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. 2B) right striatum. After 2 h of equilibration, dialysates were collected every C 2+ or DAT (Fig. 2 ) as determined by HPLC. That Oct3 accumulated 30 min for 1 h before PQ injection (15 mg/kg, i.p.) for baseline meas- more PQ+ than did DAT could relate to transporter expression urements (pooled for 0 time point) and for an additional 3 h after the level, the affinity and maximal transport rates of the transporters, injection, followed by HPLC analyses for DA. n = 7 animals per group. Area under the curve was generated using GraphPad Prism followed by and the different driving forces. To rule out the possibility that the atwo-tailedt test. *P < 0.05 compared with the Oct3+/+ group. (E: observed increased uptake in the presence of SDT was due to redox transport study) Uptake of tritiated MPP+ was assessed in EM4 cells action of this agent on the transporters, we performed a transport + (modified human embryonic kidney cells; SI Materials and Methods)with study using stable DAT cells and MPP as a substrate because this stable expression of DAT or empty vector control. Uptake reaction me- molecule is not affected by SDT (Fig. S2). Intracellular levels of diated by DAT was assessed in cells preincubated with 500 μMPQ2+ up to MPP+, as measured by HPLC, were comparable between the 2+ 60 min and compared with the control group (“C”) without PQ pre- groups of cells treated with or without SDT, indicating that this – incubation. n =3 5 independent experiments in quadruplicate. reducing agent did not affect the function of DAT itself. Together, our results strongly support that PQ2+,onceconvertedtoPQ+,is Oct3−/− capable of entering cells through DAT and Oct3. comparable loss of DA neurons was also observed in + mice treated with PQ2+, we also observed a significant reduction To determine whether higher uptake of PQ in cells would (∼40%) in immunoreactivity of striatal TH (the rate limiting lead to more oxidative stress and cytotoxicity, we assessed the −/− +/+ levels of reactive oxygen species (ROS) and cell viability. Cells enzyme for DA production) in Oct3 but not in Oct3 mice were treated with PQ2+ for 20 min with or without SDT as per- (Fig. 1B). HPLC analysis confirmed a corresponding reduction in formed for Fig. 2 A–C. This short treatment of SDT with PQ2+ total striatal DA content (Fig. 1C). This lack of DA reduction in dramatically increased intracellular levels of ROS in stable cells wild-type mice despite a loss of DA neurons is proposed to be with Oct3 or DAT expression, but not in those with empty vector related to the compensatory up-regulation of TH activity in the D–F 2+ (Fig. 2 ). As expected, the control treatment of H2O2,which striatum after PQ injection (9). On the basis of our previously does not need a transporter to enter cells, increased ROS pro- proposed function of Oct3 in mediating the neurotoxicity of duction in all cell types. The increase in ROS levels induced by methamphetamine (12), it is possible that the striatal neurotox- + G–I Oct3−/− PQ subsequently led to higher cytotoxicity (Fig. 2 ). These icity observed in the mice was due to a reduced PQ results indicate that PQ+, but not PQ2+, enters cells through both buffering capacity in non-DA cells, and, hence, more PQ was Oct3 and DAT to induce oxidative stress and cell death. available for DAT-mediated transport into DA terminals. To lend further support for this argument, we performed in vivo DA Enhances Cytotoxicity Induced by PQ+. PQ+ induced the same microdialysis to compare the functional effects of PQ2+ treatment −/− +/+ extent of toxicity in EM4 cells regardless of whether it entered cells in freely moving Oct3 and Oct3 mice. Using DA overflow as through Oct3 or DAT (Fig. 2). These results raise the question of 2+ a functional response of DA neurons to PQ (15, 16), we why in animal models DA neurons are more vulnerable to PQ detected an increase in extracellular DA ∼120 min after a single −/− +/+ toxicity (7). DA itself has been suggested to contribute to the en- PQ2+ injection (i.p.) in Oct3 mice, but not in their Oct3 hanced cytotoxicity of PQ in DA neurons as DA is known to littermates (Fig. 1D). These results suggest that PQ2+ exerted generate reactive species through processes such as auto-oxida- −/− a more dramatic effect on striatal DA terminals in Oct3 mice. tion, enzymatic metabolism, or interactions with the products To test whether this enhanced DA overflow was the result of (such as superoxide) generated from the redox cycling of PQ2+/ an interaction between PQ2+ and DAT, we used stable cells PQ+ (18). To test this hypothesis, ROS and cell viability was expressing DAT to assess the ability of PQ2+ to compete with measured with or without the addition of 50 μM DA. In the

