Role of ferroptosis iron-dependent cell death in neurodegenerative processes.

Accumulation or overload of bioavailable redox active metals causes protein overexpression-aggregation, mitochondrial dysfunction, nucleus alterations, inflammation-immune responses and high oxidative stress that directly disrupt normal neuronal cell functions. Significantly, a novel form of iron-dependent regulated cell death process termed ferroptosis was recently identified. Misregulated ferroptosis pathways have been speculated to trigger neurodegeneration through programmed cell death that is distinct from classical mechanisms, although the underlying mechanisms remain only partially understood.We induced ferroptosis (erastin, buthioninesulfoximine and sulfasalazine, ammonium iron(III) citrate) on in vitro cultured neurons. Neuronal responses were characterized by bioassays (ROS, GSH, Iron, DPPP), RNA-seq and confocal microscopy.We induced ferroptosis using compounds that target the main ferroptotic pathways and analyzed the biochemical responses. We selected buthionine-sulfoximine targeting GPX4, erastin targeting system Xc- , and iron (III) citrate targeting iron transporters/trafficking . We dosed neurons with 0-200μM to determine IC50, dose- and time-dependent responses. Notably, the ferroptosis inducers caused a time-dependent increase in cytosolic and lipid ROS, as assessed by microplate assay and fluorescent microscopy using fluorescent probes H2 DCFDA and DPPP, respectively. After 24 h, the increase in ROS was accompanied by cell detachment, cell death and change in morphology. Intracellular glutathione pool suffered depletion, whereas labile iron accumulated over the course of the treatment. ROS accumulation and cell death were suppressed by cotreatment with the iron chelator deferoxamine (100 μM). Furthermore, we performed RNA-seq to determine pathways altered by ferroptosis, and morphological characterization by confocal and electron microscopy.Our data show marked alteration in metabolism of iron, glutathione depletion, ROS overproduction, and marked lipid peroxidation in neurons dosed with ferroptosis inducers, confirming susceptibility of neurons to iron-dependent ferroptosis cell death.