The role of persistent currents generated by sodium channel splicing in neurological disorders

Lead Research Organisation: University College London
Department Name: UNLISTED

Abstract

Neurons in the brain communicate using electrical signals. These signals are generated by a particular set of molecules called sodium channels. Many drugs used to treat neurological illnesses, such as epilepsy and chronic pain, bind to sodium channels. These drugs do not simply block the channels (that would be fatal to the patient). Instead they allow the channels to make the electrical signals, but they ensure that in between the signals the channels are firmly closed. We have discovered that the genes that code for sodium channels can often be read in two alternate ways in order to make two slightly different channels from each gene. These different channels are called splice variants, because they are made by splicing the genes differently. We have shown that the splice variants from one gene are much more likely to open in between electrical signals, and consequently they may be the most important target of many drugs.
We have also found that some diseases, such as epilepsy, cause the sodium channel genes to generate more of the splice variants that open between electrical signals. This means the disease itself may lead to neurons in the brain having more of these channels that require drugs to keep closed between signals. Many diseases are thought to change the number of channels that open between signals, including Parkinson s disease, motor neurone disease and neuropathic pain, as well as epilepsy. We ask whether any or all of these diseases change the amount of sodium channel splice variants, and whether these sodium channel splice variants may represent a more specific target for drugs that would produce fewer side effects than drugs that affect all sodium channel splice variants.
By looking at the behaviour of neuronal cells grown in petri dishes, and examining how drugs bind to the sodium channels and how the genes are being spliced we will determine whether the splice variants are specifically targeted by drugs and how removing the splice variants will change the neurons. We will also investigate whether conditions that mimic diseases lead to changes in the splicing. It is possible that by preventing the neurons from making the splice variants that open between electrical signals, we will be able to directly alleviate some of the changes in neuronal behaviour linked to neurological disorders. This may lead to a new approach to treating some of these difficult and persistent disorders.

Technical Summary

Voltage-gated sodium channels generate the action potentials that define virtually all excitable cells. The rapidly inactivating transient sodium currents from these channels controls the duration of action potentials, but a second type of sodium current, the persistent current, modulates the timing, spacing and threshold of action potentials. Although the persistent current is much smaller in amplitude than the transient current, it has profound effects on neuronal properties. In fact, subtle changes in persistent currents are associated with many mutations in sodium channels that cause inherited epilepsies, and seem to be sufficient to lead to seizures.
The molecular basis of persistent currents remains mysterious. They are modulated by G-protein pathways, but may be generated by the same physical channels that generate transient sodium currents. We have recently shown that a conserved site of alternative splicing in TTX-sensitive sodium channels has dramatic effects on the amount of persistent current one type of channel (SCN1A) produces when heterologously expressed in mammalian cell lines. This alternative splicing is dynamically regulated during development, and also in epilepsy, most likely leading to changes in persistent currents in neurons and their behaviour.
We will ask how this splicing affects closely-related sodium channels, whether the changes in persistent currents seen in other neurological disorders are also associated with changes in splicing, and how directly manipulating the levels of the splice variants in vitro affects neuronal behaviour. We have also recently shown that certain sodium channel beta subunits are capable of masking the effects of alternative splicing in SCN1A, however there is evidence that these beta subunits are down-regulated in the same conditions that promote the splicing that leads to persistent currents. Consequently we will also investigate how the regulation of the beta subunits complements the changes in splicing in the alpha subunits, and whether directly down-regulating the beta subunits will lead to unmasking of low levels of alternatively spliced alpha subunits in otherwise healthy cells.
We are combining our biophysical, physiological and molecular expertise to approach the question of the regulation, functional impact and consequences of alternative splicing for these important channels. Finally, because the persistent currents are an important target of many drugs that target sodium currents, understanding the nature, generation and regulation of these currents and their link to alternative splicing should provide new insights into drug effects and may provide a more specific target for future drug design.

