Small molecule FTO inhibitor MO-I-500 protects differentiated SH-SY5Y neuronal cells from oxidative stress

Denise Greco^1*Zuzana Čočková¹Debanjan Das²Akash Shivling Mali¹Jiri Novotny¹Mark Jon Olsen³Petr Telensky^1*

• ¹Faculty of Science, Charles University, Prague, Czechia
• ²First Faculty of Medicine, Charles University and General University Hospital., Prague, Czechia
• ³Midwestern University, Glendale, United States

Abstract Oxidative stress is a central driver of brain aging, impairing cellular function and increasing susceptibility to neurodegenerative diseases. Recent studies suggest that the RNA demethylase FTO regulates N6-methyladenosine (m6A) RNA modification, a key pathway in modulating oxidative stress in the brain. However, the precise mechanisms underlying FTO's role remain unclear. This study examines the neuroprotective potential of MO-I-500, a small-molecule FTO inhibitor, against oxidative stress induced by tert-butyl hydroperoxide (TBHP) in neuron-like SH-SY5Y cells differentiated with retinoic acid and BDNF (dSH-SY5Y). dSH-SY5Y cells were treated with MO-I-500 alone for 72 hours or with TBHP alone for 24 hours. Alternatively, cells were pretreated with 1 µM MO-I-500 for 48 hours, followed by co-treatment with MO-I-500 and 25 or 50 µM TBHP for an additional 24 hours, for a total treatment duration of 72 hours. Cellular metabolism was assessed using a Seahorse XF MitoStress assay, and oxidative stress markers, including ROS and superoxide levels, were quantified with DCFDA and MitoSOX probes. ATP content was measured using a bioluminescence assay. FTO inhibition by MO-I-500 induced a metabolic shift toward an energy-efficient state, enhancing cellular resilience to oxidative stress. Pretreatment significantly reduced TBHP-induced oxidative damage, lowering intracellular ROS levels and preserving ATP content. Together with our previous findings demonstrating the protective effects of MO-I-500 in astrocytes and recent studies supporting the importance of astrocyte function in neurodegeneration, these results suggest a dual protective role of MO-I-500 in neurons and astrocytes. This dual action positions MO-I-500 as a promising therapeutic strategy to mitigate oxidative damage and reduce the risk of neurodegenerative diseases, including Alzheimer's disease.