miRNA regulation of developmental sodium channel isoform transition and its implications for Dravet syndrome

Project Information

miRSodium

Grant agreement ID: 101064343

DOI

10.3030/101064343

Project closed

EC signature date 20 June 2022

Start date 1 September 2022

End date 31 August 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 215 534,40

Coordinated by

ROYAL COLLEGE OF SURGEONS IN IRELAND
Ireland

Periodic Reporting for period 1 - miRSodium (miRNA regulation of developmental sodium channel isoform transition and its implications for Dravet syndrome)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

The miRSodium project focused on understanding how the levels of sodium ion channels are controlled in the developing brain and how this could be used for new therapies. Sodium ion channels are specialised proteins that allow the flow of ions in and out of cells and thus control how active nerve cells are and how they work together. Mutations in the gene SCN1A, which gives rise to one of these channels (sodium channel 1.1) cause a severe and rare form of epilepsy in children called Dravet syndrome (DS). Dravet syndrome leads to recurrent seizures, learning difficulties, developmental delay, other neurological problems and often sudden unexplained death in epilepsy (SUDEP) in children. Even though we know it is caused by a mutated gene SCN1A, we still do not understand a lot about how epilepsy works in people with DS, and there is no cure available. DS patients have the functional gene SCN3A producing a different version of the same channel. Around the age of 6 months, the expression of the channel from the defective SCN1A gene becomes dominant over SCN3A and causes abnormal neuronal function resulting in DS. Since children develop normally before the transition from functional SCN3A to non-functional SCN1A, we hypothesise that understanding how this transition in expression occurs can open new options for DS therapy.
To explore this hypothesis, the miRSodium project focused on the involvement of small molecules called microRNAs (miRNAs) in the regulation of sodium channel production and their potential contribution to the transition between SCN1A and SCN3A. MicroRNAs are small naturally occurring molecules that control the activity of genes - they are molecular switches that can turn genes on or off. As the transition between sodium channel versions occurs across mammalian species, we studied its mechanism in the mouse model recapitulating DS symptoms (Scn1a+/-) and control animals without the mutated gene. Investigation in the animal model allows us to explore the activity of miRNAs and the targets under their control in young brains before and during the onset of DS symptoms (typically on day 17 in mice). We aim to investigate how specific miRNAs influence ion channels, with the goal of developing a novel treatment approach that can relieve symptoms of DS.

Project Objectives:
• Enhance our understanding of the molecular processes ongoing in DS.
• Identify miRNAs that regulate sodium ion channels involved in DS.
• Explore the impact of manipulating these miRNAs on the function of brain cells.
• Test in a mouse model if the manipulation of these miRNAs leads to the reduction of DS symptoms.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The overall scientific goals of the project were achieved. The active miRNA participation in gene expression regulation was evaluated in the brains of young mice – both healthy and in the context of mutation causing Dravet Syndrome. Before the onset of DS symptoms, miRNA regulation did not impact genes differently in mice with the mutated Scn1a gene compared with controls. Interestingly, we observed altered regulation of six genes in post-DS-onset mice. Our analysis confirmed that miRNAs regulate multiple versions of sodium channels (including those expressed from Scn1a and Scn3a genes) before and during the onset and progression of DS. Based on these results, we identified and validated miRNAs governing the protein production from Scn1a and Scn3a genes. As miRNAs act as negative regulators of sodium channels, we tested if we could increase the expression of these proteins by miRNA inhibition. Our analyses in cell cultures of mouse neurons showed that inhibition of miRNAs controlling expression of genes Scn1a and Scn3a leads to increased amount of sodium channels. This inhibition did not show a negative effect on the cell viability or excitability in cultured neurons. Finally, the inhibition of selected miRNA led to increased sodium channel levels in young mouse brains. While this increase did not lead to suppression of DS symptoms in mice with mutated Scn1a gene at the tested dose, these results indicate the capacity of miRNAs to manipulate sodium channel levels in the mammalian brain and encourage further investigation of this phenomenon.
The work completed by the fellow in this MSCA PF project has to date resulted in project results disclosed at 3 conferences with different audiences, 3 institutional talks and 3 poster presentations, and a journal article currently in preparation. We generated a list of miRNAs with validated regulation of sodium channels Scn1a and Scn3a and two large datasets describing miRNA activity in young mouse brains covering the period from the birth of the mice, processes preceding the onset of DS (including the transition in sodium channel dominance) until the age 22 days – shortly after the onset of DS symptoms in mice. Both datasets were uploaded to an online database and will be freely available for reuse by other researchers upon publication of our results.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The miRSodium project’s findings on how miRNAs control sodium ion channels is important for DS and other epilepsies. Our analyses disclosed that miRNAs control the gene expression differently in the brains of mice at different ages between birth and day 22 (in both DS and control mice). This brings us closer to the understanding of gene expression regulation in the young postnatal brain. The differences in miRNA regulation observed in animals after DS onset (and the detected sex-specific alterations) point towards altered mechanisms in the DS brain beyond the sodium channel mutation. Importantly, we confirmed that sodium channel subunits as well as other genes relevant for epilepsy are regulated by miRNAs in young brains before and during the onset of DS. While miRNAs exert control over sodium channels, our data indicate that rather than triggering the transition between Scn1a and Scn3a, miRNAs fine-tune the expression of these genes. To unlock the potential to intervene with their transition, additional mechanisms of gene expression regulation (e.g. epigenetic manipulations, long RNAs with regulatory functions or activity of protein regulators) need to be addressed by future research.
We validated the control over sodium channels Scn1a and Scn3a for five miRNAs, opening up their potential use for manipulation of sodium channel levels in DS and other sodium channel-related pathologies (e.g. neuropathic pain). While we did not observe the improvement of DS symptoms in mice undergoing treatment with miRNA inhibitors, we detected an increase in sodium channel expression in these mice, proving that miRNA manipulation can adjust levels of these channels in the brain. Further investigation is needed to explore if the miRNA-based increase of sodium channel expression can achieve favourable outcomes in DS. We will continue this work and apply for funding for a follow-up project(s) focused on the deeper scrutiny of miRNA manipulation of sodium channels in DS (testing a wider range of miRNA inhibitor doses, testing additional routes of administration or investigating additional miRNA candidates) and investigation of both short-term and long-term outcomes in the mouse model.
Via completion of the miRSodium MSCA PF, the postdoctoral fellow gained experience with the state-of-the-art research methodology in the area of neuroscience as well as skills and competencies essential for research career progression, including project management, leadership and innovation and established a rich network of (potential) collaborators.