Impact of oxytosis on the cross-talk of mTORC with mitochondrial proteins in drug-resistant cancer stem cells.

By delivering the environmental inputs to transport nutrients and growth factors, Mechanistic Target of Rapamycin (mTOR) plays a significant role in the growth and metabolism of eukaryotic cells through the regulation of numerous elementary cellular processes such as autophagy, protein synthesis, via translation of mitochondrial protein transcription factor A mitochondrial, mitochondrial ribosomal proteins, and mitochondrial respiratory complexes I &V that are encoded in the nucleus with the help of translation initiation factor 4E-BP. These mitochondrial proteins are involved in cell signaling to regulate proper cell growth, proliferation, and death which are essential for tumor growth and proliferation. This suggests that tumor cells are dependent on mTORC1 for various metabolic pathways. However, this crucial regulator is activated and regulated by calcium homeostasis. Mounting evidence suggests the role of calcium ions in regulating mitochondrial enzymes and proteins. Hence, disrupting calcium homeostasis leads to calcium-dependent cell death called "Oxytosis" through hampering the expression of various mitochondrial proteins. "Oxytosis" is a novel non-apoptotic cell death characterized by glutamate cytotoxicity and ferritin degradation. The present review focuses on the crosstalk between mTORC1 and mitochondrial proteins in the cancer pathophysiology and the impact of calcium ions on disrupting mTORC1 leading to the induction of "Oxytosis."

Future Perspectives of Oxytosis/Ferroptosis Research in Neurodegeneration Diseases.

The current report briefly summarizes the existing hypotheses and relevant evidence of oxytosis/ferroptosis-mediated cell death and outlines future perspectives of neurodegeneration research. Furthermore, it highlights the potential application of specific markers (e.g., activators, inhibitors, redox modulators, antioxidants, iron chelators) in the study of regulatory mechanisms of oxytosis/ferroptosis. It appears that these markers may be a suitable option for experimental investigations targeting key pathways of oxytosis/ferroptosis, such as the inhibition of the cystine/glutamate antiporter/glutathione/glutathione peroxidase 4 axis, glutamate oxidative toxicity, glutathione depletion, iron dyshomeostasis, iron-mediated lipid peroxidation, and others. From a clinical perspective, an innovative research approach to investigate the oxytosis/ferroptosis pathways in cells of the central nervous system and their relationship to neurodegenerative diseases is desirable. It is necessary to expand the existing knowledge about the molecular mechanisms of neurodegenerative diseases and to provide innovative diagnostic procedures to prevent their progression, as well as to develop effective neuroprotective treatment. The importance of preclinical studies focused predominantly on oxytosis/ferroptosis inhibitors (iron chelators or lipoxygenase inhibitors and lipophilic antioxidants) that could chelate iron or inhibit lipid peroxidation is also discussed. Specifically, this targeted inhibition of neuronal death could represent a potential therapeutic strategy for some neurodegenerative diseases.

Various Forms of Programmed Cell Death Are Concurrently Activated in the Population of Retinal Ganglion Cells after Ischemia and Reperfusion.

Retinal ischemia-reperfusion (IR)-which ultimately results in retinal ganglion cell (RGC) death-is a common cause of visual impairment and blindness worldwide. IR results in various types of programmed cell death (PCD), which are of particular importance since they can be prevented by inhibiting the activity of their corresponding signaling cascades. To study the PCD pathways in ischemic RGCs, we used a mouse model of retinal IR and a variety of approaches including RNA-seq analysis, knockout animals, and animals treated with an iron chelator. In our RNA-seq analysis, we utilized RGCs isolated from retinas 24 h after IR. In ischemic RGCs, we found increased expression of many genes that regulate apoptosis, necroptosis, pyroptosis, oxytosis/ferroptosis, and parthanatos. Our data indicate that genetic ablation of death receptors protects RGCs from IR. We showed that the signaling cascades regulating ferrous iron (Fe2+) metabolism undergo significant changes in ischemic RGCs, leading to retinal damage after IR. This data suggests that the activation of death receptors and increased Fe2+ production in ischemic RGCs promote the simultaneous activation of apoptosis, necroptosis, pyroptosis, oxytosis/ferroptosis, and parthanatos pathways. Thus, a therapy is needed that concurrently regulates the activity of the multiple PCD pathways to reduce RGC death after IR.

