Ferroptosis Inhibition Underlies Oleamide's Neuroprotective and Antiseizure Effects

Ferroptosis Inhibition Underlies Oleamide's Neuroprotective and Antiseizure Effects | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 May 2025 V1 Latest version Share on Ferroptosis Inhibition Underlies Oleamide's Neuroprotective and Antiseizure Effects Authors : Eun Jung NA , Daseul Lee , Nayoung Hwang , and Hwa-Jung Kim 0009-0000-8514-2902 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174784696.68816435/v1 467 views 288 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Oleamide, an endogenous fatty acid amide, had been previously reported to exert various neuromodulatory effects; however, its therapeutic potential in neurological disorders remained unclear. Previously our study demonstrated that oleamide attenuated kainic acid (KA)-induced seizures and excitotoxicity via calpain inhibition. This study aimed to determine whether oleamide exerted neuroprotective and antiseizure effects by inhibiting ferroptosis. Experimental Approach: We investigated the effects of oleamide using HT22 hippocampal neuronal cells and mouse models of epilepsy induced by KA and pentylenetetrazole (PTZ). Mice were administered oleamide either before or after seizure induction to evaluate both prophylactic and therapeutic potential. Molecular and histological analyses were conducted to assess ferroptosis-related markers, oxidative stress levels, and synaptic protein integrity. Key Results: Pre- or post-treatment with oleamide significantly reduced lipid reactive oxygen species (ROS) and restored the expression of key ferroptosis-related proteins, including ACSL4, HO-1, and FTH1. These changes were accompanied by a substantial reduction in seizure severity. Additionally, oleamide preserved synaptic integrity by maintaining the levels of CRMP2, synapsin-1, synaptophysin, and PSD95 in the hippocampus. Moreover, oleamide reversed seizure-induced dysregulation of the mTOR and GSK3β signaling pathways, indicating a multifaceted mechanism of action. Conclusion and Implications: Oleamide exerted both neuroprotective and antiseizure effects in experimental epilepsy models by inhibiting ferroptosis and preserving synaptic function. The results highlighted oleamide as a promising candidate for epilepsy therapy with potential for both prophylactic and therapeutic application. Ferroptosis Inhibition Underlies Oleamide’s Neuroprotective and Antiseizure Effects Eun Jung Na a,1 , Daseul Lee a,1 , Nayoung Hwang a,1 , Hwa-Jung Kim a, * a College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of Korea *Corresponding author: Hwa-Jung Kim, College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea Telephone: 82-2-3277-3021 E-mail: [email protected] (H.-J. Kim) 1 Eun Jung Na, Daseul Lee and Nayoung Hwang contributed equally to this work. ABSTRACT Background and Purpose: Oleamide is an endogenous fatty acid amide with diverse neuromodulatory properties, however, its therapeutic utility in neurological disorders, including epilepsy, remains insufficiently defined. Emerging evidence implicates ferroptosis in the pathogenesis of epilepsy, making it as a compelling therapeutic target. This study aimed to evaluate whether inhibition of ferroptosis contributes to oleamide’s preventive and therapeutic antiseizure effects, as well as its neuroprotective actions. Experimental Approach: The effects of oleamide were investigated in vitro using HT22 hippocampal cells and in vivo using murine models of epilepsy induced by kainic acid and pentylenetetrazole. Oleamide was administered either prior to or following seizure induction. Neuronal viability, oxidative lipid damage, and the expression of ferroptosis-related and synaptic proteins were assessed through molecular analyses. Key Results: Oleamide significantly suppressed ferroptosis-induced neuronal death, and reduced seizure severity in both preventive and post-treatment paradigms. These effects were accompanied by reduced lipid peroxidation and modulation of key ferroptosis markers, including ACSL4, HO-1, and FTH1. Additionally, oleamide preserved synaptic integrity by maintaining levels of CRMP2, synapsin-1, synaptophysin, and PSD95. Notably, oleamide also mitigated seizure-induced alterations in mTOR and GSK3β signaling pathways. Conclusion and Implications: Oleamide conferred significant neuroprotective and antiseizure effects in experimental epilepsy models, primarily via inhibition of ferroptosis and preservation of synaptic integrity. Its ability to restore dysregulated mTOR and GSK3β signaling further supports a multifactorial mechanism of action. These findings position oleamide as a promising candidate for the development of novel therapeutic interventions for epilepsy, with potential applications in both preventive and treatment contexts. Keywords : oleamide; ferroptosis; kainic acid; pentylenetetrazol; mTOR; synaptic proteins Abbreviations: ACSL4, acyl-CoA synthetase long-chain family member 4; FSP1, ferroptosis suppressor protein 1; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; GSK3β, glycogen synthase kinase 3β; HO-1, heme oxygenase-1; KA, kainic acid; mTOR, mammalian target of rapamycin; PTZ, pentylenetetrazole; PSD95, postsynaptic density protein 95; S6, ribosomal protein S6 kinase; 4-HNE, 4-hydroxynonenal What was already known? • Oleamide had previously been shown to exert various neuromodulatory and sleep-inducing effects in the central nervous system. • Prior studies had demonstrated oleamide’s ability to reduce KA-induced seizures and excitotoxicity via calpain inhibition, but its broader therapeutic mechanisms remained unclear. What did this study add? • The first comprehensive evidence that oleamide exerted both neuroprotective and antiseizure effects by inhibiting ferroptosis in vitro and in vivo. • Oleamide preserved synaptic integrity and normalized mTOR/GSK3β signaling, highlighting its multifaceted mechanism in seizure control. What is the clinical significance? • Oleamide could serve as a dual-function agent for epilepsy treatment by both preventing and reversing seizure-induced neuronal damage. • Given its efficacy in models of drug-resistant epilepsy and its action on ferroptosis and synaptic preservation, oleamide emerged as a promising candidate for novel antiepileptic therapy development. 