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1115141108 Rappold et al. Downloaded by guest on September 27, 2021 Fig. 2. PQ+ is transported by both Oct3 and DAT to induce cytotoxicity. Stable EM4 cells expressing empty vector control (A), Oct3 (B), or DAT (C) were cultured in 24-well plates for 24 h. Cells were then washed and incubated with varying concentrations of PQ2+ with or without 0.5 mM sodium dithionite (SDT) in degassed Krebs Ringer Hepes (KRH) buffer to convert PQ2+ to PQ+. After 20 min, cells were washed and then collected for HPLC analysis. To determine the effects of PQ+ on the formation of reactive oxygen species (ROS) (D–F), these cell types were treated for 24 h with the indicated concentrations of PQ2+ with or without 0.5 mM SDT. After 20 min, cells were washed and grown in regular medium. After 24 h, cells were incubated with 5 μM dihydroethidium and analyzed for ROS levels using ﬂow cytometry (AFU: arbitrary ﬂuorescence unit).

H2O2 (30 μM) was incubated with cells for 20 min as a positive control for ROS production. To assess whether the observed ROS production would lead to cytotoxicity, cell viability was performed (G–I). Cells were treated with PQ2+ and SDT as shown in D–F, washed, and grown in cell culture medium for another 48 h before a 3-(4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. n = 4 independent experiments in quadruplicate, analyzed by two-way ANOVA followed by the Newman–Keuls post hoc test. *P < 0.05 compared with

the respective control groups without SDT. NEUROSCIENCE

presence of this nontoxic concentration of DA, PQ+ induced EM4 cells expressing Oct3 or DAT. After 24 h of treatment with a dramatic increase in ROS (Fig. 3A) and cell death (Fig. 3C)in PQ2+, EM4 cells were collected for the measurement of PQ DAT cells, but not in EV cells (Fig. 3B). This enhanced ROS uptake using HPLC. As demonstrated in Fig. 4 A–C, higher production and cell death was blocked by the DAT inhibitor uptake was detected in Oct3- and DAT-expressing cells when GBR12909 and was absent when DA was replaced by the DAT- cocultured with microglia, suggesting that microglia are capable substrate tyramine, which has a similar structure to DA (but only of converting PQ2+ to PQ+, which is then transported into cells one hydroxyl substituent on the phenyl ring). The metabolism of through these transporters. The patterns of uptake in these cells tyramine requires monoamine oxidase; however, EM4 cells con- are similar to those in Fig. 2, which shows the conversion of PQ2+ tain only catechol-O-methyltransferase, and thus, only DA can be to PQ+ by SDT. This conversion was mediated at least partly metabolized to reactive species in these cells. Consistent with the by NADPH oxidase on microglia because inhibiting the activity fi low affinity of Oct3 for DA (Km = 1.5 mM) (19), we observed that of this enzyme by apocynin signi cantly attenuated the uptake of addition of 50 μM DA did not further increase ROS (Fig. 3A)in PQ+ through Oct3 and DAT (Fig. 4 D–F). The reduction of Oct3-expressing cells. Together, these results support the signifi- intracellular PQ levels resulted in significant attenuation of cell cant role that DA contributes to selective cell death in PQ toxicity. death (Fig. 4 G–I). Because NOS is also capable of reducing PQ2+ and blocking this enzyme in mouse microglia decreases ROS Human Microglia Are Capable of Converting PQ2+ to PQ+ Through production (18), we assessed the role of NOS in converting PQ2+ Their Intrinsic NADPH Oxidase Activity. To demonstrate that the to PQ+ in our cell culture paradigm. Our results indicate that, conversion of PQ2+ to PQ+ can take place in the brain and is after treating human microglia with the same concentration of relevant to humans, we cocultured human microglia with stable N-nitro-L-arginine methyl ester (L-NAME) (a NOS inhibitor) as