Synthesis and Biological Evaluation of Flavonoid-Cinnamic Acid Amide Hybrids with Distinct Activity against Neurodegeneration in Vitro and in Vivo.

Flavonoids are polyphenolic natural products and have shown significant potential as disease-modifying agents against neurodegenerative disorders like Alzheimer's disease (AD), with activities even in vivo. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in several phenotypic screening assays related to neurodegeneration and AD. Therefore, we have exchanged the flavonoid part of the hybrids with different flavonoids, which show higher efficacy than taxifolin or silibinin, to improve the activity of the respective hybrids. Chemical connection between the flavonoid and cinnamic acid was realized by an amide instead of a labile ester bond to improve stability towards hydrolysis. To investigate the influence of a double bond at the C-ring of the flavonoid, the dehydro analogues of the respective hybrids were also synthesized. All compounds obtained show neuroprotection against oxytosis, ferroptosis and ATP-depletion, respectively, in the murine hippocampal cell line HT22. Interestingly, the taxifolin and the quercetin derivatives are the most active compounds, whereby the quercetin derivate shows even more pronounced activity than the taxifolin one in all assays applied. As aimed for, no hydrolysis product was found in cellular uptake experiments after 4 h whereas different metabolites were detected. Furthermore, the quercetin-cinnamic acid amide showed pronounced activity in an in vivo AD mouse model at a remarkably low dose of 0.3 mg/kg.

Cobalt nanoparticles trigger ferroptosis-like cell death (oxytosis) in neuronal cells: Potential implications for neurodegenerative disease.

The neurotoxicity of hard metal-based nanoparticles (NPs) remains poorly understood. Here, we deployed the human neuroblastoma cell line SH-SY5Y differentiated or not into dopaminergic- and cholinergic-like neurons to study the impact of tungsten carbide (WC) NPs, WC NPs sintered with cobalt (Co), or Co NPs versus soluble CoCl2 . Co NPs and Co salt triggered a dose-dependent cytotoxicity with an increase in cytosolic calcium, lipid peroxidation, and depletion of glutathione (GSH). Co NPs and Co salt also suppressed glutathione peroxidase 4 (GPX4) mRNA and protein expression. Co-exposed cells were rescued by N-acetylcysteine (NAC), a precursor of GSH, and partially by liproxstatin-1, an inhibitor of lipid peroxidation. Furthermore, in silico analyses predicted a significant correlation, based on similarities in gene expression profiles, between Co-containing NPs and Parkinson's disease, and changes in the expression of selected genes were validated by RT-PCR. Finally, experiments using primary human dopaminergic neurons demonstrated cytotoxicity and GSH depletion in response to Co NPs and CoCl2 with loss of axonal integrity. Overall, these data point to a marked neurotoxic potential of Co-based but not WC NPs and show that neuronal cell death may occur through a ferroptosis-like mechanism.

Two single nucleotide polymorphisms in IL13 and IL13RA1 from individuals with idiopathic Parkinson's disease increase cellular susceptibility to oxidative stress.

The human genes for interleukin 13 (IL-13) and its receptor alpha 1 (IL-13Rα1) are in chromosomal regions associated with Parkinson's disease (PD). The interaction of IL-13 with its receptor increases the susceptibility of mouse dopaminergic neurons to oxidative stress. We identified two rare single SNPs in IL13 and IL13RA1 and measured their cytotoxic effects. rs148077750 is a missense leucine to proline substitution in IL13. It was found in individuals with early onset PD and no other known monogenic forms of the disease and is significantly linked with PD (Fisher's exact test: p-value = 0.01, odds ratio = 14.2). rs145868092 is a leucine to phenylalanine substitution in IL13RA1 affecting a residue critical for IL-13 binding. Both mutations increased the cytotoxic activity of IL-13 on human SH-SY5Y neurons exposed to sublethal doses of hydrogen peroxide, t-butyl hydroperoxide or RLS3, an inducer of ferroptosis. Our data show that both rs148077750 and rs145868092 conferred a gain-of-function that may increase the risk of developing PD.

