The role of mTORC2 in the in vitro neurotoxicity of Parkinsonian mimetics 6-OHDA and MPP+

Abstract

The mechanistic target of rapamycin complex 2 (mTORC2), a key regulator of cellular metabolism, growth, and survival, remains poorly characterized in the context of dopaminergic neurotoxicity. In this study, we investigated the role of mTORC2 signaling in the survival of SH-SY5Y neuroblastoma cells exposed to the Parkinsonian neurotoxins 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA), and examined its interplay with oxidative stress and major stress-responsive signaling pathways. Both neurotoxins induced oxidative stress and mitochondrial damage, accompanied by PINK1 accumulation and culminating in caspase-3 activation, PARP1 cleavage, and apoptotic cell death. These effects were associated with reactive oxygen species (ROS)-dependent phosphorylation of the mTORC2 components Rictor and SIN1, as well as the downstream mTORC2 target Akt (Ser473), indicating activation of mTORC2 signaling in response to neurotoxic insult. RNA interference-mediated depletion of the mTORC2 subunits Rictor, SIN1, or mLST8 reduced Akt phosphorylation and potentiated 6-OHDA-induced cytotoxicity by exacerbating oxidative stress, mitochondrial damage, PINK1 accumulation, and the apoptotic cleavage of caspase-3 and PARP1. In contrast, only Rictor depletion, but not SIN1 or mLST8 knockdown, increased the susceptibility of SH-SY5Y cells to MPP⁺-induced toxicity. Genetic inactivation of mTORC2 reduced basal phosphorylation of the cellular energy sensor AMP-activated protein kinase (AMPK), but did not alter neurotoxin-induced phosphorylation of AMPK or the mitogen-activated protein kinases ERK and JNK. Together, these findings demonstrate a protective role of mTORC2 components against 6-OHDA-induced, and to a lesser extent, MPP⁺-induced, mitochondrial damage and apoptotic cell death.