MicroRNAs in epilepsymiRCURY LNA™ microRNA in vivo Inhibitors
Dr. David C. Henshall
Dr. Eva M. Jimenez-Mateos
Eva M. Jimenez-Mateos and David C. Henshall work at the Centre for the Study of Neurological Disorders at the Royal College of Surgeons in Ireland. Here, they study how microRNAs influence epilepsy. Read about their results, some of the challenges involved and how they were overcome.
What is the main focus of your research?
Epilepsy is a serious, chronic neurological disorder characterized by recurrent spontaneous seizures that affects about 50 million people worldwide. Antiepileptic drugs typically control seizures in two-thirds of patients but probably do not alter the underlying pathophysiology1
. The development of symptomatic epilepsy is thought to involve altered expression of ion channels, synaptic remodeling, inflammation, gliosis and neuronal death, among other factors2
. However, few antiepileptogenic interventions targeting these processes have shown sufficient efficacy in vivo1
, and our understanding of the cell and molecular mechanisms of epileptogenesis is incomplete. We looked at miRNA because they are meta-controllers of protein levels in cells and a previously missed aspect of the pathophysiology
How did you come to be interested in microRNAs?
Evidence was emerging that microRNAs may be crucial to the pathogenesis of several neurological disorders3
, including epilepsy. In our study, we decide to focus on the role of microRNA-134 (miR-134), a brain-specific, activity-regulated microRNA that had been implicated in the control of neuronal microstructure4
What is the aim of your current project?
We wanted to know the role of miR-134 in seizures and in the development of epilepsy together with the molecular mechanism involved in the regulation of miR-134, particularly its target gene Limk1, a known regulator of dendritic spines
Which specific questions did you want to address?
Here we got interested whether blocking miR-134 might alter excitability and so we explored the in vivo
effect of silencing miR-134 using LNA™ microRNA inhibitors. We wanted to know if we could block evoked or spontaneous seizures in epilepsy models.
Which experiments had you performed leading up to this project?
First we analyzed levels of miR-134 in experimental and human epilepsy. We observed that miR-134 was up-regulated in each situation. We also used in situ
hybridization to confirm that the miR was present in neurons, not glia. Then, we decided to analyze if inhibition of miR-134 affects spontaneous seizures, development of seizures and pathophysiology of epilepsy.
What, if any, was your previous experience with microRNA inhibition?
Previously we used microRNAs inhibitors to target miR-132. We found that this protected the mouse brain against damage caused by a prolonged seizure, but it actually prevent the seizures themselves.
How did you perform the experiments and analyze the results?
For the in situ
hybridization we used 5’ DIG-labeled LNA™ microRNA detection probes against miR-134 and a scramble control. The results were visualized using Phosphatase Alkaline substrate. The inhibition of miR-34 was carried out using intra-cerebroventricular injection of a cholesterol-modified LNA™ microRNA inhibitor or a scrambled control.
What were some specific challenges in your experiments?
A main challenge in the project was finding the optimal dose of inhibitor and scramble control to get specific inhibition and avoid off-target effects.
How did you overcome the challenges?
We performed dose-response studies at varying doses of scramble or microRNA inhibitor (0.12nmol, 0.5 nmol and 1.0nmol) and analyzed the results using qPCR. In addition, we performed western-blot analysis of several known target genes of miR-134, Limk1 and CREB 24h after a single injection of scramble or inhibitor, to confirm the effects of the inhibitor.
What is the main advantage of the LNA™ microRNA inhibitors from Exiqon?
For us the main benefits of Exiqon’s microRNA inhibitors have been potency and high stability. Just a single injection of the antagomir produced an effect lasting 2 months. The potency also meant that only very small quantities were needed. Solubility was also important, the antagomir could be dissolved in a suitable vehicle for injection into the mouse brain.
What would be your advice to colleagues about getting started with microRNA inhibition and in particular in vivo inhibition?
Particularly for in vivo
injection we recommend checking the dose for each inhibitor. Our experience is that each microRNA may need a different dose of antagomir. Getting the wrong dose will likely cause off target effects that will affect your results.
What are the future perspectives for this research?
We still have several questions to answer: Do the antagomirs stop seizures in already epileptic mice? What is the mechanism? Can we deliver via other routes?
When and where will we hear more about your studies?
Original publication will follow soon.
1. Pitkänen, A. & Lukasiuk, K. Mechanisms of epileptogenesis and potential treatment targets. Lancet Neurol. 10, 173–186 (2011)
2. McNamara, J.O., Huang, Y.Z. & Leonard, A.S. Molecular signaling mechanisms underlying epileptogenesis. Sci. STKE 2006, re12 (2006)
3. Eacker, S.M., Dawson, T.M. & Dawson, V.L. Understanding microRNAs in neurodegeneration. Nat. Rev. Neurosci. 10, 837–841 (2009)
4. Schratt, G.M. et al. A brain-specific microRNA regulates dendritic spine development. Nature 439, 283–289 (2006).