1 INTRODUCTION Oleamide (cis-9,10-octadecenoamide) was first identified as a sleep-inducing signaling molecule in the brain (Cravatt et al., 1995) and is a prototype of fatty acid primary amide (FAA) lipid messengers abundant in the central nervous system (CNS) (Hiley and Hoi, 2007). Although oleamide has been recognized for its various CNS effects (Leggett et al., 2004), its therapeutic potential and role in neurological diseases remain unclear. Endocannabinoids represent a well-known FAA lipid transmitter system in the brain, and their activation through the inhibition of metabolism by FAA hydrolase (FAAH) has been suggested as a potential antiepileptic mechanism (Karanian et al., 2007). Because oleamide is a known substrate of FAAH (Hiley and Hoi, 2007), some studies have investigated its potential antiepileptic effects. We reported that oral oleamide administration significantly blocked behavioral seizure activity in a rat epilepsy model induced by kainic acid (KA). Furthermore, oleamide exhibited neuroprotective effects against KA-induced excitotoxic damage in striatal brain tissue through calpain inhibition (Nam et al., 2017). Another study reported that intraperitoneal (i.p.) administration of oleamide delayed seizure onset in mice with pentylenetetrazole (PTZ)-induced epilepsy (Wu et al., 2003). Furthermore, several studies have explored the mechanisms underlying the neuroprotective effects of oleamide. Oleamide was shown to prevent apoptotic cell death by attenuating caspase-3 activity induced by K + deprivation in cultured rat cerebellar granule neurons (Yang et al., 2002). Furthermore, it exerted antioxidant activity by preventing lipid peroxidation and increasing the reduced-to-oxidized glutathione ratio in rat cortical slices (Reyes-Soto et al., 2022). However, the additional mechanisms underlying oleamide’s neuroprotective and antiepileptic actions and its potential as a drug candidate for both the prevention and treatment of epilepsy remain unclear. Epilepsy is a prevalent neurological disorder worldwide, with incidence rates increasing because of increased life expectancy and the growing occurrence of severe head trauma, stroke, and brain tumors (Falco-Walter, 2020). The most concern is that approximately one-third of epilepsy cases are drug-resistant (Perucca et al., 2023). Although the precise mechanisms underlying epileptic pathogenesis are not fully understood, recent studies have suggested that ferroptosis contributes to the pathogenesis of epilepsy, making ferroptosis a promising therapeutic target (Jin et al., 2023). Several contributing factors have been identified, including an inadequate antioxidant defense system in the mammalian brain (Jin et al. , 2023), excessive iron accumulation (Zecca et al., 2004), overproduction of reactive oxygen species (ROS), and elevated levels of polyunsaturated fatty acids (PUFAs) in neuronal membranes (Halliwell, 2006), all of which exacerbate oxidative damage. In epilepsy, where recurrent seizures occur, these oxidative stress-related factors may contribute to progressive neuronal loss. Ferroptosis is a form of regulated cell death caused by excessive lipid-derived ROS accumulation resulting from iron overload (Dixon et al., 2012). Growing evidence suggests that ferroptosis is implicated in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s disease (Wang et al., 2023), and epilepsy. Ferroptosis can occur via at least three major pathways. Disruptions in iron metabolism can cause abnormal accumulation of labile iron in neurons, which may trigger ferroptosis (Ru et al., 2024). Heme oxygenase 1 (HO-1) is a key marker of such disruptions; HO-1 is usually upregulated during ferroptosis, reflecting an increase in intracellular labile iron that promotes iron-dependent lipid peroxidation (Kwon et al., 2015). Furthermore, the elevated levels of ferritin heavy chain 1 (FTH1) reflects the burden on cellular iron homeostasis during ferroptosis (Feng et al., 2024). One key regulator is glutathione peroxidase 4 (GPX4), which prevents ferroptosis by converting lipid hydroperoxides into nontoxic lipid alcohols (Seibt et al., 2019). In addition to GPX4-dependent mechanisms, GPX4-independent antioxidant systems contribute to ferroptosis. For example, ferroptosis suppressor protein 1 (FSP1) inhibits lipid radicals through a GPX4-independent pathway; therefore, its reduction can increase the susceptibility to ferroptosis (Doll et al., 2019). In the ferroptosis pathway, lipid peroxidation upregulates acyl-CoA synthetase long-chain family 4 (ACSL4), leading to increased incorporation of PUFAs into membrane phospholipids. This, in turn, enhances lipid peroxidation at the cell membrane and accelerates ferroptosis progression (Doll et al., 2017). In this study, we propose that oleamide possesses both preventive and therapeutic effects against epilepsy and exerts neuroprotective effects by inhibiting ferroptosis as its novel intracellular mechanism. We demonstrated the anti-ferroptotic effects of oleamide in both in vitro neuronal systems and in vivo epileptic mouse models induced by KA or PTZ. 2 MATERIALS AND METHODS 2.1 Chemicals and reagents Oleamide, RAS-selective lethal 3 (RSL3), arachidonate, glutamate, Fe 2 (SO 4 ) 3 (iron(III)), iron(II) sulfate heptahydrate (FeSO 4 ) , 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt (ferrozine), deferoxamine, ethylene-diamine-tetraacetic acid (EDTA), valproic acid sodium salt (valproate), PTZ, C 11 -BODIPY 581/591 , and H 2 DCFDA were purchased from Sigma-Aldrich (USA). KA was purchased from MedChemExpress (USA). Antibodies against p-GSK3β, p-mTOR, mTOR, CRMP2, p-S6, S6, HO-1, FTH, and β-actin were purchased from Cell Signaling Technology (USA), PSD-95 from Santa Cruz (USA), AIFM2/FSP1 from Proteintech (USA), GPX4 from Abcam (USA), and ACSL4 from ABclonal Technology (USA). Quanti-Max™ WST-8 Cell Viability Assay Kit and Protein Quantification Assay Kit were purchased from Biomax (Korea). 