Nitration of microtubules blocks axonal mitochondrial

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Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson’s disease Morgan G. Stykel,* Kayla Humphries,* Mathew P. Kirby,* Chris Czaniecki,* Tinya Wang,* Tammy Ryan,* Vladimir Bamm,* and Scott D. Ryan*,†,1

*Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada; and †Neurodegenerative Disease Center, Scintillon Institute, San Diego, California, USA

Neuronal loss in Parkinson’s disease (PD) is associated with aberrant mitochondrial function in dopaminergic (DA) neurons of the substantia nigra pars compacta. An association has been reported between PD onset and exposure to mitochondrial toxins, including the agrochemicals paraquat (PQ), maneb (MB), and rotenone (Rot). Here, with the use of a patient-derived stem cell model of PD, allowing comparison of DA neurons harboring a mutation in the a-synuclein (a-syn) gene (SNCA-A53T) against isogenic, mutation-corrected controls, we describe a novel mechanism whereby NO, generated from SNCA-A53T mutant neurons exposed to Rot or PQ/MB, inhibits anterograde mitochondrial transport through nitration of a-tubulin (a-Tub). Nitration of a-Tub inhibited the association of both a-syn and the mitochondrial motor protein kinesin 5B with the microtubules, arresting anterograde transport. This was, in part, a result of nitration of a-Tub in the C-terminal domain. These effects were rescued by inhibiting NO synthesis with the NOS inhibitor Nv-nitro-L-arginine methyl ester. Collectively, our results are the first to demonstrate a gene by environment interaction in PD, whereby agrochemical exposure selectively triggers a deficit in mitochondrial transport by nitrating the microtubules in neurons harboring the SNCA-A53T mutation.—Stykel, M. G., Humphries, K., Kirby, M. P., Czaniecki, C., Wang, T., Ryan, T., Bamm, V., Ryan, S. D. Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson’s disease. FASEB J. 32, 000–000 (2018). www.fasebj.org

ABSTRACT:

KEY WORDS: neurodegeneration



isogenic hiPSCs



anterograde transport



reactive nitrogen species



tubulin

nitration

ABBREVIATIONS: 3-NT, 3-nitrotyrosine; a-syn, a-synuclein; a/b-Tub,

a/b-tubulin; APO, apochromat; CHIR, CHIR99021; Corr, Corrected; CTT, C-terminal tubulin; CTT-Y, C-terminal amino acids of de novosynthesized a-Tub; DA, dopaminergic; DAF-FM, 4-amino-5-methylamino29,79-difluorofluorescein; DIC, differential interference contrast; EPA, U.S. Environmental Protection Agency; FRET, fluorescent resonance energy transfer; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hN, A9-type dopaminergic neuron; KIF5B, kinesin 5B; L-NAME, Nv-nitro-L-arginine methyl ester; LED, light-emitting diode; LMX1, LIM homeobox transcription factor 1; MANOVA, multivariate ANOVA; MAP2, microtubule-associated protein 2; MB, maneb; MitoDSRed, mitochondrial-targeted DsRed; MPP+, 1-methyl-4-phenylpyridinium; Nurr1, nuclear receptor-related 1 protein; O22, superoxide anion; Oct3/4, octamerbinding transcription factor 3/4; ONOO2, peroxynitrite; PD, Parkinson’s disease; PLA, proximity ligation assay; PQ, paraquat; RNS, reactive nitrogen species; ROS, reactive oxygen species; Rot, rotenone; SNCA, a-synuclein gene; SNpc, substantia nigra pars compacta; Sox2, sex-determining region Y-box 2; SYN1, synaptophysin I; TH, tyrosine hydroxylase; Ty-Tub, tyrosinated a-tubulin; VIS-IR, visible-infrared; WT, wild type

1

Correspondence: Department of Molecular and Cellular Biology, University of Guelph, Science Complex (Room 3456), 50 Stone Rd. East, Guelph, ON N1G 2W1, Canada. E-mail: [email protected]

doi: 10.1096/fj.201700759RR This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

0892-6638/18/0032-0001 © FASEB

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). The loss of neurons in PD is preceded by the accumulation of intracellular proteinaceous aggregates, known as Lewy bodies and Lewy neurites, that contain an array of misfolded protein, the primary constituent of which is a-synuclein (a-syn) (1). Accumulation of oligomeric species of a-syn has been implicated in both sporadic and familial cases of PD, including the A53T (G209A) mutation in the a-syn gene (SNCA-A53T), which is associated with early disease onset (2–4). Whereas SNCA mutations are causal in rare familial forms of PD and dramatically increase the risk of both motor and nonmotor disease symptoms, the prevalence of a-syn aggregates in Lewy neurites found in the brains of idiopathic disease cases (5, 6) emphasizes the need to understand how a-syn accumulation leads to neuropathology. In addition to PD cases arising from familial mutation, there is a growing body of evidence to suggest that agrochemical exposure is linked to PD etiology (7–13). Indeed, 1

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exposure to the agrochemicals paraquat (PQ) and maneb (MB) is associated with a 200% increase in risk (9, 12). In fact, the highest correlation is observed following combined PQ/MB exposure (14). Moreover, in those with causal familial mutations, such as the SNCA-A53T mutation, agrochemical exposure correlates with disease onset at an earlier age (9). Whereas the underlying reason for this association remains unclear, there is strong evidence linking pesticide exposure with mitochondrial stress (15). Indeed, whereas the epidemiologic association between pesticide exposure and PD onset is controversial, there is definitive causality with regard to pesticides and mitochondrial dysfunction (10). Impairments in mitochondrial function result in excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as superoxide anion (O22) and NO. NO is a soluble gas that diffuses throughout the cell and reacts with tyrosine residues on neighboring proteins, thereby altering protein structure and stability through an oxidation process known as nitration (16). In addition, O22 reacts rapidly with NO to yield a highly toxic RNS product, peroxynitrite (ONOO2), which can in turn, promote further protein nitration (16). Emerging evidence suggests that redoxbased modification may be of critical importance to loss of function observed in PD. Indeed, several recent reports have implicated aberrant post-translational modification of transport machinery with defective mitochondrial trafficking in PD (17–19). Neurons are particularly vulnerable to deficits in mitochondrial transport, which functions to deliver ATP to the synapse and maintain neurotransmission. The distribution of mitochondria at the nerve terminal can modulate both synaptic transmission and neuronal architecture (20, 21). Overexpression of the human SNCA-A53T disease variant in human neurons and transgenic animals is associated with multiple aspects of mitochondrial dysfunction, including abnormal distribution of mitochondria and impaired mitochondrial dynamics (22, 23) as well as impaired mitochondrial complex I function (24–26). As a result, the SNCA-A53T mutation also leads to excess production of ROS/RNS (27, 28). How this, in turn, impacts aspects of mitochondrial dynamics, such as mitochondrial transport, however, is not well understood. The two are nonetheless critically linked, as impairments in mitochondrial trafficking, in and of itself, can lead to abnormal distribution of fragmented mitochondria, decreased ATP synthesis, and increased oxidative stress, exacerbating cellular pathology (29, 30). Here, we test whether exposure to the agrochemicals PQ/MB or rotenone (Rot), at levels below the U.S. Environmental Protection Agency (EPA)-reported lowest observed effect level, impacts on mitochondrial transport in predegenerative neurons harboring the SNCA-A53T mutation. Recently, the generation of human isogenicinduced pluripotent stem cell (hiPSC) models of familial PD has facilitated the analysis of PD pathology in human neurons at the cellular level. We are now able to generate and contrast A9-type DA neurons (hNs) of PD patient origin, harboring the SNCA-A53T mutation, with isogenic, genome-corrected controls (27, 31). With the use of this 2

