Paraquat Neurotoxicity Is Mediated by the Dopamine Transporter and Organic Cation Transporter-3

Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3 Phillip M. Rappolda,1, Mei Cuia,b,1, Adrianne S. Chessera, Jacqueline Tibbetta, Jonathan C. Grimaa, Lihua Duanc, Namita Senc, Jonathan A. Javitchc, and Kim Tieua,2 aDepartment of Neurology in the Center for Translational Neuromedicine, University of Rochester, Rochester, NY 14642; cCenter for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University, New York, NY 10032; and bDepartment of Neurology, Huashan Hospital, Fudan University, Shanghai 200433, China Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 8, 2011 (received for review September 14, 2011) The herbicide paraquat (PQ) has increasingly been reported in addition, PQ2+ induces α-synuclein up-regulation and aggregation epidemiological studies to enhance the risk of developing Parkin- (10), a neuropathological feature detected in PD patients. In a son’s disease (PD). Furthermore, case-control studies report that recent case-control study (2), PQ2+ was reported to increase the individuals with genetic variants in the dopamine transporter risk of PD in subjects with certain genetic variants in the dopamine (DAT, SLC6A) have a higher PD risk when exposed to PQ. However, transporter (DAT). Together, these studies support the neurotoxic it remains a topic of debate whether PQ can enter dopamine (DA) role of PQ2+ in the nigrostriatal system and highlight the need neurons through DAT. We report here a mechanism by which PQ is to understand the mechanism by which PQ2+ induces toxicity. transported by DAT: In its native divalent cation state, PQ2+ is not However, to date, the very fundamental questions of whether and, a substrate for DAT; however, when converted to the monovalent if so, how PQ2+ enters DA neurons remain unanswered (11). cation PQ+ by either a reducing agent or NADPH oxidase on micro- In the present study, we describe a mechanism by which PQ2+ glia, it becomes a substrate for DAT and is accumulated in DA enters DA neurons. We propose that, in the brain, PQ2+ is re- neurons, where it induces oxidative stress and cytotoxicity. Im- duced to PQ+ extracellularly by enzymes such as NADPH-oxi- paired DAT function in cultured cells and mutant mice significantly dase on microglia. In contrast to its parent compound, PQ+ is + + attenuated neurotoxicity induced by PQ . In addition to DAT, PQ a DAT substrate and is therefore accumulated in DA neurons NEUROSCIENCE is also a substrate for the organic cation transporter 3 (Oct3, where it establishes a new redox cycle intracellularly, leading to Slc22a3), which is abundantly expressed in non-DA cells in the the generation of superoxide and DA reactive species and, ulti- nigrostriatal regions. In mice with Oct3 deficiency, enhanced stria- mately, neurotoxicity. Blocking DAT function abolished PQ+ tal damage was detected after PQ treatment. This increased sen- neurotoxicity in both cells and living mice. In addition to DAT, sitivity likely results from reduced buffering capacity by non-DA PQ+ is also a substrate for the organic cation transporter-3 + cells, leading to more PQ being available for uptake by DA neu- (Oct3), a bidirectional transporter that is highly expressed in rons. This study provides a mechanism by which DAT and Oct3 astrocytes and GABAergic neurons in the nigrostriatal regions 2+ + modulate nigrostriatal damage induced by PQ /PQ redox cycling. (12, 13). Together, these two transporters function in a con- certed manner to mediate nigrostriatal damage. Collectively, our neurodegeneration | extraneuronal monoamine transporter | astrocytes | data point to an interplay between DA and non-DA cells me- in vivo microdialysis diated, respectively, by DAT and Oct3, which modulate the function and viability of the nigrostriatal pathway. arkinson’s disease (PD) is characterized primarily by the loss Pof dopamine (DA) neurons in the substantia nigra pars Results compacta (1). Although in past decades discoveries of genetic PQ2+ Induces Striatal Neurotoxicity and DA Overflow in Mice with mutations linked to PD have significantly impacted our current Oct3 Deficiency. We recently reported that Oct3 can modulate understanding of the pathogenesis of this devastating disorder, it toxicity in the dopaminergic system through its bidirectional is likely that the environment plays a critical role in the etiology transport capability of various toxic cations (12). In the present study, we asked whether this mechanism was also relevant to PQ2+. of sporadic PD. Human epidemiological studies indicate that −/− +/+ exposure to herbicides, pesticides, and heavy metals increase the To this end, we injected Oct3-null (Oct3 )miceandtheirOct3 2+ risk of PD. One such environmental toxicant is paraquat (PQ2+, wild-type littermates intraperitoneally (i.p) with PQ . Consis- 2+ N,N′-dimethyl-4–4′-bipiridinium) (2, 