Oxidative stress (OS) stimulates autophagy in different cellular systems, but it remains controversial if this rule can be generalized. We have analyzed the effect of chronic OS induced by the parkinsonian toxin paraquat (PQ) on autophagy in astrocytoma cells and primary astrocytes, which represent the first cellular target of neurotoxins in the brain. PQ decreased the basal levels of LC3-II and LC3-positive vesicles, and its colocalization with lysosomal markers, both in the absence and presence of chloroquine. This was paralleled by increased number and size of SQSTM1/p62 aggregates. Downregulation of autophagy was also observed in cells chronically exposed to hydrogen peroxide or nonlethal concentrations of PQ, and it was associated with a reduced astrocyte capability to protect dopaminergic cells from OS in co-cultures. Surprisingly, PQ treatment led to inhibition of MTOR, activation of MAPK8/JNK1 and MAPK1/ERK2-MAPK3/ERK1 and upregulation of BECN1/Beclin 1 expression, all signals typically correlating with induction of autophagy. Reduction of OS by NMDPEF, a specific NQO2 inhibitor, but not by N-acetylcysteine, abrogated the inhibitory effect of PQ and restored autophagic flux. Activation of NQO2 by PQ or menadione and genetic manipulation of its expression confirmed the role of this enzyme in the inhibitory action of PQ on autophagy. PQ did not induce NFE2L2/NRF2, but when it was co-administered with NMDPEF NFE2L2 activity was enhanced in a SQSTM1-independent fashion. Thus, a prolonged OS in astrocytes inhibits LC3 lipidation and impairs autophagosome formation and autophagic flux, in spite of concomitant activation of several pro-autophagic signals. These findings outline an unanticipated neuroprotective role of astrocyte autophagy and identify in NQO2 a novel pharmacological target for its positive modulation.
Keywords: AVs, autophagic vacuoles; Ab, antibody; BNAH, benzyldihydronicotinamide riboside; CA-DCF-DA, 5(6)-carboxy-2′,7′ dichlorofluorescein diacetate; CQ, chloroquine; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FACS, flow cytometry; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; K3, menadione; MAPK, mitogen-activated protein kinase; MFI, mean fluorescence intensity; MPTP, 1-methyl 4-phenyl 1,2,3,6-tetraidro-piridine; MitoSOX, 3,8-phenanthridinediamine, 5-(6′-triphenylphosphoniumhexyl)-5,6 dihydro-6-phenyl; NFE2L2, nuclear factor, erythroid 2-like 2; NMDPEF, N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide]; NQO2; OS, oxidative stress; PBS, phosphate-buffered saline; PQ, paraquat; ROS; ROS, reactive oxygen species; RT, room temperature; SN, substantia nigra; TTBS, Tween-Tris buffered saline; WB, western blotting; astrocytes; macroautophagy; p-, phosphorylated; paraquat; parkinson disease; shRNA, short harpin ribonucleic acid; siRNA, small interfering ribonucleic acid.