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system, in combination with human embryonic stem cell (hESC)-derived neurons, where the SNCA-A53T mutation has been introduced by genome editing, we describe a novel mechanism, whereby NO, generated from SNCAA53T mutant neurons exposed to the agrochemicals PQ/ MB or Rot, inhibits anterograde mitochondrial transport. This occurred, in part, though nitration of a-tubulin (a-Tub) in C-terminal domain, which inhibited the association of both the mitochondrial motor protein kinesin 5B (KIF5B) and a-syn with the microtubules. The effect of SNCA mutation and pesticide exposure on both tubulin nitration and KIF5B/a-syn/tubulin binding was additive, and only combinatorial stress arrested mitochondrial transport. These effects were rescued by inhibition of NO synthesis with the NOS inhibitor Nv-nitro-L-arginine methyl ester (L-NAME). Collectively, our results demonstrate a gene by environment interaction in PD, whereby neurons harboring the SNCA-A53T mutation are sensitive to agrochemical-induced oxidative stress and deficits in mitochondrial transport. MATERIALS AND METHODS Reagents All chemicals were purchased through MilliporeSigma (Burlington, ON, Canada), and all cell culture reagents were obtained from Thermo Fisher Scientific (Burlington, ON, Canada), except where indicated. hPSC cultures With the exception of the wild-type (WT) BGO1 hESCs, the cell lines used in this study were generated and kindly shared by Dr. Rudolf Jaenisch (Whitehead Institute, Cambridge, MA, USA) (31). BGO1 hESCs were derived by Bresagen (Athens, GA, USA). Genotypes of WT/corrected and A53T cell lines were confirmed by restriction digest of genomic DNA, as previously described (31). hiPSC and hESC cultures were routinely cultured and maintained in our laboratory using a protocol previously described (27), with slight modifications. In brief, hiPSCs/hESCs were plated on Matrigel-coated plates (Corning, Corning, NY, USA) and cultured using mouse embryonic fibroblast conditioned medium, changed daily. The colonies were manually passaged weekly. hN differentiation Differentiation of hiPSC and hESC cultures into hNs was performed, as previously described (32). Growth factors were purchased from PeproTech (Quebec, QC, Canada), whereas small molecules were purchased from Tocris (Minneapolis, MN, USA). Immediately preceding differentiation, the colonies were dissociated into a single-cell suspension using HyQTase and replated at 4 3 104 cells/cm2 on Matrigel (Becton Dickinson, Mississauga, ON, Canada)-coated tissue-culture dishes for differentiation. Floor-plate induction was carried out using hESC medium containing knockout serum replacement (20%), LDN193189 (100 nM), SB431542 (10 mM), Sonic Hedgehog C25II (100 ng/ml), purmorphamine (2 mM), fibroblast growth factor 8 (100 ng/ml), and CHIR99021 (CHIR; 3 mM). On d 5 of differentiation, hESC medium was incrementally shifted to N2 medium (25, 50, 75, 100%) containing DMEM/F12, N2 (13), L-Glut (13), Anti/Anti

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(13), 7.5% bovine serum albumin fraction V, and 2-ME;, supplemented with CHIR (3 mM) and LDN193189 (100 nM). On d 11, the medium was incrementally shifted (50, 100%) to neuronal induction medium containing DMEM/F12/B27/glutamax, ascorbic acid (0.2 mM), brain-derived neurotrophic factor (20 ng/ml), glial-derived neurotrophic factor (20 ng/ml), TGF-b3 (1 ng/ml), and dibutyryl cAMP (0.5 mM), supplemented with CHIR (3 mM) and dual anti-platelet therapy (10 mM). On d 13, CHIR and (2S)-N-[(3,5-difluorophenyl)acetyl]L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT) were removed. On d 21, cells were dissociated using HyQTase and replated at 4 3 105 cells/cm2 in neuronal induction medium, on dishes precoated with polyornithine (15 mg/ml)/laminin (1 mg/ml)/fibronectin (2 mg/ml). Stable mitochondrial-targeted DsRed (MitoDSRed) expression was achieved by lentiviral infection (lentiviral constructs were kindly donated by Dr. Ruth Slack, University of Ottawa, ON, Canada) at d 11 of differentiation. Virus was pseudotyped by polyethylenimine transfection of pMD2.G and psPAX2 packaging plasmids, as well as pWPXLDMitoDsRed plasmid in human embryonic kidney 293T cells. Three days post-transfection, viral supernatant was filtered and stored at 280°C until viral titer was calculated. Where the effect of agrochemicals or NOS inhibition was assessed, cells were exposed to PQ/MB (2.8/1 mM), Rot (200 nM), 1-methyl-4phenylpyridinium (MPP+; 1 mM), or DMSO (vehicle) in the presence or absence or 1 mM L-NAME for 16 h before analysis.

and microtubule-associated protein 2 (MAP2; 1:1000) were obtained from Thermo Fisher Scientific; C-terminal a-Tub (CTT; 1:1000), ubiquitin (1:1000), and a-syn (1:1000) were obtained from Abcam (Toronto, ON, Canada); anti-mouse octamer-binding transcription factor 3/4 (Oct3/4; 1:200), nuclear receptorrelated 1 protein (Nurr1; 1:500), and sex-determining region Y-box 2 (Sox2; 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA); LIM homeobox transcription factor 1 (LMX1; 1:1000) and neuroectodermal stem cell marker (nestin; 1:1000) were from MilliporeSigma; and anti-tyrosine hydroxylase (TH; 1:1000) was from Pel-Freeze Biologicals (Rogers, AR, USA). Cells were counterstained with DAPI (1:500) from Thermo Fisher Scientific. For fluorescent resonance energy transfer (FRET) analysis, cells were labeled with donor (CTT) and acceptor (3-NT) fluorophores alone to establish bleedthrough constants before colabeling and analysis. Analysis was performed using the FRET analysis tool of Volocity 6.3 (PerkinElmer). Proximity ligation assay (PLA) was performed using the DuoLink in situ Red detection platform (MilliporeSigma) with mouse and rabbit probes. Imaging was performed using either an Axio-observer LSM 800 with Airyscan (Carl Zeiss) or an Axioobserver Live-cell imaging microscope with LED-based illumination and optical sectioning by structured illumination (Carl Zeiss). Objectives used were Plan-APO 340/1.4 Oil DIC VIS-IR or PlanAPO 363/1.4 Oil DIC M27. Immunoprecipitation and protein analysis

