Alleviation of hippocampal necroptosis and neuroinflammation by NecroX-7 treatment after acute seizures

Temporal lobe epilepsy (TLE) is one of the most common neurological disorders, but still one-third of patients cannot be properly treated by current medication. Thus, we investigated the therapeutic effects of a novel small molecule, NecroX-7, in TLE using both a low [Mg2+]o-induced epileptiform activity model and a mouse model of pilocarpine-induced status epilepticus (SE). NecroX-7 post-treatment enhanced the viability of primary hippocampal neurons exposed to low [Mg2+]o compared to controls in an MTT assay. Application of NecroX-7 after pilocarpine-induced SE also reduced the number of degenerating neurons labelled with Fluoro-Jade B. Immunocytochemistry and immunohistochemistry showed that NecroX-7 post-treatment significantly alleviated ionized calcium-binding adaptor molecule 1 (Iba1) intensity and immunoreactive area, while the attenuation of reactive astrocytosis by glial fibrillary acidic protein (GFAP) staining was observed in cultured hippocampal neurons. However, NecroX-7-mediated morphologic changes of astrocytes were seen in both in vitro and in vivo models of TLE. Finally, western blot analysis demonstrated that NecroX-7 post-treatment after acute seizures could decrease the expression of mixed lineage kinase domain-like pseudokinase (MLKL) and phosphorylated MLKL (p-MLKL), markers for necroptosis. Taken all together, NecroX-7 has potential as a novel medication for TLE with its neuroprotective, anti-inflammatory, and anti-necroptotic effects.


Introduction
Epilepsy is a common neurological disorder affecting people of all ages (World Health Organization, 2022). Among various types of epilepsies, temporal lobe epilepsy (TLE) is often refractory to anti-epileptic drugs with no cure (Tellez-Zenteno and Hernandez-Ronquillo, 2012), requiring the development of novel drugs that can ameliorate key pathologies of TLE. Abrupt and excessive neuronal excitability in the brain is thought to contribute to hippocampal sclerosis, a characteristic pathological finding in TLE patients (Steve et al., 2014;Tai et al., 2018). In animal models recapitulating TLE, prolonged seizure activities called status epilepticus (SE) can trigger multifaceted processes in the hippocampus, resulting in neuronal deaths that feature both apoptosis and necrosis (Sloviter et al., 1996;Sankar et al., 1998;Fujikawa et al., 2000b). Necroptosis, an inflammationassociated novel mode of cell death, has been proposed as one of the complicated mechanisms for neuronal death after SE (Dingledine et al., 2014;Choi et al., 2021). Given that neuroinflammation, including reactive astrocytosis and microglial activation, is frequently observed in the hippocampus after SE (Vezzani et al., 2011;Cho et al., 2019), inflammation and neuronal death can have intricate relationships in neurological diseases such as epilepsy . NecroX-7 (also known as MIT-001), an indole-derived small molecule that belongs to the NecroX compound family, primarily inhibits the generation of mitochondrial reactive oxygen species (ROS) . NecroX-7 has demonstrated therapeutic efficacies in a wide range of diseases including graft-versus-host disease (GVHD) (Im et al., 2015), chemotherapy-associated mucositis (Im et al., 2019), acetaminophen-induced liver injury (Park et al., 2013), and myocardial (Hwang et al., 2018), renal (Jin et al., 2016), and hepatic ischemia-reperfusion injuries (Choi et al., 2010;Lee et al., 2016). These significant protective effects shown in multiple cell types are mainly derived from its antinecrotic and anti-inflammatory properties, which are important pathological findings also shown in TLE. However, there has been no report evaluating possible applications of NecroX-7 in central nervous system (CNS) diseases including TLE.
Here we investigated whether NecroX-7 could have an impact on SE-induced hippocampal neurodegeneration using in vitro low [Mg 2+ ] o -induced neurotoxicity and in vivo pilocarpine-induced SE models of epilepsy. We found that posttreatment of NecroX-7 after seizures, but not pre-treatment, had neuroprotective effects in the hippocampus. NecroX-7 posttreatment could alleviate low [Mg 2+ ] o -induced hippocampal neuronal synaptic losses in a dose-dependent manner. In addition, reactive gliosis was significantly downregulated by NecroX-7 treatment after acute seizures in both in vivo and in vitro. Finally, the hippocampal expression of mixed lineage kinase domain-like pseudokinase (MLKL) and phosphorylated MLKL (p-MLKL) was significantly attenuated by NecroX-7 post-treatment, demonstrating an anti-necroptotic effect of NecroX-7 after acute seizures.

