KA-mediated excitotoxicity induces neuronal ferroptosis through activation of ferritinophagy.

OBJECTIVES: This study aims to elucidate the role of Fe2+ overload in kainic acid (KA)-induced excitotoxicity, investigate the involvement of ferritinophagy selective cargo receptor NCOA4 in the pathogenesis of excitotoxicity. METHODS: Western blotting was used to detect the expression of FTH1, NCOA4, Lamp2, TfR, FPN, and DMT1 after KA stereotaxic injection into the unilateral striatum of mice. Colocalization of Fe2+ with lysosomes in KA-treated primary cortical neurons was observed by using confocal microscopy. Desferrioxamine (DFO) was added to chelate free iron, a CCK8 kit was used to measure cell viability, and the Fe2+ levels were detected by FerroOrange. BODIPY C11 was used to determine intracellular lipid reactive oxygen species (ROS) levels, and the mRNA levels of PTGS2, a biomarker of ferroptosis, were measured by fluorescent quantitative PCR. 3-Methyladenine (3-MA) was employed to inhibit KA-induced activation of autophagy, and changes in ferritinophagy-related protein expression and the indicated biomarkers of ferroptosis were detected. Endogenous NCOA4 was knocked down by lentivirus transfection, and cell viability and intracellular Fe2+ levels were observed after KA treatment. RESULTS: Western blot results showed that the expression of NCOA4, DMT1, and Lamp2 was significantly upregulated, while FTH1 was downregulated, but there were no significant changes in TfR and FPN. The fluorescence results indicated that KA enhanced the colocalization of free Fe2+ with lysosomes in neurons. DFO intervention could effectively rescue cell damage, reduce intracellular lipid peroxidation, and decrease the increased transcript levels of PTGS2 caused by KA. Pretreatment with 3-MA effectively reversed KA-induced ferritinophagy and ferroptosis. Endogenous interference with NCOA4 significantly improved cell viability and reduced intracellular free Fe2+ levels in KA-treated cells. CONCLUSION: KA-induced excitotoxicity activates ferritinophagy, and targeting ferritinophagy effectively inhibits downstream ferroptosis. Interference with NCOA4 effectively attenuates KA-induced neuronal damage. This study provides a potential therapeutic target for excitotoxicity related disease conditions.

Regulation of FSP1 myristoylation by NADPH: A novel mechanism for ferroptosis inhibition.

Excitotoxicity is a prevalent pathological event in neurodegenerative diseases. The involvement of ferroptosis in the pathogenesis of excitotoxicity remains elusive. Transcriptome analysis has revealed that cytoplasmic reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels are associated with susceptibility to ferroptosis-inducing compounds. Here we show that exogenous NADPH, besides being reductant, interacts with N-myristoyltransferase 2 (NMT2) and upregulates the N-myristoylated ferroptosis suppressor protein 1 (FSP1). NADPH increases membrane-localized FSP1 and strengthens resistance to ferroptosis. Arg-291 of NMT2 is critical for the NADPH-NMT2-FSP1 axis-mediated suppression of ferroptosis. This study suggests that NMT2 plays a pivotal role by bridging NADPH levels and neuronal susceptibility to ferroptosis. We propose a mechanism by which the NADPH regulates N-myristoylation, which has important implications for ferroptosis and disease treatment.

TIGAR plays neuroprotective roles in KA-induced excitotoxicity through reducing neuroinflammation and improving mitochondrial function.

Excitotoxicity refers to the ability of excessive extracellular excitatory amino acids to damage neurons via receptor activation. It is a crucial pathogenetic process in neurodegenerative diseases. TP53 is confirmed to be involved in excitotoxicity. It is demonstrated that TP53 induced glycolysis and apoptotic regulator (TIGAR)-regulated metabolic pathway can protect against neuronal injury. However, the role of TIGAR in excitotoxicity and specific mechanisms is still unknown. In this study, an in vivo excitotoxicity model was constructed via stereotypical kainic acid (KA) injection into the striatum of mice. KA reduced TIGAR expression levels, neuroinflammatory responses and mitochondrial dysfunction. TIGAR overexpression could reverse KA-induced neuronal injury by reducing neuroinflammation and improving mitochondrial function, thereby exerting neuroprotective effects. Therefore, this study could provide a potential therapeutic target for neurodegenerative diseases.