Mitochondrial transport and motility analysis Mitochondrial transport was assessed by live cell imaging of MitoDSRed expressing human neurons using an Axio-observer Live-cell imaging microscope with light-emitting diode (LED)based illumination, piezo stage with triggering for high-speed acquisition, and environmental control. Objectives used were Plan apochromat (APO) 340/1.4 Oil differential interference contrast (DIC) visible-infrared (VIS-IR) or Plan APO 363/1.4 Oil DIC M27. Images were captured every 100 ms. For live-cell imaging of axonal mitochondria, coverslips were transferred to phenol-free medium to maintain cell viability. Kymographs were generated from 1 to 3 min-long time-lapse movies and analyzed using Volocity (PerkinElmer, Guelph, ON, Canada) for percent time in motion, velocity, total distance traveled, and direction. Detection of NO by DAF-FM fluorescence intensity For 4-amino-5-methylamino- 29,79-difluorofluorescein (DAFFM) analysis of NO, cells were loaded with 2.5 mM DAF-FM (Thermo Fisher Scientific) in recording buffer: 5 mM D-glucose, 10 mM Hepes, 135 mM NaCl, and 5 mM KCl. In brief, cells were loaded for 15 min at room temperature, washed, and then incubated for 15 min at 37°C for de-esterification. Images were acquired using an Axio-observer Live-cell imaging microscope with LED-based illumination and optical sectioning by structured illumination. Objectives used were Plan-APO 340/1.4 Oil DIC VIS-IR or Plan-APO 363/1.4 Oil DIC M27. Quantification of fluorescent intensity was performed using Zen 2.3 (Carl Zeiss, North York, ON, Canada).

For immunoprecipitations assays, protein lysate from hiPSC- or hESC-derived neurons was precleared overnight with Dynabeads. Protease and phosphatase inhibitors (NaF, PMSF, NaV, aprotinin) were added to lysis buffers just before use. Protein concentration was determined using the DC Protein Assay (BioRad, Mississauga, ON, Canada). Samples were subsequently incubated with magnetic Dynabeads protein G (Thermo Fisher Scientific), conjugated to 5 mg of KIF5B antibody (Abcam), b-Tub (BioLegend, San Diego, CA, USA), a-syn (BD Bioscience, La Jolla, CA, USA), or mouse-IgG (Santa Cruz Biotechnology) antibody. Samples were subsequently centrifuged or placed on a magnetic rack where appropriate. Proteins were eluted in either elution buffer (50 mM glycine, pH 2.8) or boiled and subjected to SDS-PAGE. Samples were separated on 4–12% gradient Bis-Tris SDS-PAGE gel and transferred onto 0.2 mm nitrocellulose. Membranes were probed with the following primary antibodies: synaptophysin I (SYN1; 1:1000; Synaptic Systems, Goettingen, Germany), KIF5B (1:500; Abcam), 3-NT (1:500; Thermo Fisher Scientific), and anti-b-Tub (1:1000; Thermo Fisher Scientific) or anti-a-syn (1:1000; Abcam). When donkey anti-mouse (1:2000; Bio-Rad) and anti-rabbit (1:2000; Bio-Rad) horseradish peroxidase-conjugated secondary antibodies were used, Clarity Western ECL blotting substrate (Bio-Rad) was used to visualize bands on blots. When anti-mouse (800) and anti-rabbit (700) Li-Cor Biosciences (Lincoln, NE, USA) infrared-conjugated secondary antibodies were used (1:1000), bands were visualized on the Li-Cor Biosciences Fc imaging platform. Densitometry was preformed to quantify band intensity using the Li-Cor Biosciences Fc program. Synthesis of recombinant proteins

Immunocytochemistry and fluorescence analysis Cells were fixed with 4% paraformaldehyde for 20 min, washed 1 time with PBS, and blocked with 3% bovine serum albumin and 0.3% Triton X-100 in PBS for 30 min. Cells were incubated with primary antibody overnight, and the appropriate Alexa Fluor (488, 594, 647)-conjugated secondary antibodies were used at 1:1000. Primary antibodies and dilutions were as follows: 3-nitrotyrosine (3-NT; 1:500), a-Tub (1:1000), RNS IMPAIRS MITOCHONDRIAL TRANSPORT IN PD hiPSCs

A cDNA sequence encoding the 38 C-terminal amino acids of de novo-synthesized a-Tub (CTT-Y) was subcloned from a CTTcontaining pET16b construct (Dr. Philippe Savarin, INSERM, Paris, France) (33) into a Champion pET SUMO expression system (Thermo Fisher Scientific). The resulting plasmid was sequenced (Laboratory Services Division, University of Guelph, ON, Canada) and transformed into Escherichia coli BL21CodonPlus (DE3)-RP cells (Stratagene, La Jolla, CA, USA). The 3

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peptide was expressed and purified using the previously published method for the purification of myelin basic protein segments (34). The plasmid pET21a containing human a-syn cDNA was purchased through Addgene (Cambridge, MA, USA; plasmid 51486) and deposited by The Michael J. Fox Foundation for Parkinson’s Research (New York, NY, USA). Mutagenesis was subsequently performed to generate the A53T variant using the Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA) with the following mutagenesis primers: for A53T forward, 59-GCATGGTGTGACAACAGTGGC-39 and reverse, 59-ACCACTCCCTCCTTGGTT-39. Plasmids with mutant and WT a-syn variants were transformed into BL21-CodonPlus (DE3)-RIPL-competent cells (Stratagene), and protein induction was performed in the same manner as for CTT-Y. Protein purification was completed via boiling, according to the method of Livernois et al. (35) with slight modifications. In brief, the cell pellet from a 500-ml culture was thawed on ice, resuspended in 50 ml water, and placed in a beaker of boiling water for 20 min with shaking every 5 min and then placed on ice for another 5 min. The lysate was centrifuged at 50,000 g for 30 min at 4°C. Supernatant was transferred to a new tube, Tris-HCl (pH 8.0) was added to a final concentration of 20 mM, and supernatant was filtered through a 0.2-mm membrane. Anion-exchange chromatography (FPLC) was performed on a 1-ml AcroSep (Pall Corp., Port Washington, NY, USA) diethylaminoethyl column connected to a DuoLogic system (Bio-Rad). Fractions were analyzed using SDS-PAGE, and those fractions containing a-syn were pooled and run through a Symmetry 300, C18, reversed-phase HPLC column on a Waters system with Millennium 32 software (Waters, Milford, MA, USA). After reversed-phase HPLC, the pure protein solution was frozen at 80°C and lyophilized until dry. Samples were stored at 220°C until further use. The A53T molar mass was validated via mass spectrometry. Influence of 3-NT modification on protein interaction To evaluate the effect of tubulin nitration on a-syn binding, microtubules were isolated from bovine brain through multiple cycles of polymerization and depolymerization, according to the method of Castoldi and Popov (36). Isolated microtubules or purified CTT-Y protein was subsequently treated with 1 mM ONOO2 or NaOH (vehicle) control in 100 ml of 25 mM purified tubulin. Samples were then diluted 1:1000 to a final volume of 200 ml in Brinkly buffer 80 mM PIPES (80 mM PIPES-KOH, 1 mM MgCl2, 1 mM EGTA, pH 6.8) and loaded into the slot blot apparatus (Bio-Rad). The sample was allowed to enter the apparatus by gravity for 10 min before vacuum was applied. Following sample loading, wells were washed using 400 ml PBS. Subsequently, the membrane was removed from the slot blot apparatus and blocked using 5% milk in PBS for 1 h. To test the binding of a-syn, membranes were incubated with 25 nM of either WT a-syn or A53T mutant a-syn for 1 h at 37°C with shaking. The membrane was then washed 3 times with PBS and incubated with primary antibody a-syn (1:1000; Abcam) or 3-NT (1:1000; Thermo Fisher Scientific). Li-Cor infrared-conjugated secondary antibodies (donkey anti-rabbit or donkey anti-mouse, 1:2000) were used, and densitometry was preformed to quantify band intensity using Li-Cor Fc. Statistical analysis All error bars reflect SEM. Data were analyzed using Student’s t test or multifactorial ANOVA, as applicable using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Following detection of a statistically significant difference in a given series of treatments by ANOVA, post hoc Dunnett’s t tests or Tukey tests were performed where appropriate. A value of P , 0.05 was 4

