Critique Of “Selective Targeting Of Scn8A Prevents Seizure Development In A Mouse Model Of Mesial Temporal Lobe Epilepsy”

Epilepsy was found to be the fourth most prevalent neurological disorder. With such high rates of occurrence in the population, it is concerning that over thirty percent of these patients receive inadequate treatment with drug therapy. Benzodiazepines are currently used as a first line therapy, but studies have shown that their potency and efficacy can be reduced over time. This calls for more effective and sufficient drug treatments. SCN8A is a gene that codes for a specific voltage gated sodium channel (VGSC), Nav1. 6, that plays an important role in regulating neuronal excitability and threshold. It was observed that mice that had mutations that resulted in an inactive Scn8a protein were less susceptible to seizures, when both electrically and chemically induced. A nonfunctional Scn8a protein also helped improve survivability and seizure phenotypes in mice with Dravet’s syndrome or GEFS+. From these findings, Wong et al. chose to investigate the effectiveness of targeting Scn8a as a possible therapeutic approach for certain types of epilepsy, such as temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy that is unresponsive to treatment, with mesial temporal lobe epilepsy (MTLE) being the most common form of TLE. Existing treatments may target VGSCs but tend to not be subtype-specific. This can worsen symptoms of subtype specific disorders, such as Dravet syndrome (SCN1A), and may impact the effectiveness of antiepileptic drugs. Additionally, no current treatments exist that target specific VGSCs, despite their highlighted roles in the pathology of epilepsy. Wong et al. hypothesize that selectively reducing Scn8a expression in a mouse model for MTLE will improve outcomes for an epileptic phenotype. Confirming this hypothesis could suggest a therapeutic target which could improve the specificity of future treatments for seizures. The adeno-associated viral vector (AAV) in the experiment expressed a small-hairpin RNA (shRNA) construct against Scn8a. The specificity of this knockdown agent was tested against both naïve and shRNA-scram mice.

Mice that received shRNA-Scn8a injections had reduced Nav1. 6 (encoded specifically by SCN8A) protein levels after 3 and 8 weeks while their Nav1. 1 or Nav1. 2 (encoded by SCN1A and SCN2A respectively) protein levels were not significantly impacted. This result supports the use of shRNA-Scn8a as an effective tool for Scn8a-specific knockdown. Two days after performing an EEG surgery and implanting guide cannula, the team administered kainic acid (KA) into the right dorsal hippocampus. After 24 hours, the mice were divided into groups that received shRNA-scram, shRNA-Scn8a or no injection. KA-only and shRNA-scram mice experienced similar frequencies of seizures on average. However, shRNA-Scn8a–treated mice experienced a dramatic reduction in seizure occurrence. 90% of the shRNA-Scn8a-treated mice did not experience seizures during the 8-week EEG recording period. This is a powerful result that strongly advocates for this treatment.

Wong et al. consider that previous studies have indicated that MTLE patients tend to share certain behavioral symptoms such as hyperactivity and impaired learning; similar observations were made with certain intrahippocampal KA mice. Following the EEG recordings, the team tested the effect of Scn8a knockdown on alleviating these behavioral symptoms using analyses that included open field and novel object recognition paradigms. A group of mice that underwent sham surgery was included to eliminate surgery as a factor influencing behavioral symptom occurrence. Figure 4 shows that the distance traveled and average speed were significantly lower in the surgery and shRNA-Scn8a mice compared to KA-only and shRNA-scram mice, suggesting that this treatment reduces hyperactivity.

A different study showed that mice that were heterozygous for Scn8a did not exhibit heightened anxiety in the open field task, which raises the question of whether this behavior is only associated with chemically-induced seizures in mice. Wong et al. also wanted to see whether downregulating the expression of Scn8a could prevent neuron and cell death in the hippocampus. Using DAPI and NeuN stains, the team compared neuron and cell counts from a surgery-only mouse group, a shRNA-scram group and a shRNA-Scn8a group. Both the shRNA groups experienced an approximately 50% cell and neuron loss when compared to surgery-only mice, suggesting that this treatment is not neuroprotective. The surgery procedure wasn’t described, so it is possible that the cannula insertion may have contributed to the observed cell and neuron death. Since the result was expected due to the time difference between damage onset and treatment effects, the reason for performing this test is unclear. It’s also interesting to note the absence of the KA-only group, which was included in prior figures. The team also investigated the treatment’s impact on reactive gliosis.

The differences in GFAP levels, which is a marker for reactive gliosis, are not significant when comparing the shRNA-Scn8a group with the shRNA-scram and surgery-only groups. Several statements in the paper, however, assume the contrary, as in the statement: “These results demonstrate that Scn8a knockdown…did result in significantly less reactive gliosis throughout the hippocampus”. A study showed that there is an increased microglial response in epilepsy model. An experiment could be conducted with BrdU and EGFP stains that uses a two-photon microscope to more directly observe the microglial response to the Scn8a knockdown to clarify the influence of this treatment over glial activation and response.

The results of these experiments indicate that the selective reduction of Scn8a could be an effective therapeutic target for MTLE. Future studies could help emphasize the benefits of this possible therapeutic target and seek means of implementing it. For example, minocycline is a microglial inhibitor that has been shown to be neuroprotective in rodents; comparing the effects of this drug and Scn8a knockdown on the hippocampal cellular landscape could clarify any neuroprotective functions of this treatment. In some humans, the SCN8A mutation can lead to intellectual disabilities, accompanied by social interaction challenges, instead of epilepsy.

Conducting an experiment that includes the social interaction test and aims to identify the expression of the Scn8a intellectual disability phenotype in mice similar to those used by Wong et al. could test whether those mice that are not exhibiting seizure behaviors are being impacted intellectually. This could show whether this knockdown treatment could be effective in treating this alternate phenotype of the SCN8A.

Overall, Wong et al. ’s study lays a strong foundation for further studies that could help determine a novel and effective treatment method to improve outcomes for epilepsy patients around the world.