2.2 HT22 mouse hippocampal cell culture HT22 cells were cultured in Dulbecco’s Modified Eagle’s medium containing 10% fetal bovine serum, 100-U/mL penicillin, and 100-mg/mL streptomycin. All cells were maintained at 37°C in a humidified incubator under 5% CO₂ and split every 2–3 day using trypsin/EDTA solution. 2.3 Cell viability assay HT22 cells were seeded in 96-well plates and pretreated with oleamide (1–30 μM) for 30 min, followed by incubation for 24 h with 50 nM RSL3 or a combination of 5 mM glutamate and 100 μM iron(III). After 24 h of compound treatment, the WST-8 solution was added to each well, and the mixture was incubated for another hour. The formazan concentration was determined spectrophotometrically using a Tecan Infinite 200 PRO (Switzerland) at 450 nm at the Ewha Drug Development Research Core Center. 2.4 Detection of ROS using flow cytometry with H 2 DCFDA and C 11 -BODIPY 581/591 HT22 cells were pretreated with oleamide (5–30 μM) for 30 min and then stimulated with 200 nM RSL3 or a combination of 10 mM glutamate and 100 μM iron(III). After 24 h of incubation, the cells were fixed with 1% paraformaldehyde for 10 min. To detect total and lipid ROS, the cells were stained with 10 μM H 2 DCFDA and 1.5 μM C 11 -BODIPY 581/591 , respectively, for 30 min in the dark. The cells were then resuspended in phosphate-buffered saline (pH 7.4), and the fluorescence from DCFDA or C 11 -BODIPY 581/591 was measured using an ACEA 2060R flow cytometer (Novocyte Acea Biosciences, USA) at the Ewha Drug Development Research Core Center. Fluorescence data were collected from 10,000 events in each sample. 2.5 Iron-binding assay Ferrous iron binding was measured using the ferrozine method, as described by Soriano-Castell et al (Soriano-Castell et al., 2021). In brief, oleamide at the desired concentration was mixed with 5 μM FeSO₄ in 100 μL of 50 mM HEPES (pH 7.5) in a 96-well plate. After 2 min, 50 μL of 2.5 mM ferrozine was added, and the optical absorbance at 562 nm was measured using an Infinite M200 Pro microplate reader at the Ewha Drug Development Research Core Center. Each sample was analyzed in duplicate in each independent experiment. The iron-binding capacity is presented as vehicle controls with or without iron. 2.6 Animal study design ICR mice (5-weeks old) were purchased from the Orient Bio Department (Korea). Food and water were provided ad libitum under a 12 h/12 h light–dark cycle. All animal experiments were performed according to the ethical guidelines and were approved by the Institutional Animal Care and Use Committee of Ewha Woman’s University (approval no. Ewha-IACUC 2013-01-041& 2021-066). To test whether oleamide exhibits both preventive and therapeutic effects against seizures, animal experiments were performed with four protocols on the seizure behaviors induced by KA (Figure 3a, b) or PTZ (Figure 3e, f). The oleamide doses used in this study were determined based on our previous study (Nam et al. , 2017). The mice were randomized into four groups according to each experimental protocol. For the study on the oleamide’s preventive effects against KA- or PTZ-induced seizures, control groups (n = 6) were orally administered with vehicle (0.2% methylcellulose in saline) once daily for 5 days before the intraperitoneal injection of saline; KA (n = 11)/PTZ (n = 11) groups were orally administered with vehicle once daily for 5 days before the intraperitoneal injection of KA (40 mg/kg) or PTZ (70 mg/kg); oleamide + KA (n = 11)/PTZ (n = 11) groups were orally administered with oleamide (2 mg/kg) once daily for 5 days before KA or PTZ injection; valproate + KA (n = 5)/PTZ (n = 5) groups were the intraperitoneal injection of valproate (30 mg/kg) once daily for 5 days before KA or PTZ injection. For the study on the oleamide’s therapeutic effects against KA- or PTZ-induced seizures, the control groups (n = 6) were orally administered with vehicle once daily for 3 days or once 24 h after saline injection; the KA (n = 10)/PTZ (n = 10) groups were orally administered with vehicle once daily for 3 days after KA (40 mg/kg) injection or once 24 h after PTZ (70 mg/kg) injection; the oleamide + KA (n = 10)/PTZ (n = 10) groups were orally administered with oleamide (10 mg/kg) once daily for 3 days after KA injection or once 24 h after PTZ injection; the valproate + KA (n = 5)/PTZ (n = 5) groups were orally administered with valproate (30 mg/kg) (10 mg/kg) once daily for 3 days after KA injection or once 24 h after PTZ injection. After KA or PTZ injection, the mice were monitored for behavioral seizures and euthanized for tissue collection at the time points indicated in each protocol scheme (Figure 3a, b, e, f). 2.7 Monitoring behavioral seizures induced by intraperitoneal injection of KA or PTZ Behavioral seizures were continuously monitored and scored every 10 min by two observers (including one blinded observer). In the pretreatment experiment, monitoring lasted 120 min, starting 30 min after KA or PTZ injection. In the posttreatment experiment, monitoring lasted 180 min: 60 min before and 120 min after the administration of vehicle or test drugs on experimental day 1. Seizure activity was assessed using a modified version of the Racine scale (Racine, 1972; Sanchez-Hernandez et al., 2022), assigning the following scores: Score 0 = no behavioral change; Score 1 = facial muscle clonus; Score 2 = head nodding; Score 3 = forelimb clonus; Score 4 = rearing, falling with forelimb clonus; Score 5 = generalized tonic–clonic seizure. 