Poster Number: 1.014

Rationale:

Epilepsy is one of the most prevalent chronic neurological disorders globally. Among its many etiologies, central nervous system (CNS) infections represent a significant and under-addressed cause. Despite this, there are currently no anti-seizure medications specifically designed to target infection-induced epilepsy. Theiler’s murine encephalomyelitis virus (TMEV) is a well-established viral model that induces seizures in mice with relatively low mortality, making it a valuable tool for investigating infection-associated epileptogenesis(1). We previously demonstrated that TMEV-infected mice exhibiting seizures have reduced levels of gut bacteria that produce the metabolite S-equol(2). Moreover, ex vivo application of S-equol to cortical brain slices from these animals increased the threshold for action potential firing and reduced the number of action potentials, suggesting that S-equol suppresses neuronal excitability. However, the specific molecular targets and mechanisms underlying this effect remain unclear. This study aimed to identify the role of S-equol’s known targets in reducing neuronal hyperexcitability.

C57BL/6 (Jax) mice were infected with TMEV and monitored for acute behavioral seizures from 3 to 7 days post-infection (dpi). Whole-cell patch-clamp recordings were performed on cortical neurons from 7–8 days post TMEV-infected mice with a seizure phenotype. Action potential number and threshold were assessed in the presence of S-equol alone and in combination with pharmacological inhibitors of S-equol’s known targets: G protein-coupled estrogen receptor 1 (GPR30/GPER1), large-conductance calcium-activated potassium (BK) channels, and estrogen receptor beta (ESRβ). Western blotting was used to assess the protein expression levels of these targets relative to sham-infected controls (PBS injected mice).

Western blot analysis showed no significant difference in ESRβ expression between TMEV seizure mice and controls. Electrophysiological recordings indicated that blocking ESRβ did not alter the S-equol-mediated effects on action potential threshold or number. Protein expression of BK channel alpha subunit was significantly reduced in TMEV seizure mice compared to controls. Electrophysiological recordings indicated that S-equol significantly increased action potential threshold, however, pharmacological blockade of BK channels with paxillin did not influence action potential properties in the presence of S-equol. Notably, GPR30/GPER1 protein expression was also decreased in TMEV seizure mice. Importantly, co-application of the GPR30/GPER1 antagonist G15 abolished the effects of S-equol on both action potential number and threshold.