Layer-Specific CREB Target Gene Induction in Human Neocortical Epilepsy

Abstract

Epilepsy is a disorder of recurrent seizures that affects 1% of the population. To understand why some areas of cerebral cortex produce seizures and others do not, we identified differentially expressed genes in human epileptic neocortex compared to nearby regions that did not produce seizures. The transcriptome that emerged strongly implicates MAP kinase signaling and CREB-dependent transcription, with 74% of differentially expressed genes containing a cyclic AMP response element (CRE) in their proximal promoter, more than half of which are conserved. Despite the absence of recent seizures in these patients, epileptic brain regions prone to seizures showed persistent activation of ERK and CREB. Persistent CREB activation was directly linked to CREB-dependent gene transcription by chromatin immunoprecipitation that showed phosphorylated CREB constitutively associated with the proximal promoters of many of the induced target genes involved in neuronal signaling, excitability and synaptic plasticity. A distinct spatial pattern of ERK activation was seen in superficial axodendritic processes of epileptic neocortex that co-localized with both CREB phosphorylation and CREB target gene induction in well-demarcated populations of layer 2/3 neurons. These same neuronal lamina showed a marked increase in synaptic density. The findings generated in this study generate a robust and spatially-restricted pattern of epileptic biomarkers and associated synaptic changes that could lead to new mechanistic insights and potential therapeutic targets for human epilepsy.

Epileptogenesis

“…For example, inducible cAMP early repressor (ICER) has been suggested to play a role in epileptogenesis as it suppresses kindling (Kojima et al 2008;Porter et al 2008). Other candidates include cAMP response element (CREB), controlling differential expression of genes in human epileptic cortex (Beaumont et al 2012), and repressor element-1 silencing transcription factor (NRSF) shown to repress epileptogenesis in a kindling model and reported to regulate target genes relevant for neuronal network remodeling in kainate-induced SE (Hu et al 2011;McClelland et al 2014).…”

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

“…Upstream of CREB activation, several known signaling pathways are rapidly activated in response to neuronal activity (Kandel 2001; reviewed in West et al 2002), including CaMKinase IV, protein kinase A, and MAPK. We have recently observed a pattern of transcriptional activation in human brain regions where seizures start that strongly implicates sustained MAPK/CREB activation and downstream coding gene activations that could underlie layerspecific changes in synaptic architecture that makes these regions prone to seizures (Rakhade et al 2005;Barkmeier et al 2012;Beaumont et al 2012).Given that human lncRNA genes tend to be less wellconserved than protein-coding genes, and can give rise to unique transcripts not found in other species, we sought out a uniquely human system to examine activity-dependent gene expression for both coding and noncoding RNAs using a pairwise comparison of human cortical regions displaying variable degrees of epileptic activities. These brain regions were removed as part of surgical treatment for intractable seizures.…”

“…The paired analysis of high-and low-spiking neocortex within each patient is also critical to isolate the variable under study, which is the degree of activity. Total RNA was prepared using a modification of the protocol described previously (Beaumont et al 2012). The difference was that only gray matter was used by pooling two to three nearby strips of gray matter that extended from the pial surface to the white matter from each block of tissue corresponding to a given electrode location.…”

“…Only repeat-free and uniquely-mapping 60mer probes were included. Specificity is assured by these sequence qualities of the probes as well as by our strategy of profiling each lncRNA gene with seven unique probe sequences (not seven replicates of the same probe).A dye-flip quadruplicate two-color microarray experiment was performed on each within-patient pair of high-spiking and low-spiking surgically resected samples on both the Agilent human genome-wide array (G4112A) and our custom lncRNA array as described, but using a different labeling method (Beaumont et al 2012). We used the Epicentre protocol to generate aminoallyl-amplified RNA (aRNA) for subsequent amplification and labeling with either cyanine or Alexa dyes.…”

Integrative analysis of gene expression associated with epilepsy in human epilepsy and animal models

“…Specifically, GRB2 (21) and CDKN1A (22) have been reported to be upregulated in human epilepsy, and APP (23) and TGF-β (24,25) were found to be upregulated following epileptic seizures in rat a model, based on DNA microarrays. Compared with wild-type mice, RT-qPCR has shown that the mRNA expression of VEGF is 1.44-fold higher in the hippocampus of VEGF Receptor-2 (Flk-1)-overexpressing mice, characterized by an elevated threshold for seizure induction (26).…”