Fig. 3. DA enhances ROS production and cytotoxicity induced by PQ+. EM4 cells with empty vector, Oct3, or DAT expression were treated with 200 μMPQ2+ plus 0.5 mM SDT for 20 min. Then the cells were washed and incubated in culture medium with or without 50 μM DA, 50

μM tyramine, or 1 μM GBR12909 (a DAT inhibitor). H2O2 (30 μM for 1 h) was used as a positive control for ROS production. After 48 h, cells were assessed for ROS levels using ﬂow cytometry (A). In a parallel set of experiments, cells with empty vector (B) and DAT (C) treated with varying concentrations of PQ2+ as described in A were assessed for cell viability using a MTT assay. n = 4 independent experiments in quadruplicate, analyzed by one-way ANOVA followed by the Newman–Keuls post hoc test. aP < 0.05 compared with respective EV; bP < 0.05 compared with the respective Oct3 groups; *P < 0.05.

Rappold et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 previously described (18), we did not see a decrease in PQ+ mutant mice, which did not require cross-fostering at birth like −/− uptake into cocultured Oct3 or DAT stable cells. These results DAT mice (25). When injected with PQ2+, mice homozygous suggest that human microglia are not capable of taking up PQ to for hypomorphic DAT exhibited no loss in nigral DA neurons interact with intracellular NOS. Alternatively, our results are (Fig. 5D) in contrast to their wild-type littermates. Because −/− consistent with previous studies reporting that human microglia DAT mice have been reported to display modest reductions of either have no or very low NOS activity (20–22), in contrast to TH in the midbrain (26) and because our in vitro data suggest rodent microglia (23, 24). Regardless of whether human micro- that cytosolic DA can modulate PQ toxicity, we assessed whether glia are capable of performing this conversion via NOS, our re- such alterations were present in this hypomorphic DAT model. sults support that, under in vivo conditions, it is likely that PQ2+ HPLC analyses of the midbrain demonstrate that DA levels (Fig. can be converted to PQ+ at least by NADPH oxidase before 5E) were comparable between genotypes, thereby eliminating being transported into DA neurons to induce neurotoxicity. the possibility that lower DA levels in the cell bodies may account for the resistance to PQ toxicity. We did observe signiﬁ- Mutant Mice with Hypomorphic DAT Are Resistant to PQ 2+ + cantly decreased striatal DA content in DAT hypomorphic mice Neurotoxicity. If PQ could be converted to PQ in vivo and (Fig. S4B), but, due to the lack of PQ toxicity observed in the then taken up by DA neurons through DAT to induce toxicity, striatum of wild-type and mutant mice (Fig. S4), the signiﬁcance then reducing DAT function should reduce neurotoxicity. of the altered DA content in this region relating to cytotoxicity Therefore, we used a genetically engineered mouse model (Fig. cannot be determined. In summary, the lack of PQ-induced A– S3) with an almost complete loss of DAT expression (Fig. 5 neurodegeneration in DAT hypomorphic mice strengthens the C < A B ). The small amount of residual DAT ( 5%) (Fig. 5 and ) role of this transporter in mediating PQ neurotoxicity. allows for normal survival rates and normal lactation in these Discussion After the discovery of MPTP as the cause of acute parkinsonism in a group of drug abusers (5) and the subsequent characteriza-