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considered statistically significant, and P , 0.01 was considered highly statistically significant.

RESULTS Agrochemical exposure impairs anterograde transport of mitochondria in PD neurons To evaluate the relationship between agrochemical exposure and PD causal mutations, we generated a homogeneous population of DA neurons of both hiPSC and hESC origins. With the use of a floor-plate induction protocol (Fig. 1A) (32), we characterized the lineage progression of these cells from Oct4/Sox2-positive stem cells to Nurr1/ LMX1/forkhead box A2-positive floor-plate progenitors to TH/MAP2-positive DA neurons by immunofluorescence (Fig. 1B–D). We subsequently characterized the effect of the SNCA-A53T mutation on a-syn pathology in these neurons by contrasting PD patient hiPSC-derived neurons, harboring the SNCA-A53T mutation with isogenic mutation-corrected controls (27, 37) (Fig. 1E). We also characterized a-syn pathology in hESC-derived neurons, in which the SNCA-A53T mutation was introduced by genome editing, against isogenic WT controls (27, 37) (Fig. 1F). Immunolabeling for both a-syn phophoserine 129 and ubiquitin showed increased a-syn-PS129 abundance and increased colabeling for ubiquitin in both hiPSC- and hESC-derived SNCA-A53T neurons relative to their respective isogenic controls (Fig. 1E, F), suggesting early deposition of a-syn pathology. To determine the effect of pesticide exposure on mitochondrial transport, we transduced neurons with MitoDSRed, which allows for stable expression of DsRed within mitochondria (Fig. 2A–F and Supplemental Fig. S1). Neurons were then exposed to agrochemicals and analyzed by high-speed live-cell imaging (Fig. 2A–F). We have previously determined that agrochemical exposure triggers apoptosis within 24 h in human DA neurons at concentrations of 28 mM PQ, 50 mM MB, and 1 mM Rot (27). Therefore, we treated neurons with agrochemicals at concentrations 10- to 50-fold lower than those that result in toxicity (2.8 mM PQ, 1 mM MB, and 200 nM Rot). These concentrations are below the EPA-reported lowest observed effect level (http://www.epa.gov/iris/subst/0183.htm) for these toxicants. Before agrochemical exposure, we found no significant difference in the percent time mitochondria spent in motion in either the anterograde or retrograde direction between either hiPSC-derived (Fig. 2G, H) or hESC-derived (Supplemental Fig. S1A, B) SNCAA53T neurons and their respective isogenic controls, suggesting no pre-existing impairment in mitochondrial transport. Nonetheless, exposure to either Rot (Fig. 2C, D, G) or PQ/MB (Fig. 2E–G and Supplemental Fig. S1A) significantly impaired anterograde transport in SNCAA53T neurons but not isogenic control neurons. This was consistent with the effect of exposure to the mitochondrial complex I inhibitor MPP+ (Supplemental Fig. S1A, C), suggesting that this was a result, at least in part, of an effect on mitochondrial function. The percent time in motion in the retrograde direction was, however, unaltered by chemical exposure (Fig. 2H and Supplemental Fig. S1B, D).

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Figure 1. Characterization of hiPSC- and hESC-derived human neurons (hNs). A) Schematic representing DA neuron differentiation. Application of the floor-plate induction protocol patterns, human stem cells (hiPSCs or hESCs) to neural precursor cells (hNPCs), and subsequently, DA neurons (hNs). B) hNs were matured for 60 d in vitro before experimental analysis. hiPSCs expressed typical markers of pluripotency; Sox2 (upper) and Oct4 (lower) and counterstained with DAPI; original scale bar, 20 mm. C ) At 9 d in vitro, cells were positive for midbrain neural precursor factors; Lmx1A/B, Nurr1, and forkhead box A2 (FoxA2); original scale bar, 100 mm. D) At 60 d in vitro, cells expressed characteristic markers of DA neurons; Map2- and TH-positive; original scale bar, 50 mm. E, F ) hiPSC-derived (E ) and hESC-derived (F ) hNs harboring the SNCA-A53T were labeled for ubiquitin (Ubq; green) and a-syn-phophoserine 129 (a-syn-PS129; pink) as markers of synuclein pathology and compared with isogenic controls. Colocalization masks show the degree of colocalization between the 2 markers. Scale bars, 10 mm.

These findings demonstrate an additive effect of agrochemical exposure and impaired a-syn proteostatis on mitochondrial transport in PD patient neurons. Agrochemical exposure inhibits KIF5B binding to the microtubules in SNCA-A53T neurons In a healthy neuron, mitochondria are transported along microtubules in an anterograde fashion via the Miro/Milton/KIF5B motor complex (38, 39). When KIF5B is tubulin bound, mitochondria are generally in motion. Inhibition of KIF5B/tubulin interaction arrests mitochondrial motility (29). As Rot and PQ/MB impaired RNS IMPAIRS MITOCHONDRIAL TRANSPORT IN PD hiPSCs

anterograde transport of mitochondria, we next sought to determine whether agrochemical exposure altered KIF5B association with the microtubules. To assess this, we immunoprecipitated KIF5B from both SNCA-A53T neurons and SNCA-Corrected (Corr) neurons, with and without exposure to agrochemicals (Fig. 3A–C). We found no difference in the baseline level of KIF5B bound to tubulin between SNCA-A53T neurons and SNCA-Corr neurons (Fig. 3A). Moreover, we found no evidence of the SYN1positive synaptic vesicle in association with KIF5B (Fig. 3A). Whereas KIF5B immunoprecipitation in SNCA-Corr neurons also showed no change in a-Tub association following exposure to agrochemicals (Fig. 3B), in SNCA-A53T 5