2.8 Sample preparation and western blotting Cells were seeded in 6-well plates overnight, followed by treatment with oleamide for 30 min and cotreatment with RSL3 or a combination of glutamate and iron(III) for 24 h. For the preparation of samples of FSP1 and HO-1 along with arachidonate and iron(III), cells were incubated for 12 h after treatment. The mice were euthanized 1 or 3 days after KA or PTZ injection. Hippocampus tissue was collected and lysed using cold lysis RIPA buffer supplemented with 1% protease inhibitor and then kept on ice for 20 min. The lysates were centrifuged at 13,000 × g for 20 min at 4°C. The protein concentrations of the supernatants were determined using a protein quantification assay. Equal amounts of the sample proteins were fractionated using SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Millipore Corporation, USA). The membranes were blocked using 5% nonfat dry milk in TBS buffer with 0.1% Tween 20 (TBST). Incubation with primary antibodies was performed overnight at 4°C in TBST containing 5% bovine serum albumin. Membrane blots were incubated with horseradish peroxide-conjugated secondary antibody for 2 h at room temperature. Membranes were developed by Chemidoc MP (Bio-rad, USA) using the chemiluminescence method, and bands were analyzed using ImageJ. 2.9 Statistical analysis All data are presented as means ± standard errors of the mean. The statistical significance of the differences between the two groups was determined by the two-tailed unpaired t -test and one-way analysis of variance (ANOVA), followed by Tukey’s Honest Significant Difference test with ranks for multiple-group comparisons or two-way ANOVA with Bonferroni post hoc analysis. Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, USA). P- values <0.05 were used to denote statistical significance. 3 RESULTS 3.1 Protective effects of oleamide against ferroptotic neuronal death and lipid ROS formation in HT22 cells Ferroptosis can be triggered by various mechanisms, including RSL3-mediated inhibition of GPX4, glutamate-induced suppression of the Xc-transporter, and increased labile iron levels, which enhance the Fenton reaction (Du and Guo, 2022). To determine whether oleamide protects neuronal cells against ferroptotic death, we assessed oleamide’s effects on HT22 hippocampal cells. Oleamide alone, at concentrations up to 100 μM, did not affect cell viability (Figure 1a), confirming that the tested concentration range was nontoxic. However, under ferroptosis conditions induced by either RSL3 or a combination of glutamate and iron(III), oleamide significantly improved cell viability at concentrations of ≥10 μM (Figure 1b, c). These findings suggest that oleamide exerts a protective effect against ferroptosis in hippocampal cells. Lipid peroxidation and lipid-derived ROS accumulation are the key features of ferroptosis. We next investigated oleamide’s ability to reduce oxidative stress caused by ferroptosis inducers. Oleamide significantly decreased RSL3-induced oxidative stress, as shown by the lower levels of intracellular total ROS (Figure 1d) and lipid ROS (Figure 1e), with notable effects observed at concentrations of ≥5 μM. Similarly, oleamide dose-dependently lowered the lipid ROS levels under ferroptotic conditions induced by glutamate and iron(III) (Figure 1f). These results indicate that oleamide effectively reduces lipid ROS, thereby contributing to its neuroprotective and anti-ferroptotic effects. 3.2 Involvement of lipid and iron metabolisms in the anti-ferroptotic effects of oleamide in HT22 cells The following primary factors increase the susceptibility to ferroptosis: GPX4 inhibition, PUFA oxidation, and excessive labile iron accumulation (Jin et al. , 2023; Yang and Stockwell, 2016). To investigate the mechanisms underlying oleamide’s anti-ferroptotic effect, GPX4 involvement was examined. Under ferroptotic conditions induced by either RSL3 or a combination of glutamate and iron(III), GPX4 protein levels were significantly reduced, whereas oleamide treatment did not restore these levels (Figure 2a, b). As shown in Figure 2c, FSP1 levels decreased under ferroptotic conditions induced by a combination of arachidonate and iron(III), and treatment with 20-μM oleamide failed to restore FSP1 levels. These results indicate that neither GPX4 nor FSP1 is involved in oleamide’s anti-ferroptotic action under these in vitro experimental conditions. When ferroptosis was induced by a combination of glutamate and iron(III) or arachidonate and iron(III), ACSL4 levels increased; however, pretreatment with 30 μM oleamide significantly reduced ACSL4 levels compared with the control (Figure 2d,e). This result suggests that oleamide interferes with the lipid metabolism pathway of ferroptosis. HO-1 is a key marker reflecting disruptions in iron metabolism. We observed that HO-1 levels, which increased under ferroptotic conditions induced by RSL3, a combination of glutamate and iron(III), or arachidonate and iron(III), were decreased by oleamide at the tested concentrations (Figure 2f, g). This finding suggests that oleamide’s anti-ferroptotic effect involves the modulation of iron metabolism. Furthermore, considering that oleamide reduced the elevated FTH1 levels induced by RSL3 or a combination of glutamate and iron(III), oleamide appears to alleviate the burden on cellular iron homeostasis (Figure 2h, i). However, a direct iron-chelation assay (Figure 2j) demonstrated that oleamide did not possess iron-chelating properties, indicating that oleamide’s anti-ferroptotic effect is not due to iron chelation. 