Functional significance of neuronal sodium channel splicing in epilepsy and pain

Many neurological disorders, including epilepsy and chronic pain, are associated with changes in the functioning of nerve cells. These cells become more active than in healthy tissue, and can even start sending signals when they should remain quiet. It is thought that these changes can lead to the uncontrolled activity in seizures, or the chronic sensation of pain, even when there is no painful stimulus present. But why do the cells become excessively active?

We are seeking to answer this question by exploring the changes in the processing of molecules which drive neuronal activity: the sodium channels. Many sodium channels exist in two versions, one predominates in healthy adult neurons, the 'adult variant', the other, almost exactly the same, predominates in very young neurons, the 'neonatal variant'. However in some diseases, such as epilepsy, the neonatal variant is increased, even in mature nerve cells. How does this increase in neonatal channels alter the behaviour of adult nerve cells?

We are especially interested in the change in the sodium channels because we know that many common drugs bind to these channels. These include common anti-epileptics, most local anaesthetics, and other drugs used to treat a growing number of disorders, including chronic pain. At present, many of these drugs are thought to bind to *all* sodium channel variants, but some drugs may show some preference for the neonatal or the adult channels. If the neonatal variant is being up-regulated in disease, is it helpful to specifically target this variant - to knock it back down? Could deliberately suppressing only the neonatal variant reduce the side-effects caused when these drugs bind to healthy sodium channels?

An additional line of evidence suggesting these variants are important is that they are extremely highly conserved in evolution, but as yet it is not known what function they play in any animal, from flies to humans. Such conservation is usually a sign that a variation is important to healthy functioning of the organism. We will investigate the roles of these highly-conserved variants healthy nerve cells, and also in models of both epilepsy and pain, and will use our findings to recommend whether pharmacological companies should consider developing compounds which are specific for either variant.

Our findings will offer new perspectives on how these fundamental channels are regulated, and whether there is an avenue for treatments that are better tolerated in humans.

While it has been known for decades that sodium channels are subject to highly conserved alternative splicing in their voltage sensing paddles, the functional role and adaptive significance of this splicing has been obscured by the challenge of expressing, clamping and comparing these channels in neurons. The sensitivity of the channels to the intracellular modulation, and their specialisation for neuronal functions, means that heterologous expression is severely limited, and even over-expression of cDNAs in neurons may not recapitulate the roles of these highly similar variants.
We have recently established tools that enable us to ask, in neurons, the functional consequences of selectively manipulating splice variants. We have developed shRNA sequences that can significantly alter the proportion of each splice variant in neurons. Using our lentiviral-delivered shRNAs we have shown in vitro, that reducing each of the two variants of SCN8A leads to distinct effects, with reducing the 'neonatal' variant significantly altering resting membrane potential, while reducing the 'adult' variant disrupts regular firing, and appears to reduce the contribution of the axon initial segment to action potentials.
We have developed four parallel approaches for measuring the functional roles and distribution of splice variants in neurons. In addition we have two models of clinically-important disease (epilepsy and pain) that are strongly associated with altered sodium channel activity. We will test whether key drugs that modulate sodium channels can distinguish between the functions of splice variants, and whether the changes in this voltage sensing paddle may be a potential target for rationally-designed drugs. Finally we will return to the question of how a human polymorphism that changes splicing might lead to altered dosage of drugs to ask how splicing impacts drug response.

Impact on Health: Epilepsy and pain are two serious neurological disorders that can have devastating effects on patients. Both are often resistant to existing pharmaceutical treatments. One problem is that many of the treatments to both of these disorders target sodium channels, but fail to distinguish between 'healthy' and 'abnormal' channels. By probing one of the physical changes that occurs in sodium channels during the progression of diseases, we will raise the possibility of treatments with fewer off-target effects, by demonstrating the impact of selectively manipulating the 'abnormal' channel splice variants. Improved treatments to chronic and serious neurological disorders would offer an improvement in quality of life.
The third sector: SS currently serves on the Science Advisory Committee of the charity Epilepsy Research UK, and this involvement means our findings will be transmitted quickly to the patient bodies and other researchers in this sector, where other researchers may adapt it to generate new hypotheses and to exploit findings for new treatment strategies.

Timescales: During the course of the project we will present data at charities and symposia that will reach our academic and clinical beneficiaries. Our publications will fall towards the end of the project, and shortly thereafter. By the end of the project (3 years) we hope that our findings will be informing other studies of drug response in epilepsy and pain, and any data that suggest an effective approach to mitigating seizures or excess excitability of pain sensing neurons will serve to underpin applications for pre-clinical studies (4 years). In the longer term, our findings may lead to rationally targeted novel anti-epileptic drugs and pain therapy.

Skills for staff on project: The staff will benefit from the development of advanced skills in slice patch clamp and use of GM viruses, a growing field for gene therapy treatments. We have found skilled electrophysiologists to be in high demand (both when we try to recruit, and when students graduate from the group), thus we anticipate that the skills they hone will place them in high demand for future positions.