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Figure 2. A–F) Agrochemical exposure inhibits anterograde transport of mitochondria in A53T hNs. Axonal mitochondrial transport was monitored in hiPSC-derived SNCA-Corr (A, C, E) and SNCA-A53T hNs (B, D, F ) in both the anterograde and retrograde directions following exposure to DMSO (A, B), 200 nM Rot (C, D), or 2.8 mM PQ and 1 mM MB. G, H ) Representative kymographs and traces resulting from particle tracking are depicted. Quantification of the percent time motile in the anterograde (G) and retrograde (H) directions shows a reduction in anterograde mitochondrial transport in A53T hNs exposed to agrochemicals. Data represent means + SEM. **P , 0.01 by multivariate ANOVA (MANOVA) with post hoc Tukey, n = 6 replicate experiment from 3 independent differentiations. Scale bars, 10 mm.

neurons, exposure to PQ/MB or Rot resulted in a dramatic reduction in a-Tub/KIF5B association (Fig. 3C). We subsequently performed the reciprocal experiment and immunoprecipitated bIII-Tub from SNCA-A53T neurons and SNCA-Corr neurons and probed for coimmunoprecipitated KIF5B in both hESC-derived (Fig. 3D) and hiPSC-derived (Fig. 3E–G) human neurons. These experiments corroborated our findings. Whereas no difference in KIF5B binding to tubulin was observed in either SNCA-A53T neurons or their respective isogenic controls (Fig. 3D, E), agrochemical exposure dramatically inhibited KIF5B binding to microtubules in SNCA-A53T neurons (Fig. 3G) but not in isogenic control neurons (Fig. 3F). To confirm this result in an intact cellular context, we next performed a PLA on a-Tub and KIF5B (Fig. 3E) using the Duolink in-cell interaction system assays. PLAs generate an immunoreactive signal only if 2 antigens (target proteins) are localized within 40 nm of each other (40). With the use of antibodies against KIF5B and a-Tub, we 6

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performed PLAs in SNCA-A53T and SNCA-Corr neurons exposed to agrochemicals (Fig. 3H). Both SNCA-A53T and SNCA-Corr showed a robust fluorescent Duolink signal under basal conditions (Fig. 3E, upper), indicating that KIF5B and a-Tub proteins are in close proximity. Indeed, the Duolink signals formed clusters in neurites, rather than in cell bodies, in keeping with transport of mitochondria along microtubules. Exposure to either PQ/MB or Rot resulted a selective loss in the Duolink signal in SNCAA53T neurons, further supporting the notion that agrochemical exposure impairs KIF5B association with tubulin. Taken together, these data suggest that agrochemical exposure impairs mitochondrial transport in SNCA-A53T neurons by inhibiting the association of the KIF5B mitochondrial motor complex with the microtubules. The association of KIF5B with the microtubules is governed, in part, by post-translational modifications to a-Tub, whereby acetylation of a-Tub increases KIF5B

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Figure 3. A–C) Agrochemical exposure results in the selective loss of KIF5B association in SNCA-A53T neurons. KIF5B was immunoprecipitated (IP) from SNCA-Corr and SNCA-A53T neurons both at baseline (A) and following exposure to DMSO, Rot, or PQ/MB (B, C ). Lysates were then probed for KIF5B, a-Tub, and either SYN1 or TH. Less a-Tub was pulled down by KIF5B from SNCA-A53T hNs than SNCA-Corr neurons following agrochemical exposure. D–G) Likewise, bIII-Tub was immunoprecipitated at baseline from hESC-derived (D) or hiPSC-derived (E ) neurons or from SNCA-Corr (F ) and SNCAA53T (G) hiPSC-derived neurons treated with DMSO or PQ/ MB, and subsequent immunoblot analysis for KIF5B and tubulin demonstrated that pesticide treatment reduced KIF5B association to microtubules. H ) PLA, using anti-KIF5B and anti-a-Tub antibodies, confirms the change in the KIF5B association with the microtubules in SNCA-A53T hNs exposed to agrochemicals; scale bars, 10 mm.

binding (41), whereas Ty-Tub has a lower affinity for KIF5B and directs mitochondria to more stable axonal microtubules (42, 43). These modifications generally govern microtubule stability, with acetylated and detyrosinated microtubules less dynamic than tyrosinated (44). Furthermore, multiple studies have shown that RNS IMPAIRS MITOCHONDRIAL TRANSPORT IN PD hiPSCs

neuronal a-Tub is a substrate of protein nitration through oxidative modification of tyrosine residues to 3-NT residues (45–48). We have previously reported that mitochondrial stress, resulting from agrochemical exposure, exacerbates NO synthesis in DA neurons (27). Elevated NO reacts rapidly with O22 to form ONOO2, leading to nitration of tyrosine residues. Therefore, we sought to determine whether increased NO synthesis in SNCAA53T neurons exposed to agrochemicals resulted in the nitration of tubulin that may alter the KIF5B interaction with the microtubules. With the use of the NO-sensitive dye DAF-FM, we first confirmed that a selective increase in NO synthesis in SNCA-A53T neurons occurs upon agrochemical exposure (Fig. 4A). We observed an increase in DAF-FM fluorescence in SNCA-A53T neurons when exposed to either Rot or PQ/MB, whereas agrochemical exposure at this concentration has no effect on NO levels in isogenic control neurons. The agrochemical-induced increase in NO was blocked by treatment with L-NAME, an inhibitor of NOS (Fig. 4A). Indeed, L-NAME treatment returned SNCA-A35T neurons to NO levels comparable to SNCA-Corr control neurons. We next assessed whether this increase in NO led to increased 3-NT-modified tubulin levels in SNCA-A53T neurons by first confirming that agrochemical exposure increased the global level of 3-NT-modified proteins, specifically in SNCA-A53T neurons (Fig. 4B), followed by a direct assessment of 3-NTmodified tubulin. As no antibody is available to measure 3-NT tubulin directly, we used FRET to determine whether tubulin was among the proteins undergoing nitration. Both SNCA-A53T and SNCA-Corr neurons were labeled with a tubulin antibody targeted against CTT as the FRET donor and with anti-3-NT antibody as the FRET acceptor (Fig. 4C). We then assessed energy transfer between donor and acceptor upon donor excitation (488 nm) by monitoring emission in the acceptor channel (594 nm). If the microtubules are nitrated in response to agrochemical exposure, then the acceptor emission intensity should increase. We found that exposure of SNCA-A53T neurons to either PQ/MB or Rot resulted in a significant increase in FRET intensity relative to SNCA-Corr neurons (Fig. 4C–F). Maximal FRET intensities clustered to neurites rather than in cell bodies, consistent with the loss of localization of KIF5B. To confirm that tubulin had been nitrated, we performed are series of tubulin immunoprecipitation experiments and measured the relative levels of 3-NT-modified tubulin in both SNCA-A53T and control neurons, with and without exposure to PQ/MB (Fig. 4G–L). Analysis of densitometry, performed on Western blots of 3-NT levels, following tubulin immunoprecipitation, showed a modest elevation in 3-NT tubulin in SNCA-A53T neurons relative to SNCA-Corr neurons (Fig. 4G, H). Whereas exposure of SNCA-Corr control neurons to PQ/MB did not result in an increase in 3-NT-modified tubulin (Fig. 4I, J), an increase in 3-NT tubulin was detectable in SNCA-A53T neurons exposed to PQ/MB relative to vehicle-treated neurons (Fig. 4K, L). Immunoprecipitation experiments confirm that exposure of a-syn mutant neurons to agrochemicals results in nitration of the microtubules. Collectively, these data argue that elevated NO levels arrest anterograde 7