Materials and methods
Animals week-old C57BL/6N mice or pregnant rats (KOATECH, Pyungtaek, Korea) were bred at a constant temperature of 22°C ± 1°C in a light-controlled room with food and water ad libitum. All animal trials were approved by the Ethics Committee of the Catholic University of Korea (CUMS-2020-0103-04, CUMS-2022 and Dankook University (DKU-22-047) and were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23, revised 1996).

Pilocarpine-induced mouse model of SE and sample preparation
A mouse SE model was generated as previously described (Jeong et al., 2011;Cho et al., 2015;Kim and Cho, 2018). In short, scopolamine methyl nitrate (2 mg/kg; Sigma-Aldrich, St. Louis, MO, United States, S2250) and terbutaline hemisulfate salt (2 mg/kg; Sigma-Aldrich, T2528) were administered intraperitoneally (i.p.) 30 min prior to pilocarpine hydrochloride (280 mg/kg; i. p., Sigma-Aldrich, P6503) injection. After pilocarpine was injected, behavioral seizures were carefully observed according to the modified Racine scale (Racine, 1972) and the time when the mouse exhibited the first grade 3 seizures (Seizure onset) and the time when the mouse started to demonstrate continuous motor seizures (SE onset) were recorded to assess the seizure kinetics in the groups. Mice with continuous generalized convulsive seizures higher than grade 3 were identified as having shown SE and were subjected to further studies. Diazepam (10 mg/kg, i. p.) was injected 3 h after the onset of SE to cease behavioral seizures. Then, they were housed in an incubator (30°C) and fed with water-moistened chow to facilitate recovery. After 2 days, they were sent back to the original environment.
After the final NecroX-7 treatment, animals were anesthetized with ketamine (50 mg/mL) and xylazine (23.3 mg/mL) mixture (4: 0.5), followed by transcardial perfusion with saline. After the brain was removed, hemibrains were postfixed in 4% paraformaldehyde (pH 7.4) for 3 days, then immersed in 30% sucrose solution for 3 days. Then, the brains were frozen in liquid nitrogen and serial hippocampal coronal sections (30 μm thick) were made with a cryostat (Leica, Wetzlar, Germany, CM 1850) for staining. For the other half of the brain, the hippocampus was frozen under liquid nitrogen for western blotting.

Drug treatment
NecroX-7 was provided by MitoImmune Therapeutics Inc. Cells were allocated into 4 groups: vehicle, 10 μM, 30 μM, or 100 μM NecroX-7. In the pretreatment groups, cells were exposed to either vehicle or NecroX-7 for 1 h prior to 0.1 mM [Mg 2+ ] o treatment (Kim et al., 2008;Kim et al., 2016). In the post-treatment group, cells started to be exposed to either vehicle or NecroX-7 at 1 h after 0.1 mM [Mg 2+ ] o medium-induced epileptiform activity until the end of the experiment. MK-801 (10 μM), an NMDA receptor blocker known to have neuroprotective effects, was pre-treated 30 min before exposure to low [Mg 2+ ] o medium as a positive control.
Pilocarpine-injected mice were allocated into 3 groups: placebo, 20 mg/kg, or 40 mg/kg of NecroX-7. One hour after diazepam injection, each group was given intraperitoneal injection of placebo or drug solutions, which was prepared by dissolving the powder in 5% dextrose. Mice were daily administered the same dose of NecroX-7 or placebo for the following 7 days after pilocarpine injection. On the 7 th day, mice were sacrificed after the final injection of the drug.