Using Plants as a Source of Potential Therapeutics for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia with the numbers expected to increase dramatically as our society ages. There are no treatments to cure, prevent, or slow down the progression of the disease. Age is the single greatest risk factor for AD. However, to date, AD drug discovery efforts have generally not taken this fact into consideration. Multiple changes associated with brain aging, including neuroinflammation and oxidative stress, are important contributors to disease development and progression. Thus, due to the multifactorial nature of AD, the one target strategy to fight the disease needs to be replaced by a more general approach using pleiotropic compounds to deal with the complexity of the disease. In this perspectives piece, our alternative approach to AD drug development based on the biology of aging is described. Starting with plants or plant-derived natural products, we have used a battery of cell-based screening assays that reflect multiple, age-associated toxicity pathways to identify compounds that can target the aspects of aging that contribute to AD pathology. We have found that this combination of assays provides a replicable, cost- and time-effective screening approach that has to date yielded one compound in clinical trials for AD (NCT03838185) and several others that show significant promise.

Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate.

BACKGROUND: Acanthus ebracteatus (AE), an herb native to Asia, has been recognized in traditional folk medicine not only for its antioxidant properties and various pharmacological activities but also as an ingredient of longevity formulas. However, its anti-neurodegenerative potential is not yet clearly known. This work aimed to evaluate the protective effect of AE leaf extract against glutamate-induced oxidative damage in mouse hippocampal HT22 cells, a neurodegenerative model system due to a reduction in glutathione levels and an increase in reactive oxygen species (ROS). METHODS: Cell viability, apoptosis, and ROS assays were performed to assess the protective effect of AE leaf extract against glutamate-induced oxidative toxicity in HT22 cells. The antioxidant capacity of AE was evaluated using in vitro radical scavenging assays. The subcellular localization of apoptosis-inducing factor (AIF) and the mRNA and protein levels of genes associated with the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant system were determined to elucidate the mechanisms underlying the neuroprotective effect of AE leaf extract. RESULTS: We demonstrated that AE leaf extract is capable of attenuating the intracellular ROS generation and HT22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with the extract significantly reduced apoptotic cell death via inhibition of AIF nuclear translocation. The increases in Nrf2 levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with the extract. CONCLUSIONS: The results suggested that AE leaf extract possesses neuroprotective activity against glutamate-induced oxidative injury and may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.

HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification.

Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species.

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl(1)-NH-Aryl(2) scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.

Oxytosis/Ferroptosis in Neurodegeneration: the Underlying Role of Master Regulator Glutathione Peroxidase 4 (GPX4).

Oxytosis/ferroptosis is an iron-dependent oxidative form of cell death triggered by lethal accumulation of phospholipid hydroperoxides (PLOOHs) in membranes. Failure of the intricate PLOOH repair system is a principle cause of ferroptotic cell death. Glutathione peroxidase 4 (GPX4) is distinctly vital for converting PLOOHs in membranes to non-toxic alcohols. As such, GPX4 is known as the master regulator of oxytosis/ferroptosis. Ferroptosis has been implicated in a number of disorders such as neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), etc.), ischemia/reperfusion injury, and kidney degeneration. Reduced function of GPX4 is frequently observed in degenerative disorders. In this study, we examine how diminished GPX4 function may be a critical event in triggering oxytosis/ferroptosis to perpetuate or initiate the neurodegenerative diseases and assess the possible therapeutic importance of oxytosis/ferroptosis in neurodegenerative disorders. These discoveries are important for advancing our understanding of neurodegenerative diseases because oxytosis/ferroptosis may provide a new target to slow the course of the disease.

