Abstracts

This is a Late-Breaking abstract.

Rationale: Early-life seizures (ELS) are associated with development of epilepsy and cognitive deficits that persist into adulthood. One potentially targetable process linking ELS to cognition is the regulation of dendritic spine development. We hypothesize that seizure-like activity disrupts actin turnover through the actin-capping protein Eps8, decreasing the spines’ capacity for structural plasticity to lead to cognitive impairment. We previously assessed Eps8 protein expression and localization in cells from 10 days in vitro (DIV10) through to DIV21 via immunohistochemistry and found increased localization throughout dendrites (especially at the dendritic spines) with age. When seizure-like activity was induced in this period, Eps8 dendritic protein expression and spine motility was reduced. Here we aimed to determine the timeline of eps8 gene regulation using an in vitro model of seizure activity in rat hippocampal neurons. Specifically, we assessed eps8 gene expression following in vitro seizure-like activity understand the molecular mechanisms underlying dysfunction following ELS.

Methods: Hippocampal neurons were cultured from embryonic day (E)18 rats in neurobasal medium + B27 supplement.  On DIV16, the cells were transfected with siRNA against eps8 or %GC-matched scramble siRNA, or were left as non-transfected controls (NTCs). After 24 hours, cells were incubated at 37C in Mg²⁺-free media for 3 hours to induce neuronal burst firing akin to seizure-like activity.  Then, cells were returned to normal growth media and allowed to recover.  RNA was collected from cells at 3 timepoints: prior to seizure induction, 1 hour into incubation in Mg²⁺-free media, and 1 hour into the recovery phase.

Results: As expected, we found less eps8 RNA expression at baseline in cells transfected with eps8 siRNA compared to cells transfected with scramble siRNA. After 1 hour of culturing in Mg²⁺-free media, eps8 mRNA expression slightly increased compared to baseline in the NTCs and in cells transfected with scramble siRNA, but not in the cells transfected with eps8 siRNA (Figure 1). However, 1 hour after return to normal media, eps8 mRNA expression decreased over 70% in the scramble siRNA-transfected cells, and nearly 98% (p = 0.04) in the eps8 siRNA-transfected cells (Figure 2).

Conclusions: Our results suggest that eps8 mRNA upregulation begins rapidly in response to seizure-like activity and rises further within the additional 2 hours of exposure and into the recovery period. Ongoing work aims to determine the role of the upregulation in eps8 gene expression and Eps8 protein localization to the dendritic to the decrease in dendritic spine motility shown previously. We will expand the current paradigm to investigate the role of Eps8 in pathogenesis of disease following in vitro seizure activity, both in the acute stage and in later time points following seizure-like activity.

Funding: Funding was provided by Center for Chronic Diseases of Aging, PCOM Division of Research.