SEARCH

SEARCH BY CITATION

Keywords:

  • Epilepsy;
  • Epileptogenesis;
  • Hippocampal sclerosis;
  • Noncoding RNA

Summary

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

MicroRNAs are an important class of noncoding RNA, which function as posttranscriptional regulators of protein levels within cells. Emerging work has revealed that status epilepticus produces select changes to microRNA levels within the brain, which may impact levels of proteins involved in neuronal structure and excitability, gliosis, inflammation, and apoptosis. Animal studies show that targeting microRNAs using locked nucleic acid–modified oligonucleotides (“antagomirs”) can have potent effects on status epilepticus, seizure-induced neuronal death, and the later emergence of recurrent spontaneous seizures. Accordingly, microRNA-based therapeutics may have potential as a future treatment of status epilepticus.

MicroRNA (miRNA) is an important class of small noncoding RNA, which is critical for network-level regulation of gene expression. Biogenesis of miRNAs begins with transcription of a primary transcript (pri-miRNA), mainly from intergenic or intragenic regions of the genome, by the action of DNA polymerases (e.g., pol II). Production of the mature miRNA results from sequential actions of the RNAse III enzymes Drosha and Dicer. One strand of the mature miRNA (approximately 22 nucleotides) is incorporated into the RNA-induced silencing complex (RISC). Within the RISC, the miRNA serves as a guide to direct Argonaute-2 (Ago2) to target messenger RNAs (mRNAs). The miRNA works by forming Watson-Crick base pairs with complementary sequences of the mRNA target. This occurs mainly within the 3′ untranslated region (UTR), but binding to the 5′ UTR or coding sequence is also possible. Reduction in protein levels is due predominantly to the miRNA promoting degradation of the mRNA target, with the reminder accounted for by translation inhibition (Bartel, 2009; Krol et al., 2010).

Multitargeting Effects of miRNAs on Pathways

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

Although there are only approximately 1,500 miRNAs in the human genome, these are predicted to regulate one-third or more of all protein-coding genes. This is because a single miRNA is typically able to target approximately 200 mRNAs. RNA sequencing, ribosomal profiling (sequencing mRNAs during translation by ribosomes), and quantitative proteomics have provided critical insight into the effect of miRNA on protein levels in cells; overexpression or silencing of a miRNA produces changes to hundreds of proteins (Selbach et al., 2008; Guo et al., 2010).

miRNA Inhibitors

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

One promising approach to manipulating miRNAs is by way of chemically modified antisense oligonucleotides. Termed “antagomirs,” these bind the miRNA, resulting in its depletion from the cell. Locked nucleic acid (LNA) modification of these oligonucleotides confers long-lasting stability in tissues and biofluids. As a result, LNA-antagomirs can produce miRNA silencing lasting many weeks and are associated with upregulation of large sets (approximately 200) of target mRNAs (Elmen et al., 2008). A further development has been the design of so-called “tiny LNAs.” These are 8-mer LNA oligonucleotides that target shared seed regions of miRNA families, thus producing more extensive silencing of miRNA functions (Obad et al., 2011). An miRNA-based therapeutic, miravirsen—an LNA-based antagomir that targets miR-122 for the treatment of hepatitis C—recently successfully completed phase 2 clinical trials, suggesting that manipulation of miRNA can be safe and tolerated in patients. MiRNAs may have unprecedented potential as future treatments of human diseases.

Status Epilepticus Produces Select Changes to miRNA Levels in the Brain

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

In 2010, the first research showing miRNAs were altered by status epilepticus appeared. Along with subsequent profiling studies, we now have a detailed understanding of the bidirectional spatiotemporal miRNA changes that accompany status epilepticus. Several display consistent change, including miR-34a, miR-132, miR-134, and miR-146a. Some anticorrelated expression between miRNA and mRNA profiles has been noted, consistent with the current model of how miRNAs mainly act to downregulate their targets. The development of antibodies suitable for Ago2 immunoprecipitation enabled RISC-loaded miRNAs to be identified after status epilepticus, including miR-132 and miR-134 (Jimenez-Mateos et al., 2011, 2012). Nevertheless, we currently have a poor understanding of which proteins are actually being controlled by miRNAs after status epilepticus. Future efforts must also focus on identifying the mRNAs within the RISC.

Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

A highly attractive quality of miRNA-based drugs is the long-lasting effects achieved; hippocampal miRNAs can be inhibited for >1 month after a single intracerebral antagomir injection (Jimenez-Mateos et al., 2012). To date, antagomirs have been used to target four miRNAs in vivo, including miR-34a, miR-132, and miR-134. Of these, inhibition of miR-34a and miR-132 was reported to reduce seizure-induced neuronal death, but no effects were found on seizure severity or duration during status epilepticus (Jimenez-Mateos et al., 2011; Hu et al., 2012). Silencing miR-134 produced the most remarkable effects. This miRNA is known to target proteins involved in the control of dendritic morphology, and thus is potentially of direct relevance to excitatory neurotransmission. We showed that pretreatment (24 h) with a single intracerebroventricular microinjection of LNA-antagomirs targeting miR-134 reduced kainate-induced status epilepticus by 50–70% (Jimenez-Mateos et al., 2012). Damage to the hippocampus was also strongly reduced in the mice that were pretreated with the antagomirs. Of potential importance as a future antiepileptogenic strategy, injection of antagomirs after status epilepticus reduced the later occurrence of spontaneous seizures by 90% or more (Fig. 1). Silencing miR-134 also reduced progressive damage to the hippocampus.

image

Figure 1. Posttreatment with antagomirs reduces later spontaneous seizures. Mice were injected with antagomirs targeting miR-134 (Ant-134) or a nontargeting scrambled control (Scram) 1 h after triggering status epilepticus, and then epilepsy monitoring commenced. Graph shows box-and-whisker plots of data on telemetry (weeks 1–2) and video-monitored (weeks 3–4) spontaneous seizures in mice (n = 5–6 per group; *p < 0.05). Data are adapted from Jimenez-Mateos et al. (2012).

Download figure to PowerPoint

Summary and Future Directions

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

MiRNAs represent an entirely new target for the treatment and prevention of status epilepticus. Important next steps include optimizing doses, formulation, and route of delivery. The overall safety of antagomirs and their effectiveness in other models must be established. Undoubtedly, other high-value miRNA targets for status epilepticus exist. Other tools for modulating miRNAs in vivo have also emerged including microRNA “sponges,” which may provide even greater capacity to control miRNAs and influence status epilepticus and its harmful effects on the brain.

Acknowledgments

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

The author would like to thank Eva M. Jimenez-Mateos for assistance with the preparation of the manuscript. The author also acknowledges funding support from Science Foundation Ireland grants 08/IN.1/B1875 and 11/TIDA/B1988, and National Institutes of Health grant R56 073714.

Disclosure

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References

The author has no conflicts of interest to declare. The author confirms that he has read the Journal's position on issues involved in ethical publication and affirms that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Multitargeting Effects of miRNAs on Pathways
  4. miRNA Inhibitors
  5. Status Epilepticus Produces Select Changes to miRNA Levels in the Brain
  6. Effects of Antagomirs on Status Epilepticus, Seizure-Induced Neuronal Death, and Epileptogenesis
  7. Summary and Future Directions
  8. Acknowledgments
  9. Disclosure
  10. References
  • Bartel DP. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215233.
  • Elmen J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A, Hedtjarn M, Hansen JB, Hansen HF, Straarup EM, McCullagh K, Kearney P, Kauppinen S. (2008) Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res 36:11531162.
  • Guo H, Ingolia NT, Weissman JS, Bartel DP. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466:835840.
  • Hu K, Xie YY, Zhang C, Ouyang DS, Long HY, Sun DN, Long LL, Feng L, Li Y, Xiao B. (2012) MicroRNA expression profile of the hippocampus in a rat model of temporal lobe epilepsy and miR-34a-targeted neuroprotection against hippocampal neurone cell apoptosis post-status epilepticus. BMC Neurosci 13:115.
  • Jimenez-Mateos EM, Bray I, Sanz-Rodriguez A, Engel T, McKiernan R, Mouri G, Tanaka K, Sano T, Saugstad JA, Simon RP, Stallings RL, Henshall DC. (2011) MicroRNA expression profile after status epilepticus and hippocampal neuroprotection by targeting miR-132. Am J Pathol 179:25192532.
  • Jimenez-Mateos EM, Engel T, Merino-Serrais P, McKiernan RC, Tanaka K, Mouri G, Sano T, O'Tuathaigh C, Waddington JL, Prenter S, Delanty N, Farrell MA, O'Brien DF, Conroy RM, Stallings RL, Defelipe J, Henshall DC. (2012) Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects. Nat Med 18:10871094.
  • Krol J, Loedige I, Filipowicz W. (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597610.
  • Obad S, dos Santos CO, Petri A, Heidenblad M, Broom O, Ruse C, Fu C, Lindow M, Stenvang J, Straarup EM, Hansen HF, Koch T, Pappin D, Hannon GJ, Kauppinen S. (2011) Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet 43:371378.
  • Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455:5863.