Confocal imaging
Primary neurons were transferred to a confocal microscope (LSM 700; Carl Zeiss, Jena, Germany) and observed through a ×60 objective (numerical aperture, 0.8). A total of eight crosssections were obtained at 1 μm intervals along the z-axis of the cell using the z-stack imaging technique and were combined to obtain one image. Using an argon ion laser, the GFP is excited at 488 nm and emits light at 519 nm (10 nm band pass). The RFP is excited at 555 nm (HeNe laser) and emitted at 565 nm.

Microscope analysis and quantification
FJB-positive or immunoreactive cells were quantified as previously described (Choi et al., 2022). The number of FJBpositive cells was counted in every 12th hippocampal section, which was summed up and multiplied by 24 to estimate the total number of FJB-expressing cells in an animal. The CA1 and CA3 pyramidal cell layer (PCL) was demarcated by the point of rapid widening of PCL thickness and the larger soma size of CA3 pyramidal cells. The hilus was defined as the triangular area formed by connecting endpoints of the upper and lower granule cell layer (GCL) but excluding the CA4 PCL. As for quantitative analysis of reactive gliosis, the percentage of Iba1-and GFAPimmunoreactive areas in the dentate gyrus (DG) was analyzed using NIH ImageJ software. Sections with damaged regions in the hippocampal subfields were excluded from the analysis and samples containing two or more sections with damaged subfields were also excluded from the analysis. Briefly, background intensity was determined by measuring the pixel intensity of non-tissue area in each image. Then, Iba1-and GFAP-immunoreactivity were determined using the background intensity as a threshold for Frontiers in Pharmacology frontiersin.org reactivity. Finally, the percentage of the marker-immunoreactive area over DG area value for all the images was calculated, and then those values were averaged for each mouse. The Iba1immunoreactive area in CA1 and CA3 subfields of the hippocampus was evaluated using the same protocol as described above. For primary cultured neurons, the integrated intensity was quantified as the sum of pixel values of target immunoreactivity in an image of GFAP-or Iba1-positive areas using ImageJ software (NIH, United States) (Hong et al., 2021). Analysis of PSD95 puncta superimposed on MAP2 immunocytochemistry was done by quantitative measurements of the number of PSD95 puncta per 0.1 mm dendrite recognized by MAP2.

Statistical analysis
Data are presented as mean ± standard error of the mean (SEM) and statistical significance was assessed using GraphPad Prism 9 software (GraphPad Software Inc.) or SPSS 12.0. Experimental groups were randomly assigned, and the exact sample size is presented in the figure legends. To reduce the experimental bias, analyses were performed by a researcher blind to the experimental groups. Body weight changes of pilocarpine-induced SE model mice were analyzed using Repeated measures one-way ANOVA, while seizure onset and SE onset after pilocarpine injection were analyzed using one-way ANOVA with Tukey's multiple comparisons and Kruskal-Wallis test with Dunn's multiple comparisons test, respectively. For FJB staining results, statistical differences were mainly determined by Kruskal-Wallis test with Dunn's multiple comparisons test as normal distribution was not assumed. For DG analysis, Brown-Forsythe one-way ANOVA with unpaired t-test with Welch's correction was performed as normal distribution was assumed but equal variance was not assumed. For analysis of Iba1 and GFAP immunohistochemistry, One-way ANOVA with Tukey's multiple comparisons test was used when normal distribution was assumed. In case normal distribution was not assumed, Iba1 in CA3 subfield, Kruskal-Wallis test with Dunn's multiple comparisons test was done. For western blot analysis, oneway ANOVA with Newman-Keuls multiple comparisons test was used. For in vitro results, one-way ANOVA with Bonferroni posthoc test was performed. The level of statistical significance was set as 95% confidence interval p < 0.05 or 99% confidence interval p < 0.01.