3). This molecule exists tent with the neurotoxic features of PQ on the nigrostriatal ∼ natively as a divalent cation, but can undergo redox cycling with system (7, 14), we detected a loss of nigral DA neurons ( 22%) A fi fi cellular diaphorases such as NADPH oxidase and nitric oxide (Fig. 1 ). Quanti cation of total Nissl-positive neurons con rmed synthase (4) (NOS) to yield the monovalent cation PQ+. From that this reduction was due to cell loss, and not to down-regula- this redox cycle, superoxide is generated, leading to oxidative tion of the phenotypic marker tyrosine hydroxylase (TH) (saline control group: 15,438 ± 532; PQ2+ treated group: 12,364 ± 522, stress-related cytotoxicity. (For clarity and brevity, the abbrevi- n ± ations PQ2+ and PQ+ will be used to signify the respective cat- = 5 mice/group; data represent mean SEM). Although ions, whereas PQ represents a general term when the valency is ambiguous.) On the basis of its structural similarity to 1-methyl-4- + Author contributions: P.M.R., M.C., and K.T. designed research; P.M.R., M.C., A.S.C., J.T., phenylpyridinium (MPP ), an active metabolite of the parkinso- and J.C.G. performed research; L.D., N.S., J.A.J., and K.T. contributed new reagents/ana- nian agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lytic tools; P.M.R., M.C., A.S.C., and K.T. analyzed data; and P.M.R., M.C., A.S.C., J.A.J., and (5), PQ2+ has been predicted to be a potential environmental K.T. wrote the paper. parkinsonian toxicant (6), and with subsequent recent epidemio- The authors declare no conflict of interest. logical studies (2, 3), there has been increasing interest in this This article is a PNAS Direct Submission. herbicide as a potential pathogenic agent in PD. 1P.M.R. and M.C. contributed equally to this study. 2+ When PQ is injected into mice, it induces a loss of nigral DA 2To whom correspondence should be addressed. E-mail: [email protected]. neurons, but the striatum is spared (7, 8), most likely due to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. compensatory striatal sprouting in the remaining neurons (9). In 1073/pnas.1115141108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115141108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 transport of the DAT substrate MPP+. Preincubation of cells with PQ2+ for up to 1 h did not affect the uptake of MPP+ mediated by DAT (Fig. 1E). Additionally, we found no difference in the −/− +/+ kinetics of PQ2+ reaching the striatum in Oct3 and Oct3 mice using in vivo microdialysis and HPLC to quantify PQ levels in mice injected with PQ2+ (Fig. S1). Thus, although the microdialysis data suggest that, in the absence of Oct3 function, PQ somehow leads to striatal DA release and subsequent neurotoxicity, the mechanism for these effects is unclear, given that PQ2+ does not appear to interact functionally with DAT on the basis of its inability to inhibit MPP+ transport by DAT (Fig. 1E). PQ2+ Is a Poor Substrate for Both Oct3 and DAT, Unless Converted to PQ+. To further investigate directly whether PQ2+ is a substrate for Oct3 and DAT, we assessed uptake of PQ2+ in stable EM4 cells (modified HEK293) expressing Oct3, DAT, or an empty vector control (12). Despite its similar structure to MPP+,which is an excellent substrate for these transporters, we found that PQ2+ was not taken up into cells through these transporters, as shown in the dose–response studies (Fig. 2 A–C). This is consistent with Fig. 1. PQ2+ injection increases striatal neurotoxicity and DA overflow in the inability of PQ2+ to interfere with MPP+ uptake (Fig. 1E)and −/− mice with Oct3 deficiency. (A–C: neurotoxicity study) Oct3 mice and a previous study reporting that PQ2+ was not transported by DAT +/+ – 2+ Oct3 littermates (10 12 wk old) were injected with PQ (10 mg/kg, (11). We hypothesized that the two positive charges on PQ2+ may i.p., every second day for a total of 10 injections) or saline. Seven days interfere with its ability to be transported by Oct3 and DAT, as after the last injection, mice were processed for stereological cell counting (A), striatal tyrosine hydroxylase immunoreactivity (B, optical both transporters favor monovalent cations as substrates. To test density), or HPLC measurement of DA content (C). n = 5 animals per this hypothesis, we performed transport studies in the presence of group for A and B and n =5–9 per group for C. *P < 0.05, analyzed by sodium dithionite (SDT). This reducing agent was used to donate 2+ + two-way ANOVA followed by the Newman–Keuls post hoc test. (D: an electron and hence convert PQ to PQ as previously described − − in vivo microdialysis study) Oct3 / and Oct3+/+ littermates (10–12 wk (17). After PQ2+ wasconvertedtoPQ+, the intracellular content of old) were stereotactically implanted with microdialysis probes into the PQ was dramatically higher in stable cells expressing Oct3 (Fig.

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