Repurposing dimethyl fumarate as an antiepileptogenic and disease-modifying treatment for drug-resistant epilepsy

Dimethyl fumarate (DMF; Sigma) was dissolved in a mixture of methylcellulose 0.5% (80%) and PEG 400 (20%) (Sigma-Aldrich) at a concentration of (50 mg/ml) and administered intraperitoneally once daily to rats at a total dose of 200 mg/kg/day for 1 week. Control animals were treated with an equivalent volume and number of injections of the vehicle as the DMF-treated animals. For biochemical and antiepileptogenic experiments, rats were treated with either DMF or vehicle for 1 week after SE. For the chronic epilepsy experiment, rats with spontaneous recurrent seizures (SRS) were treated with either DMF or vehicle for 1 week after a 3-week baseline recording period.

Anxiety-like behavior was evaluated in rats using Elevated plus maze (EPM) and open-field (OF) tests. The spontaneous alternation T-Maze was used to assess spatial learning and decision-making processes.

For immunohistochemistry studies, rats were euthanized on day 7 post-SE, 2 h after the final dose of DMF or VEH, with intraperitoneal administration of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). The rats were then perfused with heparinized ice-cold PBS, followed by transcardial perfusion with 4% paraformaldehyde (PFA; Sigma) in PBS (pH 7.4). The brains were removed, fixed overnight with 4% paraformaldehyde solution (PFA, Sigma) in PBS (pH 7.4), and cryoprotected in sucrose phosphate buffer (10%, 20%, and 30% solutions for 24 h each) at 4 \(^\circ\)C. After freeze-mounting, brain samples were immersed in O.C.T compound (Scigen) and stored at − 80 \(^\circ\)C. For immunohistochemical studies, coronal sections of 20 μm selected from the hippocampus were cut in a cryostat (Leica CM1950) at − 20 °C, fixed on poly L-lysine coated slides (Thermo Fisher Scientific), and left to dry for 2 h. Each slide contained 2–3 sections per animal. The primary antibody was added after washing thrice with PBS for 10 min each. Washed sections were incubated overnight at 4 °C with rabbit primary antibody against the neuronal marker NeuN (1:500, ab177487, Abcam, Cambridge, UK) in a solution of PBS, 0.1% Triton X-100 and BSA 1%. Following three washes with PBS for 10 min each, the sections were incubated with Alexa Fluor^® 488 goat anti-rabbit secondary antibody (1:500; ab150081, Abcam, Cambridge, UK) for 2 h at room temperature in the dark. The sections were washed three times with PBS for 10 min each and mounted with Vectashield and 4′, 6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Labs). Images were obtained at a resolution of 1024 × 1024 using a Nikon confocal A1R microscope with 20X and 40X objectives. Images were acquired at 405 nm excitation wavelength and 455 nm emission wavelength for DAPI and at an excitation of 495 nm and emission of 519 nm for NeuN. Image analysis was performed using ImageJ software with a manual cell counting image-based tool, and the investigators were blinded to the treatment. The results are expressed as cell density, that is, the average number of NeuN + cells per mm², for 2-brain sections per rat in 2–3 images of ROIs of CA1 and CA3 subfields of the hippocampus.

Rats were sacrificed 2 h after the final dose of DMF or vehicle at 7 days post-SE. The rats were euthanized by intraperitoneal administration of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). Frozen brains were immediately extracted and stored at − 80 °C until they were thawed on ice, resuspended in RIPA buffer (Thermo Fischer), homogenized, and incubated at 4 °C for 2 h under constant rotation. Protein samples were centrifuged and prepared, and 25 µg of each sample was transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Nonspecific binding sites on the membrane were blocked with SuperBlock buffer in TBS containing 0.1% Tween-20, 0.05% Tris-Chloride, and 0.03% 5 M NaCl (TBS-T) for 1 h at 22–24 °C. Membranes were subsequently incubated overnight at 4 °C with primary anti-NFE2L2 antibody (1:5000, 16,396–1-AP, Proteintech), anti-NQO1 antibody (1:1000, ab34173), and β actin (1:2000, ab8226). The preparative membranes were probed with appropriate secondary antibodies conjugated to HRP. Immunological complexes were visualized using electrochemiluminescence (ECL, Bio-Rad). Densitometry analysis was performed using Image Lab software (5.1, Bio-Rad, Hercules, CA, USA). Data were normalized to the loading control (β-actin).