Effect of NecroX-7 on low [Mg 2+ ] o -induced synapse loss in cultured rat hippocampal neurons
To investigate the effects of NecroX-7 on synapse loss that precedes excitotoxicity, we performed PSD95 immunocytochemistry, which labels excitatory synapses. The number of PSD95 puncta per 0.1 mm dendrite recognized by MAP2 was quantitatively measured (

NecroX-7 treatment reduces hippocampal necroptosis marker expression
Since NecroX-7 alleviated neuroinflammation and neuronal death, we tried to evaluate the nature of NecroX-7-mediated neuroprotection after acute seizures in vivo (Figure 4Aa) and in vitro (Figure 4Ab). On the 7 th day after SE, our western blot analysis demonstrated that the hippocampal MLKL expression (placebo: 1.00 ± 0.15 vs. 20 mg/kg: 0.67 ± 0.08 vs. 40 mg/kg: 0.46 ± 0.12), a marker for necroptosis, was significantly decreased in groups of both 20 mg/kg and 40 mg/kg of  Collectively, our results suggest that NecroX-7 treatment after SE can contribute to the reduction of seizure-induced hippocampal necroptosis.

Discussion
Despite longstanding rigorous efforts to develop effective antiepileptic drugs, epilepsy remains intractable in one-third of its patients, thereby still requiring novel drugs. In this study, we investigated a small molecule, NecroX-7, as a potential therapeutic agent for TLE. Administration of NecroX-7 after acute seizures demonstrated neuroprotective and anti-inflammatory effects on hippocampal neurons in both in vivo and in vitro models of TLE. Additionally, we demonstrated that NecroX-7 could reduce seizureinduced necroptosis marker expressions in the hippocampus.
In the present study, we proposed that necroptosis could play a role in seizure-induced cell deaths, which was prevented by NecroX-7 treatment. Necroptosis, a type of cell death also known as programmed necrosis, can be induced when TNFα binds to its receptor, TNFR1, and activates RIP1/3 to phosphorylate MLKL, an important marker of necroptosis . Phosphorylated MLKL then forms an oligomer and binds to the cell membrane, causing Ca 2+ and Na + influx, and increasing extracellular permeation of cellular components including DAMPs, which eventually leads to cell death (Du et al., 2022). Since SE is an inflammatory condition and cell deaths in SE in part showed necrotic morphologies (Fujikawa et al., 2000a;Dingledine et al., 2014;Wang et al., 2017b;Cai et al., 2017;Fricker et al., 2018;Du et al., 2022), involvement of necroptosis has been assumed. Indeed, we reported the first evidence that acute seizures can cause MLKLmediated necroptosis in the hippocampus, in addition to the involvement of truncated neogenin in this process (Choi et al., 2021). We further sought to find an anti-necroptotic agent and found a novel small molecule, NecroX-7, that could decrease MLKL and p-MLKL expression after acute seizures. In other studies, inhibition of MLKL reduced SE-related brain damage and improved cognitive performance (Wang et al., 2017a;Jia et al., 2019), supporting our data. Taken all together, we provide a new lead compound, NecroX-7, as a potential anti-epileptic drug by demonstrating the alleviation of excitotoxic necroptosis.
NecroX-7 has an advantage when it comes to drug development because it is bioavailable with oral administration and is non-toxic. Studies of IV administration of single and multiple doses of NecroX-7 to a healthy male population showed no significant side effects and it was confirmed to have optimal pharmacokinetic properties, such as dose proportionality, fast tissue distribution, and long t ½ , for a once-daily regimen (Kim et al., 2017;Kim et al., 2020). Since first-inhuman trials have been conducted, a future direction will be to test the efficacy of NecroX-7 in TLE patients. A more elaborate study design will be required to determine the appropriate dosage of NecroX-7 for TLE as wide dose ranges of 0.3-30 mg/kg (i.v.), and 20-50 mg/kg (p.o.) in mice have been reported to show therapeutic efficacy in various diseases (Park et al., 2013;Chung et al., 2015;Im et al., 2019). We also reported that 20-40 mg/kg of NecroX-7 had anti-necroptotic and anti-inflammatory effects in a mouse model of TLE. Given that the blood-brain barrier (BBB) hinders efficient drug delivery to