ameliorated the neurotoxicity in 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. 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
影响因子：
3.7
作者：
Yu Q;Wang T;Zhou X;Wu J;Chen X;Liu Y;Wu D;Zhai Q
通讯作者：
Zhai Q