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Figure 4. A) Accumulation of NO following agrochemical exposure results in excess tubulin nitration in A53T-hNs. DAF-FM signal intensities were quantified from SNCA-Corr and -A53T hNs, following exposure to 200 nM Rot or 2.8 mM PQ/1.0 mM MBrespective to baseline levels. SNCA-A53T mutant hNs have a higher level of DAF-FM fluorescence than SNCA-Corr hNs at baseline, and this is exacerbated by agrochemical exposure. Cotreatment with L-NAME significantly reduces NO accumulation. Data represent means + SEM. **P , 0.01 by MANOVA with post hoc Tukey, n = 6 replicate experiment from 3 independent (continued on next page) 8

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mitochondrial transport through oxidative modification of tubulin, resulting in chronic elevation of 3-NT levels, thereby impairing binding of the KIF5B mitochondrial motor complex. 3-NT modification of CTT regulates a-syn interaction with the microtubules To gain mechanistic insight into how mutation in a-syn acts, in combination with environmental exposures that promote microtubule nitration to arrest mitochondrial transport, we turned to a cell-free system. With the use of microtubules isolated from brain through repeated rounds of polymerization and depolymerization, we assessed the ability of both native microtubules and nitrated microtubules to bind to recombinant WT or A53T a-syn in an affinity-capture assay, subsequently analyzed by immunoblot. Tubulin isolated from microtubules was incubated with 1 mM ONOO2 for 1 h to mediate tubulin nitration, and the fold change in 3-NT modification was measured by immunolabeling for anti-3-NT (Fig. 5A). ONOO2 treatment led to a 15-fold increase in 3-NT tubulin, showing that microtubules isolated from the brain are highly sensitive to protein nitration (Fig. 5A). We next assessed binding 3-NT tubulin and unmodified tubulin to a-syn. We found that tubulin nitration caused a significant decrease in WT a-syn binding relative to the vehicle treated (Fig. 5B). This decrease in binding was approximately equivalent to the decrease in binding to tubulin observed in A53T mutant a-syn, relative to WT (Fig. 5B). Interestingly, the effects of tubulin nitration and a-syn mutation were synergistic, with A53T a-syn having almost no affinity for nitrated microtubules. Ty-Tub refers to the presence of a tyrosine residue at the C-terminus of de novo-synthesized a-Tub. Posttranslational removal and readdition of this C-terminal tyrosine govern microtubule stability (44). This C-terminal tyrosine has been previously reported to be important for KIF5B interaction with microtubules (42, 43). Therefore, we sought to assess whether the tubulin C-terminal tyrosine was sensitive to nitration and whether this site regulates a-syn interaction with the microtubules in a similar manner to KIF5B. Therefore, we synthesized a recombinant peptide consisting of the 38 C-terminal amino acids of de novo-synthesized a-Tub (CTT-Y). The C-terminal tyrosine is the only tyrosine present in this peptide and therefore, the only available site for 3-NT incorporation. With the use of the CTT-Y peptide, we

repeated the a-syn-binding experiments outlined above. Incubation of CTT-Y with 1 mM ONOO2 for 1 h led to a 50% increase in 3-NT incorporation, confirming that the CTT-Y residue is indeed sensitive to nitration (Fig. 5C). We next assessed binding of either native or 3-NTmodified CTT-Y to WT and A53T a-syn. We found that the CTT-Y peptide interacted with WT a-syn (Fig. 5D), showing that this region of the tubulin protein mediates, at least in part, the interaction between a-syn and the microtubules. Moreover, CTT-Y nitration caused a significant decrease in WT a-syn binding relative to native CTT-Y, and A53T a-syn had a similar low affinity for both native and nitrated CTT-Y (Fig. 5D). Collectively, these data show that the C-terminal tyrosine residue of a-Tub may regulate binding of a-syn to microtubules in a similar manner to that which has been reported for KIF5B and that this interaction is sensitive to tubulin nitration. a-Syn has been previously reported to interact with both KIF5 (49, 50) and the microtubules (51, 52); however, the exact nature of this interaction and whether a-syn mutation promotes or inhibits KIF5B binding to the microtubules have not been determined. Therefore, we sought to determine whether the KIF5B, a-syn and tubulin existed in complex in human DA neurons. Therefore, we immunoprecipitated a-syn from both SNCA-A53T and SNCA-Corr neurons and probed the resulting lysates for KIF5B, tubulin, and a-syn (Fig. 5F–I). Whereas we saw a ratiometric decrease in the level of a-syn bound to tubulin in SNCA-A53T neurons relative to SNCA-Corr neurons (Fig. 5E, H) at baseline, we observed no difference in the baseline level of KIF5B bound to a-syn (Fig. 5E, I). To determine whether perturbations in binding of the KIF5B/ a-syn complex explained how mutation in a-syn acts, in combination with environmental exposures that promote microtubule nitration to arrest mitochondrial transport, we repeated these immunoprecipitation experiments following exposure of neurons to agrochemicals. Exposure to PQ/MB resulted in an even greater ratiometric inhibition of a-syn binding to tubulin in SNCA-A53T neurons (Fig. 5G, H), whereas agrochemicals had only a modest effect on the genome-corrected control neurons (Fig. 5F, H). Importantly, PQ/MB exposure inhibited KIF5B binding to a-syn, only in SNCA-A53T neurons (Fig. 5G–I). Collectively, these results show an additive effect of agrochemical exposure and a-syn mutation on the ability of a-syn to interact with the microtubules, likely a result, in part, of an increase in 3-NT modification of tubulin upon agrochemical exposure. Given our observations that KIF5B exists in

differentiations. B) Western blot of global 3-NT-modified proteins shows more 3-NT-modified protein in SNCA-A53T mutant hNs than SNCA-Corr hNs following PQ/MB exposure. C–F ) FRET, from Alexa-488-labeled a-Tub C-terminus to Alexa-594-labeled 3-NT in hiPSC-derived SNCA-A53T and SNCA-Corr neurons (C ), was assessed following exposure to 200 nM Rot (D) or 2.8 mM PQ and 1 mM MB (E ), respective to baseline levels (C ), and mean FRET intensity was quantified (F ). Data represent means 6 SEM. **P , 0.01 by MANOVA, post hoc Tukey, n = 6 coverslips over 3 independent differentiations. Scale bars, 10 mm. G) Tubulin was immunoprecipitated from SNCA-Corr and SNCA-A53T neurons, and lysates were then probed for 3-NT and a-Tub. H ) Quantification of the ratio of 3-NT-modified tubulin relative to total tubulin on input was performed by densitometry. I–L) Tubulin was immunoprecipitated from SNCA-Corr (I ) and SNCA-A53T (K ) neurons exposed to DMSO or 2.8 mM PQ and 1 mM MB. Quantification of the ratio of 3-NT-modified tubulin relative to total tubulin on input by densitometry (J, L). Data represent means 6 SEM. **P , 0.01, Student’s t test, n = 3 replicate experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RNS IMPAIRS MITOCHONDRIAL TRANSPORT IN PD hiPSCs