3.3 Preventive and therapeutic effects of oleamide against KA- and PTZ-induced behavioral seizures in mice In mice, KA induces epileptic seizures that resemble human temporal lobe epilepsy (Ben-Ari, 1985), whereas PTZ triggers various seizure types, including generalized tonic–clonic, myoclonic, and absence seizures (Monteiro et al., 2024). Our previous study showed that pretreatment with oleamide prevented behavioral seizures induced by intra-striatal injection of KA in rats (Nam et al. , 2017). In this study, the preventive and therapeutic effects of oleamide in mice were evaluated; seizures were induced by intraperitoneal injection of either KA or PTZ. Valproate, a widely used broad-spectrum antiepileptic drug, served as a positive control (van der Meer et al., 2021). Figure 3 outlines four in vivo experimental protocols detailing the timelines for seizure induction (using KA or PTZ), pre- or post-treatment with test drugs, seizure monitoring, and tissue collection (Figure 3a, b, e, f) and shows the corresponding time course of behavioral seizure monitoring (Figure 3c, d, g, h). In the KA-induced seizure model, vehicle, oleamide (2 mg/kg), or valproate (30 mg/kg) was administered once daily for 5 consecutive days before KA injection. The average KA-induced seizure score was 4.1 ± 0.4. Pretreatment with oleamide significantly reduced the seizure score to 1.2 ± 0.4 30 min after seizure induction—a greater reduction than that observed with valproate (2.3 ± 0.3), with low scores maintained throughout the monitoring period. Valproate further reduced the score to 1.0 ± 1.0 at 50 min, with consistently low scores thereafter (Figure 3c). In posttreatment experiments, high seizure severity was observed before drug administration. Seizure monitoring began 60 min before and continued for 120 min after the first drug administration. Following treatment with either oleamide (10 mg/kg) or valproate (30 mg/kg), the KA-induced seizure score began to significantly decrease from 20 min onward, with maximal inhibition achieved at 80–90 min after treatment (oleamide: 1.46 ± 0.51 at 80 min; valproate: 0.2 ± 0.2 at 90 min) (Figure 3d). In the PTZ-induced seizure model, the initial PTZ-induced seizure scores reached >3. In the pretreatment experiments, pretreatment with oleamide significantly reduced the score to a lower value at 30 min, reaching 0 by 80 min after PTZ injection. Valproate completely prevented seizures throughout the monitoring period. In the posttreatment experiments, the severity of PTZ-induced seizures was significantly reduced 20 min after oleamide administration, with complete seizure blockade observed at 60 min onward. Similarly, posttreatment with valproate completely blocked seizures 50 min after administration. These results indicate that oleamide exerts preventive and therapeutic anticonvulsive potential in KA- and PTZ-induced epileptic seizure models. Notably, oleamide demonstrated significant antiseizure effects at lower concentrations than valproate under both pre- and posttreatment conditions. 3.4 Ferroptosis inhibition by pre- and post-treatment with oleamide in the hippocampus of mice with KA-induced epilepsy Recent studies have implicated that disrupted lipid metabolism and iron overload can trigger oxidative stress and contribute to epileptogenesis and chronic seizure activity (Chen et al., 2020; Wang et al. , 2023). To confirm the anti-ferroptotic effects of oleamide under in vivo conditions that mimic epilepsy, ferroptosis-related markers were analyzed in hippocampal tissues from mice with KA-induced epilepsy after behavioral testing. We first measured the expression levels of GPX4 and ACSL4. In the hippocampus of mice with KA-induced epilepsy, GPX4 expression was markedly reduced, whereas ACSL4 expression was significantly increased, indicating severe ferroptotic stress under this epileptic condition. These KA-induced changes in GPX4 (Figure 4a, b, c) and ACSL4 (Figure 4a, b, d) were reversed in mice that received either a 5-day pretreatment or a 3-day posttreatment with oleamide. Oleamide’s inhibitory effect on lipid peroxidation inducing the reduction of lipid-ROS was confirmed through 4-HNE analysis. 4-HNE is a major byproduct of lipid peroxidation and can induce autophagy, ultimately promoting ferroptosis (Liu et al., 2020). The 4-HNE level increased by KA was significantly decreased by pre- and post-treatment with oleamide (Figure 4a, b, e). These findings indicate that oleamide counteracts oxidative stress and ferroptotic damage caused by lipid metabolism disruptions. Next, we assessed whether oleamide’s antiepileptic effects also involve the normalization of iron metabolism, a process known to contribute to epileptogenesis. HO-1 was significantly elevated, and FTH1 expression was significantly decreased in the hippocampus of mice with KA-induced epilepsy, indicating disruptions in iron metabolism. These changes in HO-1 (Figure 4a, b, f) and FTH1 (Figure 4a, b, g) were fully restored by pre- and post-treatment with oleamide. Collectively, these results indicate that oleamide modulates lipid and iron metabolism as part of its anti-ferroptotic mechanism, consistent with our in vitro observations. To further assess oleamide’s effect on synaptic activity, the markers essential for synaptic integrity, including CRMP2, PSD95, synaptophysin, and synapsin-1, were evaluated. In KA-induced seizure brains, marked reductions in synaptophysin, synapsin-1, CRMP2, and PSD95 were observed; these markers were downregulated, indicating a decline in the overall synaptic activity. Notably, oleamide administration restored all of these synaptic markers, protecting against KA-induced synaptic deterioration under pre- and post-treatment conditions (Figure 4a, b, h, j), which is consistent with the behavioral improvements observed in Figure 3. These findings support the neuroprotective role of oleamide in maintaining synaptic function. 