Immediately following euthanasia, total RNA was extracted from brain tissue using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) and kept at − 80 °C following animal sacrifice. One microgram of RNA was used as Complementary DNA (cDNA) using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) with an oligo-dT15 primer. Real-time PCR was carried out in a final volume of 15 μL (7.5 μL of SYBR Green, 3 μL of primers (500 nM each), 1.5 μL of DEPC water, and 3 μL of cDNA template was added to the mixture) with SYBR Green (PerfeCTa SYBR Green FastMix, Quantabio) and monitored by CFX Connect Real-Time PCR Detection System (Bio-Rad). The following cycling parameters were used: 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 5 s, and 60 °C for 15 s. Finally, 5 s at 65 °C, and 30 s at 95 °C. Primer sequences are reported in Table 1. Each RT-PCR was performed in duplicate. The level of target mRNA was quantified relative to the housekeeping gene (GAPDH) using the ΔΔCT method [37]. GAPDH expression was not significantly different between treatments.

Data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using the GraphPad Prism 9.5.0 software. Data were analyzed using ordinary one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test or generalized log-linear mixed model, followed by the Sidak posthoc test.

Sample sizes were chosen based on our previous calculations of experimental variability. The number of animals used is described in the corresponding figure legend.

The statistical significance threshold was determined as α error with a value of P < 0.05.

We first determined the effect of DMF on mRNA and protein levels of Nrf2 in the brain following kainic acid-induced status epilepticus (KA-SE). After SE induction, the rats were treated with either vehicle or DMF for 7 days and then sacrificed 2 h after the final dose. The mRNA and protein levels of Nrf2 were examined in the cortex and hippocampus.

In agreement with our previously published study [38], a positive non-significant trend to increase Nrf2 mRNA level was observed in the cortex (Fig. 1A), and significantly increased in the hippocampus 1 week after KA-SE (Fig. 1D). DMF treatment was associated with a significant increase in Nrf2 protein levels in the cortex (Fig. 1B, C), and a higher increase in the hippocampus (Fig. 1E, F).

The functional effect of DMF on the activation of the Nrf2 pathway was evaluated. We measured the mRNA and protein levels of the downstream genes of Nrf2, NQO1, HO-1, and GCLC-1. We found that NQO1 and HO-1 mRNA levels followed a similar regional increase as that observed for Nrf2 in the cortex (Fig. 2A, B; NQO1, Sham vs. SE + DMF: p = 0.0113; HO-1, Sham vs. SE + DMF: p = 0.0283), and the hippocampus (Fig. 2D, E; NQO1, Sham vs. SE + DMF: p < 0.0001; HO-1, Sham vs. SE + DMF: p < 0.0001).

Importantly, the protein levels of NQO1 and HO-1 also increased in both cortex (Additional file 1: Fig. S2), and the hippocampus (Additional file 1: Fig. S3).

Interestingly, the GCLC-1 mRNA level in the cortex significantly decreased following SE compared to sham animals (p = 0.0474), and treatment with DMF prevented this decrease (Fig. 2C; Sham vs. SE + DMF, p = 0.5897). Moreover, the mRNA levels of GCLC-1 in the hippocampus were not reduced following SE (p = 0.6370). However, DMF treatment increased the expression of both mRNA (Fig. 2F; SE + Veh vs. SE + DMF, p = 0.0201), and protein levels (Additional file 1: Fig. S2, S3).

The induction of KA-SE has been previously reported to result in notable hippocampal neuronal damage [39‐41]. We previously reported that the KA-SE model caused significant neuronal loss in the CA1 and CA3 subfields of the hippocampus 1 week after SE [24, 34].

In light of these findings, we measured neuronal cell densities in the CA1 and CA3 subfields of the dorsal hippocampus 1 week after SE in rats treated with vehicle or DMF (Fig. 3A). In agreement with previous findings, our results indicated that SE caused a significant loss of neurons in the CA1 and CA3 regions 1-week post-SE, as evidenced by the decrease in cell density (Fig. 3B–E). Treatment with DMF for one week after SE significantly ameliorated neuronal cell death (Fig. 3B–E; CA1, SE + Veh vs. SE + DMF: p = 0.0052; CA3, SE + Veh vs. SE + DMF: p = 0.0023).