Fig. 4. Microglia promote the conversion of PQ2+ to PQ+ through NADPH oxidase. Stable EM4 cells expressing Oct3, DAT, or empty vector control were Fig. 5. Mutant DAT hypomorphic mice are resistant to PQ neurotoxicity. (A) grown on glass coverslips for 24 h. These coverslips were then transferred to Coronal striatal sections from wild type (Wt), heterozygous (Het), and ho- six-well plates on which a monolayer of human microglial immortalized cells mozygous (Hom) mutant DAT mice were immunostained with a DAT anti- (CHME5) had been plated for 24 h. Different concentrations of PQ2+ were body (brown, diaminobenzidine chromogen) and counterstained with Nissl added to these cocultures. After 24 h of PQ2+ addition, the uptake of PQ+ to reveal the structure of other brain regions. (B) For a more quantitative into EM4 cells was evaluated by removing the coverslips from the cocultures comparison, Western blotting was performed. Lanes 1–3 (Wt), lanes 4–6 and collecting cells for HPLC measurement (A–C). To determine whether (Het), and lanes 7–8 (Hom) are from eight animals. (C) Immunofluorescence increased uptake of PQ+ was mediated by NADPH oxidase or nitric oxide of TH and DAT in coronal midbrain sections containing the substantia nigra synthase (NOS), apocynin and L-NAME, respectively, were added to the and ventral tegmental area. [Scale bars: 400 μm(a′–c′, g′–i′), 100 μm(d′–f′, j′– cocultures (D–F). Inhibition of NADPH oxidase by apocynin, but not NOS by l′).] No apparent differences in morphology of midbrain DA neurons was + L-NAME, significantly reduced PQ transport into Oct3 or DAT cells. n =4 noted between mutant and wild-type mice. Stereological cell counting independent experiments, analyzed by one-way ANOVA followed by the confirmed comparable population of nigral DA neurons (D) and midbrain Newman–Keuls post hoc test. *P < 0.05 compared with the PQ2+ alone DA levels (E) in these mutant mice. (D) To assess the effects of PQ toxicity in group; #P < 0.05 compared with PQ2+ plus microglia group as well as PQ2+ these animals, DAT wild-type mice and their homozygous mutant littermates + 2+ plus microglia plus the L-NAME group. The reduced uptake of PQ in the (∼10 wk old) were injected with PQ (10 mg/kg, i.p., every second day for presence of apocynin resulted in less cytotoxicity (G–I) as assessed by using a total of 10 injections) or saline. Seven days after the last injection, mice an MTT assay 48 h later. n = 4 independent experiments, analyzed by one- were processed for stereological cell counting. n =4–5 animals per group, way ANOVA followed by the Newman–Keuls post hoc test. *P < 0.05 com- analyzed by two-way ANOVA followed by the Newman–Keuls post hoc test. pared with all other groups. *P < 0.05 compared with the control saline-treated group.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1115141108 Rappold et al. Downloaded by guest on September 27, 2021 tion of MPP+ as the active metabolite of MPTP (27), a search for cell vulnerability of nigral DA neurons (37). Although the com- environmental contaminants with a similar structure to MPP+ plex mechanisms of selective cell death in genetic or toxicant- was initiated. The widely used herbicide PQ2+ was identified as induced models of PD will likely remain a topic of research for such an agent and was suggested to be a potential environmental years to come, our demonstration of a mechanism through which parkinsonian toxicant (6). Supported by epidemiological studies, PQ2+ enters DA cells is a critical step in elucidating the path- especially in recent years (2, 3), there has been an interest in ogenic mechanism of this toxicant and perhaps other toxic redox- further investigating the mechanism by which this molecule in- cycling molecules as well. duces dopaminergic neurodegeneration. Our results also demonstrate that PQ+ is a substrate for Oct3. In the present study, we demonstrated that, when PQ2+ was The significance of Oct3 in PQ2+ toxicity is illustrated by the ob- − − reduced to the monovalent cation PQ+, it was efficiently taken servation that, in Oct3 / mice, PQ2+ injection induced striatal up by cells through DAT and Oct3. This conversion took place in damage in DA terminals. As discussed, this is an atypical toxic the presence of either a reducing agent or NADPH oxidase on profile in the PQ2+ mouse model of PD. That PQ+ is a substrate for microglia. The increase in intracellular content of PQ resulted in both Oct3 and DAT suggests opportunities for crosstalk between higher ROS production and cytotoxicity. When PQ+ was com- glia, non-DA neurons, and DA neurons. As proposed in Fig. S6, bined with a nontoxic concentration of DA, significant increases when PQ2+ reaches the striatum after systemic injection, it can be in ROS levels and cell death were detected, suggesting that DA convertedtoPQ+ by microglia and subsequently taken up into DA itself may contribute to the vulnerability of DA neurons to PQ neurons by DAT and into astrocytes as well as medium spiny toxicity. Either blocking the uptake of PQ+ through DAT or neurons by Oct3. Therefore, the initial direct target of PQ is not blocking the conversion of PQ2+ mediated by NADPH oxidase only DA neurons. Because Oct3 is bidirectional, the neighboring resulted in lower intracellular content of PQ and its subsequent non-DA cells may act as a “sink” to store the excess of PQ right after ROS production and cell death. In aggregate, these results injection and then act as a “source” to slowly release this cation (in demonstrate that, although the initial divalent form of PQ2+ is the monovalent form) to neighboring DA neurons. Therefore, this not toxic to cells, its monovalent metabolite can enter DA neu- slow release could induce a constant chronic state of oxidative stress rons through DAT to induce oxidative damage. This scenario is to DA neurons. Consistent with this theory, for cell death to occur, analogous to the fact that, although MPTP itself is not toxic, its animals have to be treated with PQ2+ for at least 3–4wk. metabolite MPP+ is the culprit in DA cell death (28). Although our results further support the role of Oct3 in me- The toxic effect of PQ2+ mediated by microglia through re- diating neurotoxicity, it highlights the intriguing question of why

