A brief exposure to rotenone alters microtubule dynamics resulting in aberrant elongation of primary cilia with impaired function

Rotenone, an environmental toxin, is widely used to model Parkinsons disease owing to its well-studied effect to inhibit mitochondrial complex I function and to increase Reactive Oxygen Species (ROS), thereby leading to dopaminergic neuron degeneration. Less appreciated is its impact on microtubules (MT), except for suggesting that it promotes microtubule depolymerization, raising the question of whether rotenone also impacts primary cilia (PC) that are microtubule-based signalling hubs, which are essential for neuronal function. Using hTERT-RPE1 (or RPE1) cells, which assemble PC upon serum starvation, we discovered that brief exposure to rotenone (2-6 h) at low concentration (100 nM) causes a striking elongation of PC and hyperacetylation of cytoskeletal microtubules. At this concentration, rotenone does not significantly alter mitochondrial dynamics or bioenergetics, and these phenotypes could not be reversed by pre-incubating cells with ROS scavenger NAC, or supplementing cells with NR. Thus, these observed effects of rotenone treatment in quiescent cells are predominantly independent of mitochondrial toxicity. Depletion of -TAT1, the -tubulin acetyltransferase, eliminated microtubule acetylation without preventing ciliary elongation, suggesting that rotenone drives PC extension through additional mechanisms. Importantly, rotenone increases soluble tubulin pools, owing to its microtubule destabilizing effect, which are likely the mechanism contributing to ciliary axoneme elongation as well as hyperacetylation of cytoskeletal microtubules. Critically, such aberrant increase in PC length impaired Sonic Hedgehog (SHH) signalling, which is exclusively transduced by PC during embryonic development and is critical for maintaining adult tissue homeostasis. Thus, our findings reveal that even brief, low-dose rotenone exposure induces aberrant elongation of PC in quiescent cells that assemble PC, while simultaneously disrupting the function of PC. Based on our observation and previous studies, we propose that such ciliary dysfunction represents an underappreciated mechanism by which rotenone contributes to the pathogenesis of neurodegenerative disease via affecting neuronal primary cilia.