3.5 Ferroptosis inhibition by pre- and post-treatment with oleamide in the hippocampus of mice with PTZ-induced epilepsy We further confirmed oleamide’s anti-ferroptotic effects in another epilepsy model. Figure 5a and 5b present representative western blots of ferroptosis-related markers in the hippocampus of mice with PTZ-induced epilepsy, following behavioral monitoring. The trends observed in mice with PTZ-induced epilepsy were consistent with those in mice with KA-induced epilepsy, with pre- and post-treatment with oleamide providing protection against ferroptotic and synaptic damage. Ferroptosis-related marker proteins, including ACSL4, 4-HNE, HO-1, and FTH1, were significantly altered by PTZ; however, oleamide reversed these changes under both pre- and post-treatment conditions (Figure 5a–f), similar to the observations in mice with KA-induced epilepsy (Figure 4). These findings further support oleamide’s role in mitigating ferroptosis-associated damage. Consistent with the findings observed in mice with KA-induced epilepsy, significant reductions in presynaptic (synaptophysin, synapsin-1 and CRMP2) and postsynaptic (PSD95) marker protein levels induced by PTZ were also reversed by pre- and post-treatment with oleamide, further supporting its neuroprotective effects on synaptic integrity (Figure 5a, b, g-i). We also investigated whether the mTOR signaling pathway, which is known to play a significant role in epileptogenesis (Hurtado Silva et al., 2024) and ferroptosis (Zhang et al., 2021), is correlated with oleamide’s antiepileptic and anti-ferroptotic effects in the PTZ-induced epilepsy model. Activation of the mTOR pathway increased the levels of phosphorylated signaling molecules, such as p-GSK3β, p-mTOR, and p-S6 (Figure 5a, b, j-l). PTZ significantly increased the levels of these phosphorylated molecules, indicating mTOR activation. However, pre- and post-treatment with oleamide significantly mitigated these increases, suggesting its potential role in modulating mTOR signaling in epilepsy prevention and treatment. These findings further highlight oleamide’s dual role in preventing and reversing ferroptotic and synaptic damage, thereby reinforcing its potential as a therapeutic agent for epilepsy. 4 DISCUSSION In this study, we demonstrated that oleamide exerts significant neuroprotective and antiepileptic effects by inhibiting ferroptosis. This was evidenced by the restoration of ferroptosis-related damage markers in both in vitro hippocampal cells and the in vivo hippocampus and the marked reduction in seizure severity in mice with KA- and PTZ-induced epilepsy. In individuals with epilepsy, the hippocampus is particularly vulnerable because hippocampal neurons are highly susceptible to aberrant discharges that contribute to seizure activity (Navidhamidi et al., 2017; Ye et al., 2024). Considering the limited research on the anti-ferroptotic properties of oleamide within the neuronal system, its protective effects against ferroptosis induced by various inducers were examined in hippocampal HT22 cells. To provide a comprehensive assessment of ferroptotic marker alterations, three ferroptosis-inducing agents were employed. RSL3 was used for its direct inhibition of GPX4, a key enzyme that regulates the primary ferroptotic pathway. A combination of arachidonate and iron(III) was selected to disrupt lipid and iron metabolism, as indicated by a significant increase in ACSL4 expression (Bouchaoui et al., 2023). Furthermore, glutamate combined with iron(III) was used to simultaneously inhibit the Xc⁻ transporter and perturb iron homeostasis (Fang et al., 2021). We found that oleamide significantly reduced lipid ROS and normalized the expression of key ferroptosis-related proteins involved in lipid and iron metabolism, including ACSL4, HO-1, and FTH1. These findings build upon those of previous studies by demonstrating that oleamide inhibits multiple ferroptotic pathways, along with its previously reported protective effects against KA-induced neuronal death in the rat striatum (Nam et al. , 2017) and glutathione imbalance due to mitochondrial dysfunction in rat cortical neurons (Reyes-Soto et al. , 2022). Considering ferroptosis as a key pathogenic mechanism in epileptogenesis, we focused on in vivo seizure models. Accumulating evidence suggests that targeting ferroptosis provides therapeutic benefits for seizure control, although the underlying regulatory mechanisms remain unclear (Jin et al. , 2023; Song et al., 2024). To investigate this, two animal models of seizures were employed: KA- and PTZ-induced seizures, which mimic different types of clinical epileptic seizures (Ben-Ari, 1985; Monteiro et al. , 2024). In our experiments, systemic administration of KA or PTZ at the indicated concentrations rapidly induced seizures, which persisted at high severity for at least 3 h before subsiding. Hippocampal tissues harvested 3 days after KA administration or 24 h after PTZ administration exhibited notable changes in ferroptotic markers (i.e., ACSL4, HO-1, and FTH1), mirroring the alterations observed in HT22 hippocampal cells exposed to canonical ferroptosis inducers. To contextualize these findings, seven publicly available datasets from the Gene Expression Omnibus were analyzed. Five datasets derived from mouse models of seizures induced by electric shock, KA, or pilocarpine (GSE60772, GSE73878, GSE88992, GSE93732, GSE100202, GSE197015, and GSE224494) exhibited no significant changes in the expression of ACSL4, HO-1, or FTH1 at the gene level, possibly because of differences in the experimental conditions. In contrast, two datasets derived from patients with drug-resistant epilepsy revealed gene-level upregulation of ACSL4 and FTH1 and downregulation of GPX4 (GSE190451 and GSE134697), with additional decreases in synaptophysin, synapsin-1 and CRMP2 transcripts in GSE134697 (Supplementary Figure S1), which are consistent with our protein-level data. Notably, treatment with oleamide at valproate concentrations reversed these molecular dysregulations and elicited dramatic antiseizure effects, suggesting its potential clinical relevance in refractory epilepsy. Our findings strongly support the dual preventive and therapeutic potential of oleamide across different seizure models. Oleamide effectively normalized dysregulated lipid and iron metabolism, as evidenced by the educed 4-HNE levels and downregulation of ferroptosis markers, such as ACSL4, HO-1, and FTH1. Interestingly, similar effects have been observed with quercetin, which suppresses seizures by reducing 4-HNE and inhibiting ferroptosis (Xie et