Estradiol- and progesterone-associated changes in microRNA-induced silencing and reduced anti-seizure efficacy of an antagomir in female mice.

About one third of individuals living with epilepsy have treatment-resistant seizures. Alternative therapeutic strategies are thus urgently needed. One potential novel treatment target is microRNA-induced silencing, which is differentially regulated in epilepsy. Inhibitors (antagomirs) of specific microRNAs have shown therapeutic promise in preclinical epilepsy studies; however, these studies were mainly conducted in male rodent models, and research into microRNA regulation in females and by female hormones in epilepsy is scarce. This is problematic because female sex and the menstrual cycle can affect the disease course of epilepsy and may, therefore, also alter the efficacy of potential microRNA-targeted treatments. Here, we used the proconvulsant microRNA miR-324-5p and its target, the potassium channel Kv4.2, as an example to test how microRNA-induced silencing and the efficacy of antagomirs in epilepsy are altered in female mice. We showed that Kv4.2 protein is reduced after seizures in female mice similar to male mice; however, in contrast to male mice, microRNA-induced silencing of Kv4.2 is unchanged, and miR-324-5p activity, as measured by the association with the RNA-induced silencing complex, is reduced in females after seizure. Moreover, a miR-324-5p antagomir does not consistently reduce seizure frequency or increase Kv4.2 in female mice. As a possible underlying mechanism, we found that miR-324-5p activity and silencing of Kv4.2 in the brain were differentially correlated with plasma levels of 17β-estradiol and progesterone. Our results suggest that hormonal fluctuations in sexually mature female mice influence microRNA-induced silencing and could alter the efficacy of potential future microRNA-based treatments for epilepsy in females. Significance Statement MicroRNA-induced silencing is currently investigated in preclinical studies as a potential novel class of treatment target for epilepsy; however, surprisingly little is known about if and how microRNA-induced silencing is regulated by biological sex and if microRNA therapeutics are as effective in females as they are in males. This study shows that microRNA silencing of the potassium channel Kv4.2 and the functional activity of its targeting microRNA, miR-324-5p, change with plasma levels of estrogens and progesterone and that an inhibitor of miR-324-5p is less effective in female mice to suppress seizures. These findings are significant as they suggest microRNA-induced silencing as a novel molecular mechanism contributing to sex differences in epilepsy that could impact future therapy development.