Mitochondrial dysfunction and glutamate toxicity are associated with neural disorders, including brain trauma. A review of the literature suggests that toxic and transmission actions of neuronal glutamate are spatially and functionally separated. The transmission pathway utilizes synaptic GluN2A receptors, rapidly released pool of glutamate, evoked release of glutamate mediated by Synaptotagmin 1 and the amount of extracellular glutamate regulated by astrocytes. The toxic pathway utilizes extrasynaptic GluN2B receptors and a cytoplasmic pool of glutamate, which results from the spontaneous release of glutamate mediated by Synaptotagmin 7 and the neuronal 2-oxoglutarate dehydrogenase complex (OGDHC), a tricarboxylic acid (TCA) cycle enzyme. Additionally, the inhibition of OGDHC observed upon neuro-inflammation is due to an excessive release of reactive oxygen/nitrogen species by immune cells. The loss of OGDHC inhibits uptake of glutamate by mitochondria, thus facilitating its extracellular accumulation and stimulating toxic glutamate pathway without affecting transmission. High levels of extracellular glutamate lead to dysregulation of intracellular redox homeostasis and cause ferroptosis, excitotoxicity, and mitochondrial dysfunction. The latter affects the transmission pathway demanding high-energy supply and leading to cell death. Mitochondria aggravate glutamate toxicity due to impairments in the TCA cycle and become a victim of glutamate toxicity, which disrupts oxidative phosphorylation. Thus, therapies targeting the TCA cycle in neurological disorders may be more efficient than attempting to preserve mitochondrial oxidative phosphorylation.

Current research suggests that promoting ferroptosis, a non-apoptotic form of cell death, may be an effective therapy for osteosarcoma, while its inhibition could facilitate bone regeneration and prevent osteoporosis. Our objective was to investigate whether the susceptibility to and regulation of ferroptosis differ between undifferentiated (UBC) and differentiated (DBC) human bone marrow stromal cells, as well as human osteosarcoma cells (MG63). Ferroptosis was induced by either inhibiting glutathione peroxidase 4 (GPX4) using RSL3 or blocking all glutathione-dependent enzymes through inhibition of the glutamate/cysteine antiporter with Erastin. Lipid peroxidation was assessed using the fluorescent probe BODIPY™581/591C11, while Ferrostatin-1 was used to inhibit ferroptosis. We demonstrate that neither Erastin nor RSL3 induces ferroptosis in DBC. However, both RSL3 and Erastin induce ferroptosis in UBC, while Erastin predominantly induces ferroptosis in MG63 cells. Our data suggest that ferroptosis induction in undifferentiated hBMSCs is primarily regulated by GPX4, whereas glutathione S-Transferase P1 (GSTP1) plays a key role in controlling ferroptosis in osteosarcoma cells. In conclusion, targeting the key pathways involved in ferroptosis across different bone cell types may improve the efficacy of cancer treatments while minimizing collateral damage and supporting regenerative processes, with minimal impact on cancer therapy.

Limited substrate availability because of the blood–brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.

Current research suggests that promoting ferroptosis, a non-apoptotic form of cell death, may be an effective therapy for osteosarcoma, while its inhibition could facilitate bone regeneration and prevent osteoporosis. Our objective was to investigate whether the susceptibility to and regulation of ferroptosis differ between undifferentiated (UBC) and differentiated (DBC) human bone mar-row stromal cells, as well as human osteosarcoma cells (MG63). Ferroptosis was induced by either inhibiting glutathione peroxidase 4 (GPX4) using RSL3 or blocking all glutathione-dependent enzymes through inhibition of the glutamate/cysteine antiporter with Erastin. Lipid peroxidation was assessed using the fluorescent probe BODIPY™581/591C11, while Ferrostatin-1 was used to inhibit ferroptosis. We demonstrate that neither Erastin nor RSL3 induces ferroptosis in DBC. However, both RSL3 and Erastin induce ferroptosis in UBC, while Erastin predominantly induces ferroptosis in MG63 cells. Our data suggest that ferroptosis induction in undifferentiated hBMSCs is primarily regulated by GPX4, whereas Glutathione S-Transferase P1 (GSTP1) plays a key role in controlling ferroptosis in osteosarcoma cells. In conclusion, targeting the key pathways involved in ferroptosis across different bone cell types may improve the efficacy of cancer treatments while minimizing collateral damage and supporting regenerative processes, with minimal impact on cancer therapy.