Toxic concentrations of glutamate induce ferroptosis without affecting mitochondrial respiration in rodent brain slice cultures

Many neuronal disorders are characterized by glutamate toxicity, which is closely associated with mitochondrial and neuronal dysfunction. The aim of this study was to investigate the link between mitochondrial function and mechanisms of cell death induced by glutamate. The study was carried out on male (C57BL/6), male Sprague Dawley acute brain slice (ABSs), male and female organotypic brain slice (BSCs). Mitochondrial respiration, activities of tricarboxylic acid (TCA) cycle enzymes, and the cell death rate (assessed by lactate dehydrogenase (LDH) release and histological analyses) were determined. Memantine was used to inhibit excitotoxicity, while Ferrostatin-1, rosiglitazone and deferoxamine were used as inhibitors of ferroptotic pathway. In astrocytes, pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDHC), showed less than 1% of the enzymatic activity observed in whole brain tissue. In BSCs, glutamate exposure induced a marked increase in oxidative phosphorylation-linked mitochondrial respiration, which was independent of OGDHC activity and disappeared when BSC homogenates were analysed. Glutamate-induced cell death was not alleviated by memantine but was significantly reduced by ferrostatin-1. The activation of ferroptosis by glutamate was also observed in ABSs, as well as the inhibition of lipid peroxidation induced by ferrostatin-1, rosiglitazone and deferoxamine. Ferrostatin-1, and rosiglitazone additionally inhibited cell death, while deferoxamine did not. The unusual response to deferoxamine requires further elucidation. In summary, our data suggest that elevated glutamate levels enhance neuronal energy metabolism in brain slices, compensating glutamate-mediated Ca²⁺ influx and reduce excitotoxicity. In contrast, persistently elevated levels of extracellular glutamate impair the cystine/glutamate antiporter, leading to ferroptosis. Significance Statement Excessive extracellular glutamate is a hallmark of many neurodegenerative and ischemic conditions. This study reveals that toxic glutamate concentrations in BSCs, modelling brain tissue, intensify neuronal energy metabolism without rescuing cells from death, shifting the predominant mechanism from excitotoxicity to ferroptosis. Such phenomenon was not observed in studies on neuronal cell cultures, also including our previous studies. These results suggest that targeting ferroptosis may offer therapeutic potential in neuronal disorders traditionally attributed to excitotoxic damage.