dox cycling of NADPH oxidase has been reported (18, 29). striatal DA terminals are usually so resistant to PQ toxicity. It NEUROSCIENCE Although the expression of this enzyme has been well docu- has been noted that different toxicants seem to affect the mented in microglia, it is also present in astrocytes (30) and DA nigrostriatal structure differentially (9). In the MPTP model, for neurons (31). In mice deficient in gp91phox (a functional subunit example, both striatal terminals and nigral cell bodies are af- required by NADPH oxidase), PQ2+ failed to induced neuro- fected. In methamphetamine toxicity, only the terminals are toxicity (29, 32). NADPH oxidase is composed of three cyto- damaged (unless a very high dose is used). Other neurotoxic solic and two membrane-bound subunits. When activated, the models such as 6-hydroxydopamine and rotenone also induce cytosolic subunits translocate and bind to the membrane-bound more toxicity in the striatum. In PQ toxicity, however, only the subunits (p22 and gp91) and produce extracellular superoxide. cell body is affected, not the striatal terminals. Addressing the Thus, this enzyme can induce redox cycling of PQ2+ extracel- issue of regional vulnerability is beyond the scope of this study; −/− lularly. Therefore, the mechanism of PQ2+ toxicity mediated by however, Oct3 mice may provide a useful model to study PQ NADPH oxidase has been suggested to be mediated by extra- toxicity when damage to both DA terminals and cell bodies is cellular superoxide (29, 32). Although it is likely that this oxi- desired. It is worthwhile to note that Oct3 immunoreactivity has dative stress pathway is involved in PQ2+ toxicity, it has been been reported to be higher in the striatum than in the niga (13). a topic of debate how PQ2+ can enter DA cells to induce oxi- A lower level of Oct3 (and hence, less buffering capacity) in dative stress intracellularly. On the basis of our data, we pro- combination with a high microglia population in the nigra might pose that PQ+ is generated from the extracellular redox cycling contribute to a higher sensitivity of the DA cell body compared and then taken up into DA neurons where it establishes a new with DA terminals when treated with PQ. This expression pat- round of redox cycling and, together with DA, induces neuro- tern is consistent with our finding that there is no damage to degeneration. Consistent with our theory that PQ2+ is a pro- striatal terminals in wild-type mice, but significant damage in −/− toxicant that must first be converted to an active metabolite, Oct3 mice when treated with PQ. when we compared DA overflow induced by PQ2+ and MPP+ In a case-control study, Ritz et al. (2) report that individuals by separately infusing equimolar amounts of these molecules with certain allelic variants belonging to clade A of the DAT gene into the striatum using a microdialysis cannula (Fig. S5), a peak are more likely to develop PD when they also have “high” ex- of DA was detected about 2 h after PQ2+ injection. This peak posure to PQ and maneb, but not in the absence of this gene/ was much delayed and less pronounced than the one produced pesticide combination, suggesting a gene–environment interac- by MPP+. tion. This study also reports that a clade A diplotype is more Although our results outline one mechanism by which PQ2+ strongly associated with the development of PD than clade B. A induces selective cell death in DA neurons, they do not address gene-dose effect is also observed. Combined with the in vitro why the group of DA neurons in the ventral tegmental area observation that clade A haplotypes have higher transcriptional (VTA) is insensitive to PQ2+ toxicity (14, 33). Di Monte and activity than clade B (38), these two studies are consistent with colleagues (33) previously reported that DA neurons expressing our observation that PQ toxicity requires DAT function. How- 2+ calbindin-D28K were resistant to neurotoxicity induced by PQ ever, Ritz et al. (2) speculated that clade A variants with increased and that the number of calbindin-D28K–containing DA neurons risk of PD in their study would result in a lower DAT expression in VTA was five times higher than in their counterparts in the (and, hence, in overall reduced function) on the basis in part of nigra. Calbindin is a Ca2+-buffering protein that has been shown the following two studies. First, clade B variants increase the level to be correlated with cell viability in PD (34, 35). Consistent with of DAT expression as measured by PET imaging and binding these observations, recent studies demonstrated that the selec- studies in humans (39). However, the impact of these genetic tive expression of L-type Ca2+ channels is responsible for higher variations on levels of functional surface-expressed DAT in cytosolic Ca2+ levels in nigral DA neurons (36). When combined neurons is unknown. Second, on the basis of a cell culture study, with synuclein and DA, these factors account for the selective PQ2+ is not a substrate for DAT (11). Although our findings are