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Figure 5. Nitration of the a-Tub C-terminus impairs binding of a-syn and KIF5B to the microtubules (MTs). A) Microtubules isolated from the brain were exposed to ONOO2 and the level of 3-NT modification was quantified by immunoblot analysis. Data represent means 6 SEM. **P , 0.01, by Student’s t test, n = 6 replicate experiments. B) Affinity capture of recombinant human WT and A53T a-syn by native vs. nitrated microtubules. Data represent means 6 SEM. **P , 0.01, *P , 0.05, MANOVA with post hoc Tukey, n = 6 replicate experiments. C ) A recombinant CTT-Y peptide consisting of the C-terminal 38 aa of de novo-synthesized a-Tub was exposed to ONOO2, and the level of 3-NT modification of the C-terminal tyrosine was measured. Data represent means 6 SEM. **P , 0.01, by Student’s t test, n = 6 replicate experiments. D) Affinity capture of recombinant human WT and A53T a-syn by native vs. nitrated CTT-Y. Data represent means 6 SEM. **P , 0.01, MANOVA with post hoc Tukey, n = 6 replicate experiments. E ) a-Syn was immunoprecipitated from hiPSC-derived SNCA-A53T and SNCA-Corr neurons, and the resulting lysates were subsequently immunoblotted for bIII-Tub, KIF5B, and a-syn. a-Syn was immunoprecipitated from either hiPSCderived SNCA-Corr neurons (F ) or SNCA-A53T (G) neurons exposed to either vehicle (DMSO) or 2.8 mM PQ and 1 mM MB, and the resulting lysates were subsequently immunoblotted for bIII-Tub, KIF5B, and a-syn. H, I ) Quantification of the ratio of tubulin (H ) and KIF5B (I ) bound to immunoprecipitated a-syn. Data represent means 6 SEM. **P , 0.01, *P , 0.05, MANOVA with post hoc Tukey, n = 3 replicate experiments.

complex with a-syn and releases from the microtubules in SNCA-A53T neurons exposed to agrochemicals, coincident with a 50% reduction in microtubule associate a-syn, these data suggest that a-syn may facilitate the interaction of KIF5B with the microtubules and therefore, play a direct role in regulating mitochondrial transport. The blocking of NO synthesis rescues agrochemical-mediated arrest of mitochondrial transport in PD neurons In our final set of experiments, we sought to assess whether excess NO could be targeted to restore mitochondrial transport in PD. First, we blocked NO synthesis with L-NAME and assessed the effect on accumulation of 3-NT-modified tubulin by FRET-mediated energy transfer from a CTT-bound donor to a 3-NT-bound acceptor. The increase in FRET intensities observed in SNCA-A53T neurons exposed to agrochemicals was reduced by LNAME treatment (Fig. 6A, B), further indicating that the nitration of tubulin was a result of agrochemical-induced accumulation of NO. As we had established that a-Tub 10

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nitration was reduced by L-NAME treatment, our last experiments evaluated whether normalizing these molecular perturbations rescued anterograde mitochondrial transport in SNCA-A53T neurons. Therefore, we exposed both SNCA-A53T hNs and their isogenic controls to agrochemicals in the presence of L-NAME and reassessed mitochondrial motility (Fig. 6D, E). Quantification of the percent time motile in the anterograde direction showed that L-NAME treatment significantly rescued anterograde mitochondrial transport in agrochemical-exposed SNCA-A53T neurons (Fig. 6F), whereas no difference in retrograde transport was observed when NO synthesis was inhibited. In summary, these data suggest that agrochemical exposure blocks mitochondrial transport to synaptic terminals by augmenting nitration of microtubules in PD neurons, specifically the tubulin C-terminal tyrosine. This reduces the affinity of the a-syn/KIF5B complex for the microtubules and is exacerbated by a-syn mutation, which acts synergistically to reduce syn binding. This culminates in the arrest of anterograde mitochondrial transport. Moreover, we demonstrated that targeted inhibition of NO synthesis reduces RNS

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Figure 6. A, B) L-NAME treatment rescues anterograde mitochondrial transport in agrochemical-exposed A53T hNs. FRET from Alexa-488-labeled a-Tub C-terminus to Alexa-594-labeled 3-NT was assessed in agrochemical-exposed SNCA-A53T neurons, with and without L-NAME treatment (A), and mean FRET intensity was quantified (B). C, D) Axonal mitochondrial transport was monitored in L-NAME-treated SNCA-Corr (C ) and -A53T hNs (D) in both the anterograde and retrograde directions following exposure to 200 nM Rot or 2.8 mM PQ and 1 mM MB relative to baseline levels. Representative kymographs and traces, resulting (continued on next page)

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levels, prevents 3-NT modification of microtubules, and rescues defective mitochondrial transport.

DISCUSSION Several environmental toxins are believed to be risk factors in the development of PD (53). Indeed, whereas controversy exists over the levels of exposure needed to induce disease, use of herbicides, such as PQ/MB and Rot, is associated with .200% higher incidence rates of PD (9, 11). Here, we have demonstrated that exposure to the agrochemicals PQ/MB or Rot at EPA-accepted levels impairs anterograde mitochondrial transport in PD neurons. An intriguing observation in the present study is the finding that agrochemical-induced arrest of mitochondrial transport is restricted to human DA neurons carrying the SNCA-A53T mutation. This result suggests that the mutant background facilitates induction of neuronal dysfunction by environmental toxins, supporting a “2-hit” hypothesis of PD pathogenesis (54). In accord with our findings in human neurons, prior studies in rodents have suggested that aberrant expression of a-syn increases the vulnerability of SNpc neurons to the neurotoxic effects of PQ/MB or Rot (55, 56). In rodents, treatment with PQ/MB or Rot causes degeneration of SNpc neurons and locomotor deficits consistent with a Parkinsonian phenotype (57–60). PQ and Rot are believed to inhibit mitochondrial complex I (8, 13, 61), whereas MB is reported to inhibit mitochondrial complex I and III, increasing oxidative stress (62, 63). Recent data suggest that a-syn is localized to mammalian neuronal mitochondria and that its expression is associated with a decrease in complex I activity (22, 23, 64). ROS/RNS, generated as a result of impaired mitochondrial function, has been reported to lead to tyrosine nitration and methionine oxidation of a-syn, thus contributing to a-syn aggregation and leading to formation of Lewy bodies and Lewy neurites (65, 66). Mitochondrial transport has been assessed in several hiPSC models of PD. In WT hiPSCs, lentiviral overexpression of WT, A30P, or A53T a-syn variants results in increased mitochondrial fragmentation but had no effect on mitochondrial transport relative to vector controls (23). Only a modest shift in the proportion of mitochondria undergoing directional movement in the a-syn A53Toverexpressing neurons is reported (23). Park2 mutant lines show a similar perturbation of mitochondrial dynamics (67), but no deficits in mitochondrial transport have been reported to date. Similar results are also described for hiPSC-derived neurons harboring the LRRK2G2019S mutation, as well as in hiPSCs from sporadic PD patients, which both show no basal deficit in mitochondrial transport (68). In fact, the authors reported that these lines showed no transport deficits until challenged with