The Neuroprotective Flavonoids Sterubin and Fisetin Maintain Mitochondrial Health under Oxytotic/Ferroptotic Stress and Improve Bioenergetic Efficiency in HT22 Neuronal Cells.

The global increase in the aging population has led to a rise in many age-related diseases with continuing unmet therapeutic needs. Research into the molecular mechanisms underlying both aging and neurodegeneration has identified promising therapeutic targets, such as the oxytosis/ferroptosis cell death pathway, in which mitochondrial dysfunction plays a critical role. This study focused on sterubin and fisetin, two flavonoids from the natural pharmacopeia previously identified as strong inhibitors of the oxytosis/ferroptosis pathway. Here, we investigated the effects of the compounds on the mitochondrial physiology in HT22 hippocampal nerve cells under oxytotic/ferroptotic stress. We show that the compounds can restore mitochondrial homeostasis at the level of redox regulation, calcium uptake, biogenesis, fusion/fission dynamics, and modulation of respiration, leading to the enhancement of bioenergetic efficiency. However, mitochondria are not required for the neuroprotective effects of sterubin and fisetin, highlighting their diverse homeostatic impacts. Sterubin and fisetin, thus, provide opportunities to expand drug development strategies for anti-oxytotic/ferroptotic agents and offer new perspectives on the intricate interplay between mitochondrial function, cellular stress, and the pathophysiology of aging and age-related neurodegenerative disorders.

Fragment-based drug discovery and biological evaluation of novel cannabinol-based inhibitors of oxytosis/ferroptosis for neurological disorders.

The oxytosis/ferroptosis regulated cell death pathway is an emerging field of research owing to its pathophysiological relevance to a wide range of neurological disorders, including Alzheimer's and Parkinson's diseases and traumatic brain injury. Developing novel neurotherapeutics to inhibit oxytosis/ferroptosis offers exciting opportunities for the treatment of these and other neurological diseases. Previously, we discovered cannabinol (CBN) as a unique, potent inhibitor of oxytosis/ferroptosis by targeting mitochondria and modulating their function in neuronal cells. To further elucidate which key pharmacophores and chemical space are essential to the beneficial effects of CBN, we herein introduce a fragment-based drug discovery strategy in conjunction with cell-based phenotypic screens using oxytosis/ferroptosis to determine the structure-activity relationship of CBN. The resulting information led to the development of four new CBN analogs, CP1-CP4, that not only preserve the sub-micromolar potency of neuroprotection and mitochondria-modulating activities seen with CBN in neuronal cell models but also have better druglike properties. Moreover, compared to CBN, the analog CP1 shows improved in vivo efficacy in the Drosophila model of mild traumatic brain injury. Together these studies identify the key molecular scaffolds of cannabinoids that contribute to neuroprotection against oxytosis/ferroptosis. They also highlight the advantageous approach of combining in vitro cell-based assays and rapid in vivo studies using Drosophila models for evaluating new therapeutic compounds.

Antiferroptotic Activities of Oxindole GIF-0726-r Derivatives: Involvement of Ferrous Iron Coordination and Free-Radical Scavenging Capacities.

