Abstract
Epilepsy Hammer MF, Krzyzaniak CT, Bahramnejad E, Smelser KJ, Hack JB, Watkins JC, Ronaldson PT. Clin Sci (Lond). 2024;138(4):205–223. doi:10.1042/CS20231572. PMID: 38348743; PMCID: PMC10881277. Background: Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models. Methods: We characterized sex differences in mice carrying a pathogenic knockin variant (p.N1768D) in the Scn8a gene that causes spontaneous tonic-clonic seizures (TCs) at ∼3 months of age and found that heterozygous females are more resilient than males in mortality and morbidity. To investigate the cellular mechanisms that underlie female resilience, we utilized blood-brain barrier (BBB) and hippocampal transcriptomic analyses in heterozygous mice before seizure onset (pre-TC) and in mice that experienced ∼20 TCs (post-TC). Results: In the pre-TC latent phase, both sexes exhibited leaky BBB; however, patterns of gene expression were sexually dimorphic. Females exhibited enhanced oxidative phosphorylation and protein biogenesis, while males activated gliosis and CREB signaling. After seizure onset (chronic phase), females exhibited a metabolic switch to lipid metabolism, while males exhibited increased gliosis and BBB dysfunction and a strong activation of neuroinflammatory pathways. Conclusion: The results underscore the central role of oxidative stress and BBB permeability in the early stages of epileptogenesis, as well as sex dimorphism in response to increasing neuronal hyperexcitability. Our results also highlight the need to include both sexes in preclinical studies to effectively translate results of drug efficacy studies.
Commentary
Sexual dimorphism refers to the biological and physiological differences between males and females of the same species. It extends beyond physical appearance and primary reproductive characteristics to include variations in brain structure and function. Emerging evidence supports that these innate differences between sexes can profoundly influence disease processes, susceptibility, and treatment responses in neurologic conditions.1–3 For instance, in the context of epilepsy, epidemiological studies indicate sexual dimorphism in epilepsy presentation and prevalence.1,4,5 Males show higher overall rates of epilepsy and are more prone to lesional forms, while females predominantly experience idiopathic generalized epilepsies and seizures preceded by auras.1–5 Females also experience higher rates of adverse reactions to antiseizure medications. 6
These marked sex-based differences in epidemiology and manifestation highlight the critical need to incorporate sex-specific factors into preclinical research and clinical approaches for comprehensive disease understanding and optimized treatment strategies. 3 Notably, preclinical epilepsy research predominantly focuses on male subjects, with comparatively fewer studies investigating females.1,3 This research gap is particularly pronounced in descriptive and mechanistic studies, excluding investigations purposely focused on female-specific epilepsies or hormone-driven neurological processes.1,3 To address this knowledge gap, Hammer et al performed a discovery-based study to identify potential mechanisms underlying sex-dependent variabilities in the pathophysiology of a mouse model of pediatric epilepsy with the Scn8a N1768D gain-of-function mutation. 7 These mice are characterized by tonic–clonic (TC) seizures by 3.5 months old and premature death associated with sudden unexpected death in epilepsy (SUDEP). 7
Mice carrying the Scn8a N1768D variant (heterozygous Scn8a D/+) exhibit sex-dependent variations in TC seizure activity and mortality.7,8 Specifically, Scn8a D/+ female mice exhibit both longer lifespans and higher resilience to TC seizures compared to Scn8a D/+ male mice.7,8 This observation led the authors to propose that sex hormones could protect against neuropathological alterations, including maintaining blood–brain barrier (BBB) integrity in Scn8a D/+ female mice, thereby enhancing seizure resilience and prolonging lifespans. Thus, to identify potential mechanisms underlying the differences in pathophysiology between male and female Scn8a D/+ mice, the authors interrogated BBB permeability and applied bulk RNAseq analysis to study hippocampal transcriptomic profiles before and after the development of TC seizures, at 6 weeks and at ∼3.5 months old, respectively, in age- and sex-matched mice.
