Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

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 ($Oct{3}^{-/-}$) 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 $10\text{-mg}/\mathrm{kg}$ i.p. ${\text{PQ}}^{2+}$ loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and $Oct{3}^{-/-}$ 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 $Oct{3}^{-/-}$ mice. Selective astrocyte DRP1 knockdown ameliorated the ${\text{PQ}}^{2+}\text{-induced}$ neurotoxicity in $Oct{3}^{-/-}$ 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.