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

Abstract

IntroductionNeuronal 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.ApproachUsing 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).ResultsAlthough 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.SignificanceCollectively, 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 InformationThis 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.

3-nitrotyrosine shortens axons of a non-dopaminergic neuron by inhibiting mitochondrial motility

“…Accordingly, a previous study suggested that 3-NT reattributes cytoplasmic dynein in A549 epithelial cells by altering microtubule association (Eiserich et al, 1999). A more recent study revealed that DA neurons with a mutation in α-synuclein, exposed to agrochemicals, resulted in the inhibition of anterograde mitochondrial transport, thought to be mediated by the nitration of α-tubulins inhibiting the association of Kif5B, the heavy chain of kinesin-1, to microtubules (Stykel et al, 2018).…”

“…Hence, the incorporation of 3-NT into αtubulin may disrupt these processes across different neuron types. Indeed, a recent study demonstrated that exposure to agrochemicals inhibits axonal transport of mitochondria in dopaminergic neurons carrying mutations associated with Parkinson's disease in the α-synuclein gene, possibly mediated by α-tubulin nitration (Stykel et al, 2018). However, whether 3-NT disrupts the regulation of axonal morphology in both dopaminergic and non-dopaminergic neurons remains to be determined.…”