Rappold et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 fully in agreement with this latter study, we have shown that PQ+ Materials and Methods + is a substrate for DAT, that PQ elevates reactive oxygen spe- See SI Materials and Methods for description of animal models, paraquat cies, and that lowering DAT expression eliminates the neuro- treatment, stereological cell counts, in vivo microdialysis, HPLC measure- toxicity of PQ. The mechanism that we have discovered makes ments of striatal DA and its metabolites, cell cultures, transport assays, and it more likely that disease-associated DAT variants have an oxidative stress assessment. + All statistical values are expressed as mean ± SEM. Differences between overall DAT-enhanced function through which more PQ is ac- means were analyzed using either one-way or two-way ANOVA followed by cumulated. Additional studies are required to directly test this Newman–Keuls post hoc testing for pairwise comparison using SigmaStat v hypothesis. 3.5. For in vivo microdialysis data, areas under the curve were generated In summary, the present study describes a mechanism by which using GraphPad Prism v 5.01 followed by a two-tailed t test. The null hy- PQ enters DA cells to induce neurotoxicity. This information is pothesis was rejected when P value was < 0.05. critical to the paradoxical observations of a higher risk of developing PD in individuals with genetic variants in DAT and in ACKNOWLEDGMENTS. We thank Dr. Baek Kim (University of Rochester) for 2+ 2+ kindly sharing the human microglia CHME5 cells. We are also thankful to individuals exposed to PQ (2), despite the lack of PQ uptake Dr. Christoph Kellendonk (Columbia University) for advice on characterizing by DAT (11). By extension, other toxic molecules with the redox- the DAT hypomorphic mice and comments on the manuscript. This work was cycling property might also use this mechanism to induce neuro- supported in part by National Institutes of Health Grants ES014899 and ES17470 (to K.T.); Grants DA022413 and DA12408 (to J.A.J.); Grant toxicity. Combined with our previous study (12) demonstrating AG040903 (to P.M.R.); Grant ES020081 (to A.S.C.); and by training Grant that both Oct3 and DAT functionally coordinate to modulate TL1RR 024135 from the National Center for Research Resources (to P.M.R.). neurotoxicity of MPTP and methamphetamine (Fig. S6), our P.M.R. and A.S.C. are trainees in the Medical Scientist Training Program funded by National Institutes of Health Grant T32 GM07356. J.C.G. is the present study has a broad mechanistic implication for neurotox- recipient of National Institutes of Health/National Institute on Environmen- icity in the nigrostriatal pathway. tal Health Sciences Undergraduate Award ES0017470-01S1.

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