The functions of repressor element 1-silencing transcription factor in models of epileptogenesis and post-ischemia.

Epilepsy is a debilitating neurological disorder characterised by recurrent seizures for which 30% of patients are refractory to current treatments. The genetic and molecular aetiologies behind epilepsy are under investigation with the goal of developing new epilepsy medications. The transcriptional repressor REST (Repressor Element 1-Silencing Transcription factor) is a focus of interest as it is consistently upregulated in epilepsy patients and following brain insult in animal models of epilepsy and ischemia. This review analyses data from different epilepsy models and discusses the contribution of REST to epileptogenesis. We propose that in healthy brains REST acts in a protective manner to homeostatically downregulate increases in excitability, to protect against seizure through downregulation of BDNF (Brain-Derived Neurotrophic Factor) and its receptor, TrkB (Tropomyosin receptor kinase B). However, in epilepsy patients and post-seizure, REST may increase to a larger degree, which allows downregulation of the glutamate receptor subunit GluR2. This leads to AMPA glutamate receptors lacking GluR2 subunits, which have increased permeability to Ca2+, causing excitotoxicity, cell death and seizure. This concept highlights therapeutic potential of REST modulation through gene therapy in epilepsy patients.

Efficient infection of organotypic hippocampal slice cultures with adenovirus carrying the transgene REST/NRSF.

Organotypic hippocampal slice cultures provide a useful platform maintaining hippocampal structure and synaptic connections of the brain over weeks in culture with ease of in vitro manipulations. Gene transfer is a particularly desirable tool for using with them but current difficulties with transformation of transgenes into these cultures is a barrier to their use in research. Previous quantifications of viral infections have shown low transformation rates and have relied upon invasive microinjections. In this paper we present an efficient way of infecting organotypic cultures with adenovirus at the acute slice stage that does not require injection. We use the adenoviral delivery system to introduce the transcription factor REST and a GFP marker, providing around 41 % cellular infection spread throughout the entire slice culture and promoting transgene expression for weeks in vitro. GFP expression was observed most intensely in the slices when they were infected just a few hours after plating and was shown to infect neurons and microglia. We decided to use the transcription factor REST/NRSF as an example transgene which was delivered into cells via the adenoviral construct, conferring overexpression of REST in addition to the GFP marker. This outlines a technique whereby adenoviral infection of organotypic cultures can infect neurons with good efficiency and confer successful manipulation of genetic factors within the cell.