Normal Development of the Perineuronal Net in Humans; In Patients with and without Epilepsy.

The perineuronal net (PN), a highly organized extracellular matrix structure, is believed to play an important role in synaptic function, including maturation and stabilization. In addition to its role in restricting plasticity, alterations in the PN are implicated in disorders such as epilepsy and schizophrenia. However, the time course of PN development is not known in humans. Therefore we set out to document the developmental timeline of the PN formation in humans in 14 frontal and hippocampal specimens from donors aged 27 days to 31 years old. Using immunohistochemistry and western blotting, we demonstrate that the PN begins to form as early as the second month of life but does not reach its robust, mature appearance until around 8 years of age, though aggrecan cleavage products are observed prior to this. A similar developmental time course was observed in specimens from epilepsy patients. Our data suggest that aggrecan is present early in development but the structured PN develops throughout early childhood, similar to what has been observed in rodents. This timeline provides information for future pathological studies on the role of the PN in disease and an additional parallel between human and rodent development.

Changes in microRNA expression in the whole hippocampus and hippocampal synaptoneurosome fraction following pilocarpine induced status epilepticus.

MicroRNAs regulate protein synthesis by binding non-translated regions of mRNAs and suppressing translation and/or increasing mRNA degradation. MicroRNAs play an important role in the nervous system including controlling synaptic plasticity. Their expression is altered in disease states including stroke, head injury and epilepsy. To better understand microRNA expression changes that might contribute to the development of epilepsy, microRNA arrays were performed on rat hippocampus 4 hours, 48 hours and 3 weeks following an episode of pilocarpine induced status epilepticus. Eighty microRNAs increased at one or more of the time points. No microRNAs decreased at 4 hours, and only a few decreased at 3 weeks, but 188 decreased 48 hours after status epilepticus. The large number of microRNAs with altered expression following status epilepticus suggests that microRNA regulation of translation has the potential to contribute to changes in protein expression during epileptogenesis. We carried out a second set of array's comparing microRNA expression at 48 hours in synaptoneurosome and nuclear fractions of the hippocampus. In control rat hippocampi multiple microRNAs were enriched in the synaptoneurosomal fraction as compared to the nuclear fraction. In contrast, 48 hours after status epilepticus only one microRNA was enriched in the synaptoneurosome fraction. The loss of microRNAs enriched in the synaptoneurosomal fraction implies a dramatic change in translational regulation in synapses 48 hours after status epilepticus.

Neurotrophin-3 mRNA a putative target of miR21 following status epilepticus.

Status epilepticus induces a cascade of protein expression changes contributing to the subsequent development of epilepsy. By identifying the cascade of molecular changes that contribute to the development of epilepsy we hope to be able to design therapeutics for preventing epilepsy. MicroRNAs influence gene expression by altering mRNA stability and/or translation and have been implicated in the pathology of multiple diseases. MiR21 and its co-transcript miR21, microRNAs produced from either the 5' or 3' ends of the same precursor RNA strand, are increased in the hippocampus following status epilepticus. We have identified a miR21 binding site, in the 3' UTR of neurotrophin-3 that inhibits translation. Neurotrophin-3 mRNA levels decrease in the hippocampus following SE concurrent with the increase in miR21. MiR21 levels in cultured hippocampal neurons inversely correlate with neurotrophin-3 mRNA levels. Treatment of hippocampal neuronal cultures with excess K(+)Cl(-), a depolarizing agent mimicking the episode of status epilepticus, also results in an increase in miR21 and a decrease in neurotrophin-3 mRNA. MiR21 is a candidate for regulating neurotrophin-3 signaling in the hippocampus following status epilepticus.