Publications

10 25 50
 
Description Appointed to Epilepsy Research UK Science Advisory Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Appointed to board of Trustees for Epilepsy Research UK
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of SACGM(CU)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Queen Square award for implemenation of 3Rs in Research
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact We are working to change our local attitudes towards working with animals in research. We presented this award to the then Cabinet Minister, and it was recognised as a high level local engagement with the 3Rs in animal work. All students presenting their scientific posters at the symposium are now aware that they must also explain their measures to improve 3Rs to our NACWO, including justifying the model used.
 
Description UCL Celebrates the 3Rs
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description FP7 grant
Amount £300,000 (GBP)
Funding ID 602130 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 09/2013 
End 08/2018
 
Description MRC Project Grant
Amount £400,000 (GBP)
Funding ID MR/L003457/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 01/2017
 
Description Royal Society Fellowship
Amount £550,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 10/2015
 
Description Wellcome Trust Project Grant
Amount £323,000 (GBP)
Funding ID 086882 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2009 
End 04/2012
 
Title COMBINED USE OF A VECTOR ENCODING A MODIFIED RECEPTOR AND ITS EXOGENOUS AGONIST IN THE TREATMENT OF SEIZURES 
Description The invention provides methods and materials for treating a seizure disorder such as epilepsy in a patient which employ a vector encoding a modified receptor, the so-called "DREADD" receptor being characterised by (i) a decreased responsiveness to its endogenous activating ligand (ii) a retained or enhanced responsiveness to an exogenous agonist. The modified receptor is expressed in neurons of a seizure focus in brain of the patient, and an exogenous agonist is administered which activates the modified receptor to reversibly alters the excitability of the neurons in the seizure focus leading to synaptic silencing or other inhibition. 
IP Reference US2016375097 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This licencing is part of a portfolio of approaches to developing gene therapy to treat epilepsy
 
Title EXPRESSION VECTORS COMPRISING ENGINEERED GENES 
Description The invention provides expression vectors, nucleic acids, vector particles and methods of treatment involving these vector particles, comprising an engineered KCNA 1 gene encoding an edited Kv1.1 potassium channel, as well as methods of confirming the presence of engineered KCNA 1 mRNA in a cell. The features of the engineered KCNA 1 gene combine to advantageously enhance the translation and activity of the Kv1.1 protein and improve detection of KCNA 1 gene expression in a cell and can be used for example in the treatment of epilepsy and similar neurological disorders. 
IP Reference WO2018229254 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact This patent is helping support our award to translate our gene therapy to a first in human clinical trial
 
Description DIS visits from students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact International undergraduate students (20-30) with the DIS programme in Copenhagen visit ION at Queen Square, hear a presentation and tour the Department, and discuss careers in science with the team.
Year(s) Of Engagement Activity 2015,2016,2017
 
Description Livery Science day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact I arranged a one day 'Introduction to the Hypothesis' which brought UCL post-graduate students to dis-advantaged London schools, to allow children to interact with scientists and to carry out simple experiments to test hypotheses.

We are invited back - and the children love the day, but without the RCUK award, it is difficult to fund. I have much stronger associations with the livery companies, and am now working to arrange fund raising on their behalf for a fellowship.
Year(s) Of Engagement Activity 2008,2009,2010
 
Description Presentation at Royal Society summer science festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I gave a presentation "Brain Hacking With Viruses" introducing the concepts of Gene therapy for epilepsy at the Royal Society Summer Science Festival. The talk was repeated 4 times, and was well attended by students from around the UK. I also raised some of the ethical and safety concerns of the approach and got very strong feedback in the questions that students were interested even if the testing involved use of animals in research.
Year(s) Of Engagement Activity 2016
URL https://royalsociety.org/science-events-and-lectures/2016/07/schools-talk-schorge-tuesday/
 
Description School Visit (Lenox) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact This was a presentation on Gene Therapy and treatments of epilepsy to a High School audience in the states.
Year(s) Of Engagement Activity 2016,2017
 
Description Work experience at ION 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Both students were well-engaged with basic and clinical research at the Institute of Neurology.

Social Mobility Fund and the Worshipful Company of Pewterers have both indicated they'd be delighted to carry on with the scheme in future years.
Year(s) Of Engagement Activity 2014