the cells in the brain, modification of the drug structure to enhance BBB penetration or high doses of NecroX-7 may be necessary to demonstrate therapeutic efficacy in the context of epilepsy. In addition, testing a variety of different cell types will be also required to provide comprehensive information about the FIGURE 3 (Continued) astrocytes, are indicated with black arrows in magnified image (a) of the placebo group. Less hypertrophic, ramified, astrocytes are indicated with white arrows in the magnified images (b, c) of NecroX-7-treated groups. Scale bar = 200 μm for low magnified images, 50 μm for higher magnified images. A graph showing GFAP-immunoreactive area in DG. Percentages of GFAP-immunoreactive area were comparable between placebo and NecroX-7-treated groups. n = 14 (placebo), n = 14 (20 mg/kg) and n = 13 (40 mg/kg). One-way ANOVA was performed with Tukey's multiple comparisons test, p = 0.45, F (2, 38) = 0.81. Data are presented as mean ± SEM. *p < 0.05. (E) Confocal images show the maximum z-stack of hippocampal neurons expressing GFAP (red) and DAPI (blue). The graph summarizes the effects of NecroX-7 (10 μM, 30 μM) post-treatment on GFAP expression caused by 0.9 mM [Mg 2+ ] o medium or 0.1 mM [Mg 2+ ] o medium. One-way ANOVA was performed with Bonferroni post-hoc test, p < 0.0001, F (5, 194) = 10.45. Scale bar = 20 μm. Data are shown as mean ± SEM. ### p < 0.001 vs. Control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. low [Mg 2+ ] o .
Frontiers in Pharmacology frontiersin.org 08 efficacy and potential side effects of NecroX-7, especially when high doses of NecroX-7 need to be treated. However, the easy administration and good tolerability of NecroX-7 make it a more promising candidate as a novel anti-epileptic drug.
Frontiers in Pharmacology frontiersin.org inhibitor, but was later found to selectively inhibit RIP3 (Sugaya et al., 2019). Finally, NSA, another small molecule, is known to block MLKL oligomerization and activation (Yan et al., 2017;Bansal et al., 2019). As our results suggest that NecroX-7 can affect the steps associated with MLKL in the hippocampus, it will be interesting to evaluate differential impacts of targeting each key signaling molecule of necroptosis on neuroprotection after acute seizures. In our study, while NecroX-7 successfully protected neuronal death in the hilus, pyramidal neurons in the CA1 and CA3 subfields could not be saved after pilocarpine-induced SE. This regional difference in cell death might be associated with region-specific alterations in microglial activation as we found that Iba1 immunoreactivity was alleviated only in the hilus. Alternatively, it may be related with different biochemical characteristics of each cell type, leading to altered response to environmental stimuli and NecroX-7 treatment. For instance, hilar interneurons express C-C motif chemokine receptor 8 (CCR8), which can inhibit dexamethasone-induced apoptosis (Spinetti et al., 2003), while pyramidal neurons do not (Liu et al., 2007). Despite speculative at the moment, NecroX-7 might boost the protective effect of CCR8, thus salvaging hilar interneurons. This hypothesis warrants further comprehensive investigation in the future as transcriptomic analysis between hilar interneurons and pyramidal neurons for the identification of differentially expressed candidates after acute seizures and then after NecroX-7 administration, respectively.
In summary, we propose NecroX-7 as a potential therapeutic agent for TLE by demonstrating the neuroprotective and anti-inflammatory effects in vitro and in vivo. In addition, we suggest that NecroX-7 after acute seizures can mediate anti-necroptosis in the hippocampus.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by The Institutional Animal Care and Use Committee at the Catholic University of Korea (Approval number: CUMS-2020-0103-04, CUMS-2022-0078-02) and Dankook University (DKU-22-047).

Author contributions
HJK and K-OC conceived and designed the study. YR, SBL, MK, and M-HK performed the experiments, analyzed the data. YR, SBL, HJK, and K-OC wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This research was funded by MitoImmune Therapeutics Inc. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.