Nitration of Microtubules Blocks Axonal Mitochondrial Transport in a Human Pluripotent Stem Cell Model of Parkinson's Disease

Introduction Neuronal loss in Parkinson's disease (PD) is associated with both accumulation of aggregated α‐synuclein (α‐syn) and impaired mitochondrial function in dopaminergic neurons of the substantia nigra. These impairments are closely associated with the accumulation of reactive nitrogen species (RNS) such as nitric oxide and peroxynitrite. Moreover, exposure to mitochondrial toxins, such as the agrochemicals paraquat, maneb and rotenone, are associated with at least a 2.5‐fold increased risk of PD. Further, in patients with a familial mutation in the α‐syn gene ( eg. SNCA ‐A53T), agrochemical exposure correlates with disease onset at an earlier age. While a mechanistic link between α‐syn aggregation and mitochondrial dysfunction has been difficult to ascertain, events seem to center on RNS generation. We thus explore a “two‐hit” hypothesis whereby an α‐syn mutation makes neurons susceptible to mitochondrial dysfunction following agrochemical exposure. Approach Using patient derived induced pluripotent stem cells harbouring the SNCA‐ A53T mutation and genetically corrected ( SNCA ‐Corr) isogenic controls, we tested whether there exists a gene‐by‐environment interaction in PD neurons that results in defective mitochondrial transport. Stem cells were transformed to stably express a mitochondria‐targeted DSRed fluorophore, allowing for live imaging of mitochondrial dynamics. Cells were then differentiated to dopaminergic neurons using a differentiation paradigm that mirrors floor plate development. Subsequently, neurons were exposed to agrochemicals at levels below EPA‐reported lowest observable effect levels (LOEL). Results Although there was no observable difference in the percentage of motile mitochondria between SNCA ‐A53T and SNCA ‐Corr neurons under basal conditions, agrochemical exposure impaired anterograde mitochondrial transport specifically in SNCA ‐A53T neurons. We further demonstrate that agrochemical exposure resulted in increased RNS production in SNCA ‐A53T neurons, leading to nitration of microtubules in SNCA ‐A53T neurons. This modification inhibited KIF5, an important member of the anterograde mitochondrial transport complex, from associating with microtubules. This impairment was rescued by blocking RNS accumulation with the nitric oxide synthase inhibitor, l ‐NAME. Significance 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 in PD patient‐derived neurons harboring the SNCA ‐A53T mutation. Support or Funding Information This work was supported in part by the Parkinson Society of Canada (2014‐685 to SDR), the Natural Sciences and Engineering Research Council of Canada (RG060805 to SDR) and an Ontario Graduate Scholarship to MGS. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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