A comprehensive analysis of survival and behavioral seizure patterns in Scn8a D/+ mice confirmed significant sex-based differences, with females demonstrating better outcomes across all measured parameters than males. Scn8a D/+ female mice lived substantially longer and experienced later TC seizure onset. Although females exhibited a higher TC seizure frequency than males, this was associated with longer post-TC survival and more frequent, extended TC seizure-free periods. However, as these studies relied solely on video monitoring, the findings may not fully capture the complete seizure characteristics, as unobserved electrographic ictal and interictal activity could significantly influence mortality and morbidity. Future video-EEG studies would be crucial in definitively confirming these sex-based variations in seizure burden and their association with SUDEP, as electrographic brain activity could reveal subtle but significant differences in seizure dynamics and interictal activity.
The study revealed valuable insights into sex-dependent differences in BBB permeability and differentially expressed genes between Scn8a D/+ and wild-type (WT) mice, both before and after TC seizure development. Blood–brain barrier leakage, measured by [C14] sucrose concentrations in the brain, was higher in Scn8a D/+ mice compared to WT mice and worsened from pre-TC to post-TC seizure periods, with male Scn8a D/+ mice showing more pronounced effects. These findings demonstrate an important connection between seizure burden and BBB leakage, though they do not yet definitively establish whether BBB instability precedes or follows seizure onset. Establishing which comes first is essential for understanding how BBB integrity and seizures interact. Going forward, as noted above, comprehensive EEG analysis across both sexes could help map this temporal relationship and potentially reveal sex-specific differences and similarities in epilepsy progression and the association to the brain molecular profile (discussed below).
Additionally, the study describes compelling evidence of profound sex-specific transcriptomic differences in hippocampi of Scn8a D/+ mice before and after the development of TC seizures. Pathway enrichment analysis revealed that, prior to TC onset, Scn8a D/+ females exhibited upregulation in energy metabolism and oxidative phosphorylation pathways, which shifted to cholesterol biosynthesis pathways following the onset of TC seizures. In contrast, Scn8a D/+ males demonstrated a broader range of upregulated pathways compared to females at both the pre-TC and post-TC seizure onset time points. Before TC seizure onset, males showed increased transcripts related to brain structure (myelination and synaptogenesis), transcriptional regulation (CREB), and glial function (S100). After TC seizures developed, males showed upregulation in pathways mainly associated with gliosis and inflammation, which are consistent with previous findings. 9 We noted that the authors used a consistent method to analyze these pathways while recognizing potential biases and limitations, such as the small sample size for the bulk RNAseq analysis (n = 3 per group). Moreover, the authors proposed a complex model explaining sex-specific mechanisms of epilepsy. In females, estrogen-related neuroprotective pathways and oxidative phosphorylation may contribute to BBB disruption, epileptogenesis, and chronic seizures. In males, neuroinflammatory pathways and gliosis appear to play similar roles. However, rigorous mechanistic studies are needed to definitively determine whether these distinct molecular landscapes are beneficial or detrimental, causal or consequential to seizure burden, and SUDEP in Scn8a D/+ mice.
In summary, few studies have explicitly explored sex differences in brain molecular profiles during epileptogenesis or epilepsy using proteomic, transcriptomic, or multiomic approaches. 9 Thus, a strength of the study by Hammer and others is that it helps fill this critical gap by comprehensively measuring gene expression patterns in a human-relevant genetic mouse model of epilepsy. The finding that female and male Scn8a D/+ mice develop epilepsy despite showing distinct gene expression profiles in the hippocampal network suggests that each sex may employ unique mechanisms to drive or modulate the neuronal hyperexcitability that leads to seizures. Thus, it is possible that seizures may not be created equal in males and females. The evidence of sex dimorphism in epilepsy, which extends beyond female-specific epilepsies (eg, catamenial epilepsy and perinatal epilepsy), challenges the “one-size-fits-all” approach in understanding epilepsy mechanisms and treatment strategies. Taken together, these observations and results emphasize the critical need to include both sexes in basic epilepsy research and in clinical studies, where female representation has historically remained inadequate.1,3