This study also determined whether the DMF-associated increase in the expression of Nrf2 and neuroprotection might translate into an anti-epileptogenic effect. We performed continuous video-ECoG recordings from rats subjected to 2 h of KA-SE and treated with vehicle or DMF. The ECoG data were analyzed for spontaneously recurring convulsive seizures, and all seizures were manually verified. Representative EEG traces from both vehicle- and DMF-treated rats are shown in Fig. 4A. The seizure frequency in vehicle-treated rats progressively increased after SE. Treatment with DMF for 1 week after SE significantly decreased seizure frequency in treated rats (Fig. 4B, F (13, 234) = 16.33, p < 0.0001, generalized log-linear mixed model). Moreover, DMF-treated rats experienced significantly fewer seizures than vehicle-treated rats (Fig. 4C, p = 0.001, unpaired t-test).

Interestingly, 10/10 vehicle-treated animals developed spontaneous seizures within 4 weeks after SE. In contrast, of 3/10 animals in the DMF-treated group remained seizure-free for the duration of the 14-week monitoring period post-SE (Fig. 4D). A log-rank test was calculated to test if DMF treatment can affect the distribution of time for seizure occurrence in all animals. Indeed, the distribution of time for seizure occurrence in the DMF treatment group significantly differs from the vehicle treated animals (p = 0.0051). Finally, the latency period (i.e., the time from SE onset until the emergence of the first spontaneous seizure) was significantly increased following DMF treatment (Median, Veh = 7 vs. DMF = 13 days, Fig. 4E).

As shown in Fig. 5, a 7-day treatment with DMF beginning shortly after attenuation of SE resulted in normalization of behavior as assessed in the open field (OF; Fig. 5A) and elevated plus-maze tests (EPM; Fig. 5B), respectively. As noted in (Fig. 5A1), DMF-treated SE rats spent more time in the center of the arena and traveled longer distances than vehicle-treated rats (Fig. 5A2, A3). However, these outcomes were not statistically different from those of sham rats. As shown in (Fig. 5B), DMF-treated rats displayed more entries into the open arm of the EPM and spent more time in the open arm than vehicle-treated animals (Fig. 5B1, 5B2).

To evaluate memory function, we used the Spontaneous Alternation T-Maze test (Fig. 5C). Rats treated with vehicle after SE showed a significant decrease in the percentage of spontaneous alterations compared to sham animals, suggesting that epileptic animals exhibited impaired spatial memory (one-way ANOVA, F (2,24) = 8.580; p = 0.0015; Sham vs. SE + vehicle: p = 0.0011; Fig. 5C2). In DMF-treated animals, there was a slight increase in the percentage of spontaneous alterations compared to vehicle-treated animals (74% vs. 59%, respectively); however, this difference was not statistically significant (p = 0.0666). Importantly, there was no significant difference between sham-operated and DMF-treated animals (p = 0.2012), suggesting that DMF treatment shortly after SE can prevent epilepsy-induced memory dysfunction.

Finally, we investigated the therapeutic potential of DMF for chronic epilepsy. Rats were first subjected to KA-SE, and 10–12 weeks later, wireless electrocorticography (ECoG) transmitters were implanted to monitor the development of spontaneous seizures. We then recorded baseline ECoG activity for 3 weeks. Rats with confirmed epilepsy were randomized to receive treatment with either vehicle or DMF (200 mg/kg/day for 7 days; n = 9 rats/group). Video-ECoG recordings were continued during the treatment period for an additional 10–12 weeks. We found that treatment with DMF significantly reduced the normalized seizure frequency compared to that in vehicle-treated animals (generalized log-linear mixed model on weeks 1–10, treatment group × week interaction effect: F (8, 59) = 20.66, P < 0.001; Fig. 6A). Furthermore, the normalized cumulative number of seizures post-treatment was significantly lower in the DMF group than that in the vehicle control group (Fig. 6B, C, P = 0.0235, Student’s t-test).