Molecular biology

Of particular interest to the laboratory are a novel class of short endogenous RNAs (MicroRNAs) that act as post-transcriptional non-coding transcripts capable of regulating gene expression. The expanding inventory of human miRNAs along with their highly diverse expression patterns and high number of potential target mRNAs suggest that miRNAs are involved in a wide variety of human diseases. Most miRNAs are conserved between humans and mice, which makes it possible to analyze their expressions with a set of selected array probes. But as of date, the gene specific role of miRNAs before and after epileptic seizures is unknown, especially in commonly used in vitro seizure models. In the current project we use microarray technology and quantitative real-time polymerase chain reaction to examine changes in miRNAs and mRNA expression in acute mouse hippocampus transverse slices (P21-25) exposed to artificial cerebrospinal fluid containing the potassium channel blocker 4-Aminopyridine (4-AP). Since this model is extensively studied in our and other laboratories, it compliments well the ongoing studies and provide underlying molecular changes of the mechanisms of seizure generation. During the experiments, slice tissue is sampled at the initial stages of acute seizure formation (20 min.) and after repetitive seizures have occurred (90 min.), as shown using electrophysiology. Our preliminary results suggest that several miRNA’s showing dynamic changes in expression are closely associated with key human ‘epilepsy genes’. We propose to perform silencing experiments using siRNA-mediated knockdown of epilepsy-associated miRNAs or their target genes in the organotypic hippocampal culture. We will test the susceptibility of the transfected slices to seizure induction by 4-AP and sustenance using multiple simultaneous extracellular recordings in the hippocampus. Novel findings of this project not only help us elucidate the significance of miRNAs mediated dynamic gene regulation, but will also allow to evaluate potential therapeutic biomarkers that can possibly be targeted in prevention or cure of epilepsy.

NOTE: Due to the sensitivity of our findings, the exact miRNA designations are not shown at present (for more information, please contact Anupam Hazra).

One of the key preliminary findings in this study indicates that miRNA can exhibit an ~ twelve fold downregulation after just twenty minutes following exposure to the convulsant solution. Furthermore, as illustrated in bar graph above (left), two key miRNAs showed differential expression at twenty versus the ninety minute timepoint. This indicates dynamic switch in miRNA role within the time frame corresponding to the formation of repetitive seizures. The microarray 'heat maps' above (right) show the overall miRNA changes at twenty and ninety minutes