al., 2022). Although a single pretreatment with oleamide in vitro provided neuroprotection without restoring GPX4 levels, multiple in vivo pre- or post-treatments led to full recovery of GPX4 and restoration of synaptic strength in mice with KA-induced epilepsy. This suggests that oleamide’s anti-ferroptotic and antiepileptic properties do not solely depend on direct GPX4 activation but rather on the sustained modulation of lipid and iron metabolic pathways. The therapeutic potential of oleamide may also be attributed to its ability to restore synaptic protein expression. In addition to attenuating ferroptotic damage, oleamide preserved synaptic integrity by maintaining the expression of key synaptic proteins, including CRMP2, synapsin-1, synaptophysin, and PSD95, which likely contributed to its antiseizure effects. Together with a previous study showing that oleamide modulated the expression of synaptophysin and PSD95 under normal physiological conditions (Tao et al., 2022), our findings suggest oleamide’s broader neuroprotective role in pathological conditions. Oleamide may also offer cognitive benefits beyond seizure control, considering the crucial roles of CRMP2 and PSD95 in learning and memory (Yuki et al., 2014; Zhang et al., 2016) and the frequently observed cognitive impairment in epilepsy (Bell et al., 2011; Khalife et al., 2022). These results extend our earlier work (Nam et al. , 2017), which demonstrated the preventive effects of oleamide against KA-induced excitotoxicity and seizure susceptibility, including reductions in CRMP2 and synapsin-2 expression. In this study, we provide additional evidence for oleamide’s therapeutic efficacy and identify ferroptosis inhibition as a key mechanism underlying oleamide’s actions in both KA- and PTZ-induced epilepsy models. Oleamide’s ability to modulate mTOR and its downstream effectors was explored, considering the established role of the mTOR pathway in epileptogenesis (Hurtado Silva et al. , 2024; Xie et al., 2023) and the growing evidence that mTOR can regulate ferroptosis through autophagy-dependent and autophagy-independent mechanisms (Xie et al. , 2023). In mice with PTZ-induced epilepsy, we observed elevated levels of p-mTOR, p-S6, and p-GSK3β (Ser9), indicating mTOR pathway hyperactivation and a concomitant reduction in GSK3β activity, which are consistent with increased ferroptotic damage. Previous studies have shown that in KA or PTZ models, seizure-induced mTOR hyperactivation leads to divergent effects on GSK3β activity (increased [decreased p-GSK3β] or decreased [increased p-GSK3β]), suggesting experimental condition-dependent bidirectional roles in seizure progression. GSK3β activation ( via decreased p-GSK3β), along with mTOR inhibition, has been associated with suppressed seizure behaviors (Urbanska et al., 2018) and neuroprotection during acute seizures (Bhowmik et al., 2015; Engel et al., 2018), as observed in this study. In contrast, other studies have shown that reducing seizure-induced GSK3β activity can lower seizure severity (Tanioka et al., 2020) and prevent neuronal loss (Goodenough et al., 2004). Ferroptosis induction is typically associated with decreased mTOR activity (Hu et al., 2024; Li et al., 2023) and increased GSK3β activation (Hu et al. , 2024). Activation of the PI3K/Akt pathway, which inhibits GSK3β, was shown to protect against ferroptotic neuronal death and enhance cell survival (Liu et al., 2022; Liu et al., 2023; Sanz-Alcazar et al., 2024). Contrary to these reports, our data demonstrate that seizures simultaneously induce mTOR hyperactivation and GSK3β inhibition, coinciding with increased ferroptotic damage. These contrasting findings highlight the context-specific roles of the mTOR/GSK3β axis in ferroptosis and epilepsy, suggesting that therapeutic strategies must be carefully tailored to avoid harmful effects. Treatment with oleamide reversed the seizure-induced changes in mTOR and GSK3β signaling, which may underlie its ability to suppress ferroptosis and alleviate seizure progression. Similar to everolimus, which has shown clinical efficacy in treating refractory seizures by inhibiting mTOR and preventing abnormal synaptogenesis (Overwater et al., 2019), we propose that oleamide may exert antiepileptic effects by modulating the mTOR pathway. Although direct evidence linking mTOR inhibition with seizure-related ferroptosis is limited, studies in oncology have suggested that mTOR activation contributes to ferroptotic cell death under metabolic stress (e.g., cystine deprivation induced by erastin) (Conlon et al., 2021). In summary, this study is the first to demonstrate that oleamide exerts both prophylactic and therapeutic effects against epileptic seizures by inhibiting ferroptosis and preserving synaptic integrity. Through comprehensive in vitro and in vivo studies, we showed that oleamide suppressed key ferroptotic processes, most notably lipid peroxidation and dysregulated iron metabolism, thereby alleviating the seizure-associated synaptic damage. Furthermore, oleamide reversed seizure-induced perturbations in the mTOR/GSK3β signaling cascade, implicating this pathway in its neuroprotective and antiseizure actions. Collectively, these results suggest oleamide as a promising candidate for the development of novel epilepsy therapeutics. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00251870) to H.-J. Kim. DATA AVALIABILITY All relevant data included in the manuscript have associated raw data and are available from the corresponding authors on reasonable request. AUTHOR CONTRIBUTIONS Eun Jung Na: writing the original draft, methodology, investigation, formal analysis, and data curation. Daseul Lee: writing the original draft, methodology, and investigation. Nayoung Hwang: writing the original draft and methodology. Hwa-Jung Kim: manuscript writing, review and editing, supervision, project administration, and funding acquisition. ORICID Eun Jung Na https://orcid.org/0000-0002-7192-4687 Hwa-Jung Kim https://orcid.org/0000-0001-5566-4611 COMPETING INTERESTS The authors have no conflicts of interest to disclose. ADDITIONAL INFORMATION Supplementary information the online version contains supplementary material available at REFERENCES Bell, B., Lin, J.J., Seidenberg, M., and Hermann, B. (2011). The neurobiology of cognitive disorders in temporal lobe epilepsy. 