Neuronal loss in Parkinson’s disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA-transgenic mice, we show that SNCA-mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA-mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA-mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn.

While mutations in theSNCAgene (α-synuclein [α-syn]) are causal in rare familial forms of Parkinson’s disease (PD), the prevalence of α-syn aggregates in the cortices of sporadic disease cases emphasizes the need to understand the link between α-syn accumulation and disease pathogenesis. By employing a combination of human pluripotent stem cells (hPSCs) that harbor theSNCA-A53T mutation contrasted against isogenic controls, we evaluated the consequences of α-syn accumulation in human A9-type dopaminergic (DA) neurons (hNs). We show that the early accumulation of α-syn inSNCA-A53T hNs results in changes in gene expression consistent with the expression profile of the substantia nigra (SN) from PD patients, analyzed post mortem. Differentially expressed genes from both PD patient SN andSNCA-A53T hNs were associated with regulatory motifs transcriptionally activated by the antioxidant response pathway, particularly Nrf2 gene targets. Differentially expressed gene targets were also enriched for gene ontologies related to microtubule binding processes. We thus assessed the relationship between Nrf2-mediated gene expression and neuritic pathology inSNCA-A53T hNs. We show thatSNCA-mutant hNs have deficits in neuritic length and complexity relative to isogenic controls as well as contorted axons with Tau-positive varicosities. Furthermore, we show that mutant α-syn fails to complex with protein kinase C (PKC), which, in turn, results in impaired activation of Nrf2. These neuritic defects result from impaired Nrf2 activity on antioxidant response elements (AREs) localized to a microtubule-associated protein (Map1b) gene enhancer and are rescued by forced expression of Map1b as well as by both Nrf2 overexpression and pharmaceutical activation in PD neurons.

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 α-synuclein (α-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 α-tubulin (α-Tub). Nitration of α-Tub inhibited the association of both α-syn and the mitochondrial motor protein kinesin 5B with the microtubules, arresting anterograde transport. This was, in part, a result of nitration of α-Tub in the C-terminal domain. These effects were rescued by inhibiting NO synthesis with the NOS inhibitor Nω-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.

Highlights d a-Syn binds LC3 on the surface of MVB membranes d Mutant a-syn draws LC3 into detergent-resistant aggregates d Loss of LC3 function at MVBs leads to a-syn secretion via exosome d Restoration of LC3 activity promotes a-syn degradation Authors

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.