Pyrrolidinophenones (PPs), a class of synthetic cathinones, have emerged as hazardous new psychoactive substances due to their high lipophilicity and potent neurotoxicity. However, the mechanisms underlying PPs-induced neuronal damage, particularly the roles of mitochondrial reactive oxygen species (ROS) and mitophagy, remain unclear. In this study, we investigated the interplay among ROS overproduction, mitochondrial dysfunction, mitophagy, and apoptosis in human neuronal cells exposed to representative PPs. Treatment with PPs induced neuronal cell toxicity in a manner dependent on the elongation of the alkyl chain, with α-pyrrolidinooctanophenone (POP) exhibiting the strongest effects. The treatment also facilitated the production of intracellular and mitochondrial ROS, including superoxide, hydrogen peroxide, and hydroxyl radical. Furthermore, the cytotoxicity was remarkably attenuated by pretreating with antioxidant, N-acetyl-L-cysteine, indicating a critical role of ROS in PPs-induced cytotoxicity. Subcellular fractionation analysis revealed an accumulation of highly lipophilic PPs such as α-pyrrolidinoheptanophenone (PHPP) and POP in mitochondria, and the treatment with PHPP or POP resulted in an increase in Bax/Bcl2 ratio, caspase-9 activation, and mitochondrial lipid peroxidation, presumably due to an activation of mitochondria-dependent apoptotic signaling. Notably, POP induced mitophagy via activation of the PINK1/Parkin pathway. Additionally, pharmacological inhibition of autophagy or mitophagy exacerbated both ROS production and cytotoxicity, suggesting a protective role of mitophagy through the removal of damaged mitochondria. Collectively, these findings demonstrate that mitochondrial accumulation of PPs promotes ROS-dependent apoptosis, while mitophagy functions as an adaptive cytoprotective mechanism. This study provides new insights into mitochondrial quality control in PPs-induced neurotoxicity and highlights mitophagy as a potential therapeutic target.