Ferroptosis and oxytosis are iron- and oxidative stress-dependent cell death pathways strongly implicated in neurodegenerative diseases, cancers, and metabolic disorders. Therefore, specific inhibitors may have broad clinical applications. We previously reported that 3-[4-(dimethylamino)benzyl]-2-oxindole (GIF-0726-r) and derivatives protected the mouse hippocampal cell line HT22 against oxytosis/ferroptosis by suppressing reactive oxygen species (ROS) accumulation. In this study, we evaluated the biological activities of GIF-0726-r derivatives with modifications at the oxindole skeleton and other positions. The addition of a methyl, nitro, or bromo group to C-5 of the oxindole skeleton enhanced antiferroptotic efficacy on HT22 cells during membrane cystine-glutamate antiporter inhibition and ensued intracellular glutathione depletion. In contrast, the substitution of the dimethylamino group on the side chain phenyl ring with a methyl, nitro, or amine group dramatically suppressed antiferroptotic activity regardless of other modifications. Compounds with antiferroptotic activity also directly scavenged ROS and decreased free ferrous ions in both HT22 cells and cell-free reactions while those compounds without antiferroptotic activity had little effect on either ROS or ferrous-ion concentration. Unlike oxindole compounds, which we have previously reported, the antiferroptotic compounds had little effect on the nuclear factor erythroid-2-related factor 2-antioxidant response element pathway. Oxindole GIF-0726-r derivatives with a 4-(dimethylamino)benzyl moiety at C-3 and some types of bulky group at C-5 (whether electron-donating or electron-withdrawing) can suppress ferroptosis, warranting safety and efficacy evaluations in animal models of disease.

COVID-19 Causes Ferroptosis and Oxidative Stress in Human Endothelial Cells.

Oxidative stress and endothelial dysfunction have been shown to play crucial roles in the pathophysiology of COVID-19 (coronavirus disease 2019). On these grounds, we sought to investigate the impact of COVID-19 on lipid peroxidation and ferroptosis in human endothelial cells. We hypothesized that oxidative stress and lipid peroxidation induced by COVID-19 in endothelial cells could be linked to the disease outcome. Thus, we collected serum from COVID-19 patients on hospital admission, and we incubated these sera with human endothelial cells, comparing the effects on the generation of reactive oxygen species (ROS) and lipid peroxidation between patients who survived and patients who did not survive. We found that the serum from non-survivors significantly increased lipid peroxidation. Moreover, serum from non-survivors markedly regulated the expression levels of the main markers of ferroptosis, including GPX4, SLC7A11, FTH1, and SAT1, a response that was rescued by silencing TNFR1 on endothelial cells. Taken together, our data indicate that serum from patients who did not survive COVID-19 triggers lipid peroxidation in human endothelial cells.

Effect of ferroptosis inhibitors oxindole-curcumin hybrid compound and N,N-dimethylaniline derivatives on rotenone-induced oxidative stress.

Oxidative stress is common to multiple cell death pathways, including apoptosis. We recently identified several compounds that protect against ferroptosis, another cell death pathway associated with oxidative stress, suggesting potential efficacy against apoptosis. The present study assessed the protective efficacies of the ferroptosis inhibitors oxindole-curcumin hybrid compound GIF-2165X-G1, N,N-dimethylaniline derivatives GIF-2014 and GIF-2115, and ferrostatin-1 against rotenone-induced apoptosis. Treatment of mouse hippocampal HT22 cells with the mitochondrial transport chain inhibitor rotenone for 24 h reduced mitochondrial membrane potential, increased reactive oxygen species production, promoted nuclear fragmentation, and ultimately impaired cell viability, consistent with apoptosis. Ferroptosis inhibitor cotreatment did not prevent any of these rotenone-induced apoptotic processes but did suppress delayed cell death associated with loss of plasma membrane integrity. These results suggest that GIF-2165X-G1, GIF-2014, GIF-2115, and ferrostatin-1 are selective for ferroptosis and do not affect apoptosis. Thus, erastin-induced ferroptosis and rotenone-induced apoptosis are distinct cell death pathways despite the common involvement of mitochondrial oxidative stress. Further, the cytoprotective efficacies of chemical antioxidants may depend on the specific source of oxidative stress.

Identification of Novel Oxindole Compounds That Suppress ER Stress-Induced Cell Death as Chemical Chaperones.

