(P18-008-24) Ferroptosis and Iron Metabolism During Neuroblastoma Differentiation

Objectives: Due to their long lifespans, neurons have altered metabolism and are subjected to age-related changes that can lead to the development of several pathologies, including Alzheimer’s Disease and Related Dementias (ADRD). Accessibility to living brains for studies of neuronal metabolism is limited, so in vitro models are of particular importance. Neuroblastoma cell lines can be differentiated into a mature neuron-like phenotype, which, similarly to the human brain, have increased iron stores. Ferroptosis is an iron-dependent form of programmed cell death that is influenced by biological processes such as iron metabolism and senescence. Ferroptosis has also been implicated in the development of age-related pathologic conditions such as ADRD. Yet, how differentiation and maturation influence neuronal iron metabolism and ferroptosis sensitivity remains unknown.

Methods: Human (SH-SY5Y) and mouse (Neuro-2A) neuroblastoma cell lines were differentiated with all-trans retinoic acid (ATRA) for 72-hr. Differentiated and undifferentiated cells were then treated with increasing doses of the iron chelator, desferrioxamine, the heme iron supplement, hemin, or the ferroptosis-inducing drug RSL3. Differences in cell viability were measured by resazurin blue. Expression of iron-related mRNA and proteins were measured by RT-qPCR and western blotting, respectively.

Results: Differentiation of both cell lines significantly increased resistance to RSL3 treatment, but did not alter their sensitivity to changes in iron availability. Moreover, rescue experiments indicated that differentiated cells do not succumb to ferroptosis. mRNA expression of the cystine importer, SLC7A11 and potent antioxidant, GPX4 was increased with differentiation in both cell lines, while iron-related mRNA and protein expression patterns were remarkably similar.

Conclusions: Differentiation of neuroblastoma cell lines enhances the expression of genes associated with ferroptosis prevention (SLC7A11 and GPX4) and confers ferroptosis resistance. Such findings shed light on how healthy neurons cope with age-related iron accumulation and provide insights for protecting against iron-mediated neurodegeneration.

Funding Sources: This research was funded by the NIH, NIA grant R03 AG07729 and NIGMS grant 5T32GM140953-03.