antimycin A, an inhibitor of mitochondrial complex III, which in addition to perturbing transport, led to excess mitophagy. These results are highly consistent with our own finding that the combined inhibition of complex I and III in SNCA-A53T neurons is needed to bring about phenotypic changes. Moreover, we have also noted increased mitophagic flux in PD-hiPSCs relative to mutationcorrected controls (69), consistent with the findings in sporadic disease cases (68). These data further support the notion that environmental exposure and genetic stress combine to trigger PD onset. Here, we report that exposure of PD neurons to PQ/MB or Rot altered microtubules via NO-mediated nitration of tubulin. Moreover, we provide evidence that this may involve the C-terminal tyrosine of a-Tub. We show that SNCA-A53T neurons had increased levels of 3-NT-tubulin relative to isogenic controls and that this accumulation of 3-NT-tubulin is exacerbated by agrochemical exposure. We have previously reported that exposure to PQ/MB and Rot increases NO synthesis in PD neurons (27). Indeed, epidemiologic studies of the effect of single nucleotide polymorphisms in the gene encoding for NOS1 have determined that organophosphate pesticide exposure is more strongly associated with PD among patients with variant genotypes in NOS1 that result in increased NO production (70). In the superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis, mitochondria accumulate iNOS in response to perturbed mitochondrial dynamics, and iNOS gene deletion significantly extends the lifespan of G93A-mutant superoxide dismutase 1 mice (71). Likewise, in PD model systems, exposure of rat ventral midbrain DA neurons to both a-synpreformed fibrils and Rot leads to increased protein nitration and cytotoxicity through iNOS induction (72). The blocking of the expression of iNOS results in a significant decline in protein nitration levels and protection against fibril-induced neuronal death (72). These data, in combination with our own, suggest an interplay between the mitochondria and cytoskeleton, wherein a-syn localization is central to the function of both. Lossof-function mutations in a-syn lead to mitochondrial stress and iNOS induction. Excess NO production then further exacerbates deficits in mitochondrial transport through 3-NT modification of tubulin, which in turn, results in loss of KIF5B/a-syn association with the microtubules, culminating in neuronal dysfunction. This mechanism may explain the association between environmental toxin exposure and increased risk of PD onset. The effect of environmental toxin exposure on mitochondrial trafficking may indeed be multifactorial. Whereas the agrochemicals Rot and PQ are potent inhibitors of mitochondrial complex I activity and have been linked to PD pathology in multiple rodent models, their effects on the cell are not restricted to complex I function.

from particle tracking, are depicted. E, F ) Quantification of the percent time motile in the anterograde (E ) and retrograde (F ) directions showed that L-NAME treatment rescued anterograde mitochondrial transport in A53T hNs exposed to agrochemicals. Data represent means 6 SEM. **P , 0.01 by MANOVA, post hoc Tukey, n = 6 coverslips over 3 independent differentiations. Scale bars, 10 mm. 12

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For instance, deletion of the NADH dehydrogenase (ubiquinone) iron-sulfur protein 4, mitochondrial gene, which abolishes complex I activity in midbrain mesencephalic neurons, does not affect the survival of DA neurons in culture, following exposure to Rot, MPP+, or PQ (73). Moreover, both Rot and PQ have been suggested to impede cytoskeletal stability that would, in turn, promote a-syn aggregation (74, 75). Rot has been suggested to bind a-Tub directly and perturb mitotic cell division through impaired tubulin assembly (76). Whereas the pathologic consequence of Rot/tubulin interaction in postmitotic neurons is unclear, most reported cellular aberrations precede detectable neuropathology (77) and therefore, likely contribute to Lewy body deposition. In conjunction with our findings that agrochemical exposure leads to the oxidative modification of microtubules, these data collectively highlight a combined role for cytoskeletal destabilization and mitochondrial dysfunction in DA neuronal loss in PD. Finally, we show that NOS can be therapeutically targeted to diminish NO levels and rescue mitochondrial dysfunction in PD neurons. Targeted NOS inhibition has been explored for pharmacological intervention in various clinical trials, including trials for spinal cord injury and cardiovascular disorders (78) (https://clinicaltrials.gov/show/ NCT00603720). In rodent models mimicking PD, L-NAME administration was sufficient to protect against behavioral and physiologic deficits (e.g., dopamine reduction) induced by either the unilateral intracerebral injection of 6hydroxydopamine or malonate to the striatum (79, 80). Here, we show that L-NAME protects PD neurons from tubulin nitration, rescuing anterograde mitochondrial transport in SNCA-A53T neurons exposed to PQ/MB or Rot. Whereas widespread inhibition of NOS would likely not be feasible in PD patients as a result of the global effect on blood pressure, it is interesting to speculate whether targeted inhibition of tubulin tyrosine ligase in SNpc neurons would slow the rate degeneration. In summary, results from this study show that low doses of PQ/MB or Rot selectively disrupt mitochondrial transport in PD neurons through oxidative modification of microtubules. We further propose that a gene, by environment interaction, occurs in PD, whereby agrochemical exposure arrests mitochondrial transport specifically in PD neurons, suggesting that environmental toxin exposure may exacerbate neuronal dysfunction in PD patients. Moreover, therapies that target the effects of excess RNS in PD improve mitochondrial dysfunction and may represent a future therapeutic pipeline.

ACKNOWLEDGMENTS The authors acknowledge Carla Coakley (University of Guelph) for technical assistance in the preparation of this manuscript, Michaela Struder-Kypke ¨ (Advanced Analysis Center, University of Guelph) for assistance with live-cell image acquisition, and Agata Zienowicz (University of Guelph) for tubulin purification. This work was supported, in part, by the Parkinson Society of Canada (2014-685 to S.D.R.), the Natural Sciences and Engineering Research Council of Canada (RG060805 and CRDPJ490841-15 to S.D.R.; Undergraduate RNS IMPAIRS MITOCHONDRIAL TRANSPORT IN PD hiPSCs

Student Research Awards to M.P.K.), and an Ontario Graduate Scholarship to M.G.S. The authors declare no conflicts of interest.

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