Endoplasmic reticulum (ER) stress and oxidative stress lead to protein misfolding, and the resulting accumulation of protein aggregates is often associated with the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion disease. Small molecules preventing these pathogenic processes may be effective interventions for such neurodegenerative disorders. In this paper, we identify several novel oxindole compounds that can prevent ER stress- and oxidative stress-induced cell death. Among them, derivatives of the lead compound GIF-0726-r in which a hydrogen atom at the oxindole ring 5 position is substituted with a methyl (GIF-0852-r), bromine (GIF-0854-r), or nitro (GIF-0856-r) group potently suppressed global ER stress. Furthermore, GIF-0854-r and -0856-r prevented protein aggregate accumulation in vitro and in cultured hippocampal HT22 neuronal cells, indicating that these two compounds function effectively as chemical chaperones. In addition, GIF-0852-r, -0854-r, and -0856-r prevented glutamate-induced oxytosis and erastin-induced ferroptosis. Collectively, these results suggest that the novel oxindole compounds GIF-0854-r and -0856-r may be useful therapeutics against protein-misfolding diseases as well as valuable research tools for studying the molecular mechanisms of ER and oxidative stress.

Structural Requirements for the Neuroprotective and Anti-Inflammatory Activities of the Flavanone Sterubin.

Alzheimer's disease (AD) is the most frequent age-associated disease with no treatments that can prevent, delay, slow, or stop its progression. Thus, new approaches to drug development are needed. One promising approach is the use of phenotypic screening assays that can identify compounds that have therapeutic efficacy in target pathways relevant to aging and cognition, as well as AD pathology. Using this approach, we identified the flavanone sterubin, from Yerba santa (Eriodictyon californicum), as a potential drug candidate for the treatment of AD. Sterubin is highly protective against multiple initiators of cell death that activate distinct death pathways, potently induces the antioxidant transcription factor Nrf2, and has strong anti-inflammatory activity. Moreover, in a short-term model of AD, it was able to prevent decreases in short- and long-term memory. In order to better understand which key chemical functional groups are essential to the beneficial effects of sterubin, we compared the activity of sterubin to that of seven closely related flavonoids in our phenotypic screening assays. Surprisingly, only sterubin showed both potent neuroprotective activity against multiple insults as well as strong anti-inflammatory activity against several distinct inducers of inflammation. These effects correlated directly with the ability of sterubin to strongly induce Nrf2 in both nerve and microglial cells. Together, these results define the structural requirements underlying the neuroprotective and anti-inflammatory effects of sterubin and they provide the basis for future studies on new compounds based on sterubin.

Haloperidol Prevents Oxytosis/Ferroptosis by Targeting Lysosomal Ferrous Ions in a Manner Independent of Dopamine D2 and Sigma-1 Receptors.

Haloperidol is a widely used antipsychotic agent that exerts antipsychotic effects through a strong antagonism of dopamine D2 receptors. In addition, haloperidol is classified as a sigma-1 receptor (S1R) antagonist that prevents endogenous oxidative stress in cultured cells. However, pharmacological activities of haloperidol against oxidative stress remain unclear. Oxytosis/ferroptosis are iron-dependent nonapoptotic oxidative cell deaths that are regarded as two names for the same cell death pathway and the potential physiological relevance of oxytosis/ferroptosis in multiple diseases is suggested. In the present study, the effects of haloperidol on oxytosis/ferroptosis were investigated in S1R-knockdown mouse hippocampal HT22 cells. The results indicate that haloperidol is a strong inhibitor of oxytosis/ferroptosis independent of S1R. Imaging of HT22 cells with a newly developed fluorescent probe showed that haloperidol was localized to late endosomes and lysosomes and reduced the accumulation of lysosomal ferrous ions, resulting in reduced production of intracellular reactive oxygen species and inhibition of cell death. These results indicate that haloperidol is useful not only as an antipsychotic agent but also as a neuroprotective agent against endogenous oxidative stress via distinct mechanisms. Furthermore, lysosome-targeting ferroptosis inhibitors could be useful for the treatment of various diseases, including cancers, ischemia-reperfusion injury, and neurodegenerative disorders, which have been associated with ferroptosis.

Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria independently of cannabinoid receptors.

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.