Background

Early-life seizures (ELS) are associated with development of epilepsy and cognitive deficits that persist into adulthood. One potentially targetable process linking ELS to cognitive disruptions is the regulation of dendritic spine development. ELS occurring during critical periods of brain maturation can disrupt dendritic spine structural plasticity and lead to development of cognitive impairment. We hypothesize that seizure-like activity disrupts actin turnover by affecting the actin-capping protein Eps8, presumably decreasing the spines’ capacity for structural plasticity. Here we prepare to test this hypothesis by determining the timeline of EPS8 downregulation in cultured hippocampal neurons.

Methods

Genomic DNA was collected from neonatal rat hippocampal neurons harvested at embryonic day 18 and cultured in vitro for 16 days (DIV16). PCR primers for Eps8 were designed, and siRNA against Eps8 was transfected into additional DIV16 rat hippocampal neurons using lipofectamine RNAiMAX. RNA was collected at 24-, 48-, and 72-hours post-transfection, and from non-transfected DIV16 cells. qPCR was performed on these samples.

Results

Following transfection, Eps8 gene expression decreased compared to age-matched controls. Specifically, gene expression decreased after 24 hours post-transfection and remained decreased 72 hours post-transfection. PCR of the genomic DNA resulted in a single product of the expected size, as seen following agarose gel electrophoresis, and melt-curve analysis suggested amplification of a single product following qPCR. Interestingly, transfection with a GAPDH siRNA showed a decrease in Eps8 gene expression, but it remains unclear if this is an effect of GAPDH knockdown or simply an effect of transfection itself.

Future Directions

We will use the current paradigm to silence Eps8 in neurons, then induce seizure-like activity at DIV17, then assess the resulting dendritic spine morphology and plasticity. We will also assess gene and protein expression. Finally, using GeneMania, an open-source online protein interaction database, we will determine and investigate potential protein interactors.