Date of Award

9-2011

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor David C. Henshall

Second Supervisor

Dr. Norman Delanty

Keywords

Epilepsy, Seizures, MicroRNAs

Abstract

The central nervous system (CNS) is a rich source of microRNAs (miRs). Within the CNS, miRs are implicated in a myriad of cellular processes and disease states. The present work is comprised of experimental and clinical investigations. We sought to characterise the role of miRs and their biogenesis machinery in seizure preconditioning (PC) and PC-induced epileptic tolerance. In parallel, we studied these processes in hippocampal and cortical tissue surgically removed from patients with pharrnaco-resistant temporal lobe epilepsy (TLE).

MiRs are a growing family of small (-22nt) non-protein coding genes which mediate translational repression through base-pairing with the 3'UTR of their target mRNAs. Core components of the miR biogenesis machinery are critical for the production of mature miRs. Following transcription of a miR gene to a primary sequence (pri-miR), Drosha generates a precursor miR (pre-miR) for export to the cytoplasm, where Dicer, the rate limiting enzyme, yields a functional miR that is guided by Ago2 to the RNA-induced silencing complex (RISC) to bind its target mRNA.

The brain possesses an inherent capacity to adapt to harmful stressors by activating endogenous programmes of neuroprotection. PC describes a seizure stimulus that protects the brain against a subsequent harmful seizure, known as tolerance. Studies suggest that significant changes to gene expression occur in the wake of seizure PC that give rise to epileptic tolerance. We profiled miR expression (CA3 subfield) in mice, in the interval (8 hours) between the PC event (systemic kainic acid [KA], 15mgIkg) and the time at which tolerance is known to be acquired (24 hours). We also characterised the spatio-temporal expression of core components of the miR biogenesis machinery (Dicer, Drosha and Ago2) in the response to seizure PC.

Expression analysis demonstrated that 155 miRs were commonly detected in control and preconditioned brain. Upregulation of miRs (25 miRs > 1.5 fold) in PC represented the predominant phenotype of the differentially expressed miRs. Expression of miR biogenesis components was consistent with these findings. MiR184 and miR204 were the two most upregulated miRs (3.26 and 2.96 fold, respectively) after seizure PC. The upregulation of miR184 was confirmed by qRT-PCR and a known target, Akt2, which showed the expected downregulation. Several others shared similar expression trends, including miR34b/c, miR7, miR448 and miR375. MiR132 was also upregulated by seizure PC. In vivo depletion of miR184 by locked nucleic acid (LNATM)- modified oligonucleotide "antagomirs" rendered an otherwise non-harmful seizure damaging, and, further, exacerbated cell death in the hippocampus in PC-induced tolerance, without altering electrographic seizures. Experimental data imply a novel, protective role for miR184 in the adaptive response to seizures.

In patients suffering with pharmaco-resistant TLE, resection of the seizure foci, often the hippocampus, represents a valuable therapeutic option to ameliorate or even eliminate seizures. Resected material offers an excellent opportunity to investigate the molecular abnormalities that underlie the pathology of this condition, namely hippocampal sclerosis. Despite evidence of miR dysregulation in some neurological disorders, miR profiling has not been undertaken in human TLE.

We carried out miR expression screens using age/gender matched sclerotic hippocampal resections from patients who underwent surgery at Beaumont Hospital, Dublin. Expression profiles were compared to autopsy control hippocampus obtained from people who died without known neurological disorders. Expression analysis demonstrated that 104 miRs were commonly detected in control and TLE patient hippocampus. Of the differentially expressed miRs, we detected significantly (> 1.5 fold) lower levels of 27 miRs, including miR132, which represented the most downregulated miR (-13 fold). In addition, 8 miRs were absent in control and TLE, while 54 miRs were uniquely absent in TLE resections. These data suggest a global collapse in miR production.

Analysis of the miR biogenesis machinery revealed full-length Dicer was significantly reduced in these patients, but present in others where miR expression was not depressed. Moreover, analysis of this pathway in resected cortical material, demonstrated that Dicer was normally expressed and several miRs, including miR132 and miR134, showed increased levels in this brain region. Bioinforrnatics predicted a widespread effect of suppressed miR signalling on biological processes including apoptosis, in the TLE brain. In support of a mechanistic explanation for these human data, status epilepticus (SEFinduced neuronal death was shown to cause Dicer cleavage and a reduction in full-length Dicer protein in the injured CA3 subfield of epileptic mice. These data suggest that altered miR signalling in TLE may be a consequence of abnormal Dicer processing within the sclerotic hippocampus.

Taken together, the present studies suggest miRs may have protective roles in the adaptive response to seizure PC, deepening our understanding of the genetic re-programming that gives rise to epileptic tolerance. Furthermore, the increased susceptibility to seizures and ongoing cell death in human TLE may be explained by this loss of miR function. This work identifies several candidate miRs with disease modifying potential.

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Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland in 2011.