NCOA4-Mediated Ferritinophagy Is Essential for HT22 Neuronal Cell Survival During Iron Deficiency (OR15-07-19)

OBJECTIVES: Iron is essential for proper cell function and development. However, mishandled iron may lead to ferroptotic cell death. NCOA4 manages the cellular labile iron pool by controlling the release of ferritin iron via ferritinophagy. The present studies examined the capacity of hippocampal cells in handling iron fluctuation, with particular focus on NCOA4 and ferritin. METHODS: HT22 mouse hippocampal cells were treated with ferric ammonium citrate (FAC) and deferoxamine (Dfo) to produce cellular iron overload and deprivation, respectively. Ferroptosis was determined by measures of ferrostatin-1 effects and Ptgs2 mRNA. For ferritinophagy studies, Ncoa4 was silenced by siRNA transfections. Functional impacts of impaired ferritinophagy were assessed via CCK-8 cell viability assays and western and qPCR analyses of iron-related genes. RESULTS: HT22 cells were highly susceptible to cellular iron overload. FAC-treated cells featured acute morphological changes, decreased viability, and elevated Ptgs2 mRNA abundance. Iron effects were prevented by ferrostatin-1, indicating ferroptosis by cellular iron overload. Dfo alone had minimal impact on cell morphology and viability. NCOA4 protein, but not mRNA, levels were acutely upregulated by Dfo treatments. Ferritin turnover by iron deficiency was impaired in NCOA4-depleted cells, presumably due to impaired ferritinophagy. Moreover, HT22 cells became sensitive to iron deficiency by loss of NCOA4. CONCLUSIONS: Our studies demonstrate iron can induce ferroptosis in neuronal cells. We also identify NCOA4-mediated ferritinophagy as an integral process for neuronal cell survival during iron deficiency. Further investigation using multi-omics approaches are in progress to determine the mechanisms by which NCOA4 depletion leads to cell death when extracellular iron supply is limited. FUNDING SOURCES: Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.