154 | Metabolomic reprogramming and ferroptosis protection by the indole-based compound 20 against Aβ (25–35) in SH-SY5Y cells

Mariapia Vietri¹, Enza Napolitano¹, Carmen Marino¹, Tania Ciaglia¹, Rodolfo Maria Abbate¹, Pietro Campiglia¹, Alessia Bertamino¹, Michele Manfra², Anna Maria D’Ursi¹, Ornella Moltedo¹, Vincenzo Vestuto¹ | ¹Department of Pharmacy, University of Salerno, Fisciano, Salerno, Italy; ²Department of Health Science, University of Basilicata, Potenza, Italy.

Amyloid-β (Aβ) accumulation is a central pathological hallmark of Alzheimer’s disease, triggering a self-perpetuating network of oxidative stress, iron dyshomeostasis, organelle dysfunction, and metabolic failure. Building on our previous work demonstrating the antioxidant and anti-amyloidogenic properties of compound 20 (1), this study provides an integrated and mechanistic characterization of its neuroprotective activity against Aβ (25–35) induced cellular stress in neuronal cells. Using SH-SY5Y neuroblastoma cells, we applied a multi-parametric approach to investigate its protective effects against ferroptosis, combining biochemical assays to detect malondialdehyde and reduced glutathione (GSH), labile iron, lipid peroxidation, mitochondrial membrane potential, and endoplasmic reticulum stress). Furthermore, gene expression profiling of redox, inflammatory, and unfolded protein response markers was investigated to confirm rationalize these data. Finally, untargeted metabolomics to assess global metabolic remodelling was performed. Co-treatment with compound 20 effectively counteracted Aβ-induced toxicity by restoring GSH levels, suppressing iron-driven lipid peroxidation, preserving mitochondrial and ER homeostasis, and normalizing the expression of critical genes such as NRF2, HO-1, iNOS, NF-κB2, IRE1, PERK, CHOP, and GPX1, associated with a ferroptosis-like cascade (2,3). Notably, untargeted metabolomics revealed a profound reversion of the Aβ-driven metabolic shift, with normalization of pathways involved in cysteine and methionine metabolism, energy production, and redox balance, resulting in a global metabolomic profile closely resembling that of control cells. Collectively, these findings demonstrate that compound 20 does not act merely as a scavenger but as a comprehensive metabolic modulator capable of interrupting the Aβ-induced ferroptotic-like cascade and restoring cellular homeostatic integrity, supporting its potential as a multitarget candidate for modulating amyloid-associated neurodegeneration.