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(* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ve; # p < 0.05, # p < 0.01, and ### p < 0.001 vs. RSL3, Glu + Fe2(III). FIGURE 2 Effects of oleamide on ferroptosis-related protein expression and iron-binding capacity in HT22 cells. ( a-i ) HT22 cells were pretreated with oleamide (0–30 μM) for 0.5 h and then exposed to 200 nM RSL3 or 10 mM/20 mM glutamate (Glu) or 100 μM arachidonic acid (AA) and 30 μM/100 μM iron(III) for 12 or 24 h. Representative western blots of GPX4 ( a-b ), FSP1 ( c ), ACSL4 ( d-e ), HO-1 ( f-g ), and FTH1 ( h-i ) and their quantified data are shown. Data are presented as means ± standard errors of the mean from 3 to 6 independent experiments. (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ve; # p < 0.05, # p < 0.01, and ### p < 0.001 vs. RSL3, Glu + Fe 2 (III), and AA + Fe 2 (III). ( j ) Fe²⁺ binding capacities measured at various concentrations of oleamide (0.5–200 µM) in a cell-free system. EDTA and deferoxamine (DFO), which are known iron chelators, were used as positive controls. Data are presented as means ± standard errors of the mean from 3 independent experiments. (*** p < 0.001 vs. Ve; ### p < 0.001 vs. ferrozine). FIGURE 3 Anticonvulsant effects of oleamide against KA- and PTZ-induced seizures in mice. ( a-b ) The protocols for in vivo seizure score testing in mice with kainic acid (KA)-induced epilepsy are schematically presented. ( c ) In the pretreatment protocol, vehicle (0.2% methylcellulose in saline), oleamide (Ole), or valproate (VPA) was administered once daily for 5 consecutive days before KA injection. Saline or KA was administered 30 min after the last drug administration. Seizure scoring was performed every 10 min from 30 to 150 min after KA injection. ( d ) In the posttreatment protocol, Veh, Ole (10 mg/kg), or VPA (30 mg/kg) was administered intraperitoneally once daily for 3 consecutive days after KA injection. Seizure scoring was performed every 10 min from 30 to 210 min after KA injection. ( e-f ) The protocols for in vivo seizure score testing in mice with PTZ)-induced epilepsy are schematically presented. ( g ) In the pretreatment protocol, veh, Ole or VPA were administered intraperitoneally once daily for 5 consecutive days before PTZ injection. Sal or PTZ (70 mg/kg) was administered 30 min after the last drug administration. Seizure scoring was performed every 10 min from 30 to 150 min after PTZ injection. ( h ) In the posttreatment protocol, Veh, Ole (10 mg/kg), or VPA (30 mg/kg) was administered 1 day after PTZ injection. Seizure scoring was performed every 10 min from 30 to 210 min after PTZ injection. Data are presented as means ± S.E.M from 3–5 separate experiments. (*** p < 0.001 vs. Control; ### p < 0.001 vs. KA or PTZ). FIGURE 4 Effects of oleamide on the expression of ferroptosis/synaptic marker proteins in hippocampal tissue from mice with KA-induced epilepsy. After the behavioral tests, hippocampal tissues from mice in the pretreatment ( a ) and posttreatment ( b ) groups were analyzed for the expression of proteins associated with ferroptosis and synaptic markers. Representative immunoblots of GPX4 ( c ), ACSL4 ( d ), 4-HNE ( e ), HO-1 ( f ), FTH1 ( g ), synaptophysin/synapsin-1 ( h ), CRMP2 ( i ), and PSD95 ( j ). Data are presented as means ± standard errors of the mean from 3 to 5 independent experiments. (*p < 0.05, **p < 0.01 vs. Vehicle; #p < 0.05, ##p < 0.01 vs. FIGURE 5 Effects of oleamide on the expression of ferroptosis/synaptic marker/mTOR pathway proteins in hippocampal tissue from mice with PTZ-induced epilepsy . After the behavioral tests, hippocampal tissues from mice in the pretreatment ( a ) and posttreatment ( b ) groups were analyzed for the expression of proteins associated with ferroptosis, synaptic markers, and mTOR pathway. Representative immunoblots of ACSL4 ( c ), 4-HNE ( d ), HO-1 ( e ), FTH1 ( f ), synaptophysin/synapsin-1 ( g ), CRMP2 ( h ), PSD95 ( i ), pGSK3β ( j ), pmTOR/mTOR ( k ), and pS6/S6 ( l ). Data are presented as means ± standard errors of the mean from 3 to 5 independent experiments. (* p < 0.05, ** p < 0.01 vs. Vehicle; # p < 0.05, ## p < 0.01 vs. PTZ alone). SUPPLEMENTARY FIGURE S1 Heat map and statistical data for differentially expressed ferroptosis and synapse-associated genes, based on 2 of 5 RNA-seq raw GEO datasets in epilepsy patients ( a ) GSE190451, hippocampus ( b ) GSE134697, neocortex and hippocampus. Horizontal axis: samples; vertical axis: genes Red: high expression; green:low expression. GPX4, FTH1, ACSL4, mTOR, synaptophysin, synapsin-1, and CRMP2 (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Normal patient group). Information & Authors Information Version history V1 Version 1 21 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords blood brain barrier learning & memory neuropharmacology Authors Affiliations Eun Jung NA Ewha Womans University View all articles by this author Daseul Lee Ewha Womans University View all articles by this author Nayoung Hwang Ewha Womans University View all articles by this author Hwa-Jung Kim 0009-0000-8514-2902 [email protected] Ewha Womans University View all articles by this author Metrics & Citations Metrics Article Usage 467 views 288 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Eun Jung NA, Daseul Lee, Nayoung Hwang, et al. Ferroptosis Inhibition Underlies Oleamide's Neuroprotective and Antiseizure Effects. Authorea . 21 May 2025. DOI: https://doi.org/10.22541/au.174784696.68816435/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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