ROS Regulate Rotenone-induced SH-SY5Y Dopamine Neuron Death Through Ferroptosis-mediated Autophagy and Apoptosis

Rotenone, a plant-derived natural insecticide, is widely used to induce Parkinson's disease (PD) models. However, the mechanisms of rotenone-induced cell death remain unclear. Here, we found that rotenone (0.01, 0.1, or 1 μmol/L) suppressed SH-SY5Y dopamine neuron viability and led to PD-like pathological changes, such as reduced tyrosine hydroxylase (TH) but increased α-synuclein. Rotenone increased the levels of intracellular Reactive Oxygen Species (ROS) and mitochondrial ROS, as well as the levels of the Antioxidants nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), ultimately resulting in oxidative stress. Moreover, rotenone significantly downregulated the expression of GPX4 and xCT but upregulated the expression of COX2 and NCOA4, which are markers of Ferroptosis. Furthermore, rotenone decreased phosphorylated mTOR level but increased Beclin-1, ATG5, LC3 and p62 expression, suggesting that rotenone enhances Autophagy and reduces Autophagy flux. Additionally, rotenone reduced Bcl-2 levels and the mitochondrial membrane potential (MMP) while promoting Bax and Caspase-3 expression, thus initiating cell Apoptosis. N-acetylcysteine (NAC), a ROS scavenger, and ferrostatin-1 (Fer-1) and deferoxamine (DFO), two Ferroptosis inhibitors, significantly eliminated rotenone-induced Autophagy and Apoptosis. Moreover, ML385, a specific inhibitor of Nrf2, suppressed rotenone-induced Ferroptosis. Our results demonstrated that ROS might mediate rotenone-induced PD-like pathological changes by regulating iron death, Autophagy, and Apoptosis. Inhibiting Ferroptosis blocked the rotenone-induced increase in Autophagy and Apoptosis. Thus, the ability of ROS to regulate rotenone-induced death through Autophagy and Apoptosis is dependent on Ferroptosis. The findings require validation in multiple neuronal cell lines and in vivo.

Apoptosis; Autophagy; Ferroptosis; Oxidative stress; Parkinson’s disease; Rotenone.