Cl-out: a novel cooperative-optogenetic strategy to control neuronal chloride

Chloride is a very important ion in the brain, because neurons use it to give negative (inhibitory) signals. It is a negatively charged ion, and for the most important type of synaptic inhibition, it provides the negative signal to neurons. For this to work, however, chloride needs to be kept very low inside neurons. On occasions, however, neurons lose the ability to remove chloride, and when this happens, synaptic inhibition no longer works well. This can present serious problems for brain function, and may give rise to seizures, spasticity, severe pain and multiple other neurological conditions.

Despite the clear importance of maintaining a good chloride balance, we know little about how this is achieved in healthy brains, or why it might go wrong. Even worse, we have very limited tools to treat this, or even do research on this important topic. We therefore designed a new way to manipulate chloride levels, both up and down, that could transform this field and even offers the potential for clinical use.

Our new technology is what is called an optogenetic protein. Optogenetics is a revolution in progress in neuroscience. It involves introducing into neurons light sensitive proteins that, when illuminated, can directly modulate the neuron's activity. Because it is such a powerful technique, allowing precise manipulation of selected neurons, it is considered to be the future of brain-machine interfaces. It is also transforming the type of research questions that can be asked. We created a new optogenetic protein with multiple beneficial effects: when illuminated, it directly stops neurons from firing, but more importantly, it also removes chloride and thereby provides a persistent protective effect by improving the brain's own inhibitory systems. We call the new protein "Cl-out", standing for "chloride (Cl) out".

So far, we have preliminary data showing Cl-out can extrude chloride. This though is just the start; our proposal describes how we will develop this further.
We have already identified a way to modify Cl-out: our original solution couples chloride movement to the removal of a hydrogen ion, but we now have identified a way of doing this differently, coupling it instead to the movement of a potassium ion. This offers a fundamentally different "quality" of effect, which will extend the utility of this tool. We will learn how to express the protein in different types of neurons, and explore what is the optimal strategy for activating it to give the best chloride-correction effect, and whether this is further boosted by the additive action of certain drugs.

We will also create a powerful set of resources for expression of Cl-out, which we will make available to the research community. These will allow Clout to be expressed in subpopulations of neurons with fine control, and will facilitate its uptake by other researchers.

In addition to the work developing Cl-out as a research tool, we will conduct parallel studies to investigate how chloride regulation affects neuronal behaviour. In this aim, we will also use another light sensitive membrane protein, Halorhodopsin, which we have demonstrated can be used to drive chloride in the opposite direction, into neurons. We thus have light-sensitive mechanisms which can drive chloride in either direction very rapidly, opening all manner of research opportunities. For instance, we will be able to learn about how ionic regulation in neurons affects whole brain behaviour. Second, and perhaps more important is our technical developments will offer simple experimental model systems which can be used for drug development, exploration and screening.

These studies build upon preliminary work to create a new optogenetic strategy to pump chloride out of cells, called Cl-out. This new technology provides the first means of reducing intracellular chloride, and complements other recent work demonstrating how a different optogenetic tool, Halorhodopsin can be used to drive chloride in the opposite direction. Together, these new tools have the potential to revolutionise research into chloride regulation, which is integral to synaptic inhibition throughout the nervous system.

Cl-out works by fusing two optogenetic proteins: the hyperpolarising proton pump Archaerhodopsin, and one of the new optogenetic chloride channels. The Archaerhodopsin serves as an optical voltage clamp, using light to clamp the cell's membrane potential below the chloride channel reversal potential. This provides the driving force to pump chloride from the cytosol by simultaneously opening Cl- channels. Because both proteins are activated by light, we can achieve Cl-clearance in millions of cells simultaneously simply by expressing the protein generally, and then bathing the tissue in light.

This proposal has two facets:
1. The validation and development of ClouT. We will first develop a variant of Cl-out, which couples Cl extrusion to the movement of a K+ ion (instead of a proton). We will further test different expression strategies, and create plasmids, viral vectors, and a floxed animal line to facilitate future work using this resource. We will investigate different illumination strategies to achieve efficient and persistent GABAergic improvement for the lowest levels of illumination (important for efficient brain-machine interface function).
2. The demonstration of the impact of chloride manipulation on neuronal function in simple in vitro model systems that can subsequently be developed for assays for drug development.

The immediate beneficiaries will be amongst the academic community. Such is the widespread importance of chloride-dependent inhibitory mechanisms across the brain, that we predict our research to have relevance to laboratories working in all areas of neuroscience. It will be of particular interest to the large and growing optogenetic community, as the first demonstration of combinatorial opsin function, whereby emergent properties arise only from co-activation of multiple opsins. And by extension, also for the field of brain-machine interfaces (BMIs), which will feed heavily off the continued developments in optogenetics; our new constructs, or derivatives of these, will, we believe, be among the principle optogenetic tools used in such BMIs. Dissemination to this interest group will be helped by the major optogenetic research initiatives in Newcastle ("CANDO").

We will make the resources generated through this project - plasmids, viral vectors, transgenic mice expressing the new optogenetic constructs - available to the research community through Addgene, Viral Vector Cores (e.g. U.Penn and UNC) and mouse line repositories (Jackson Laboratories and Harwell, MRC), and promote their use by collaborative efforts.

The Biopharma Industry can also benefit from the utilising our new techniques for manipulating chloride levels in neurons, for future drug development in this field. The successful completion and publication of our research will be the basis for engaging with the Biopharma Industry through future collaborative funded projects including CASE studentships and industry placements.

Novel research findings will be incorporated into the various teaching and outreach activities of the team. There will be obvious benefits for the postgraduate and early career researchers in the Institute, where there is growing interest in implementing optogenetics into research programmes.

We will engage with the wider academic and non-academic communities through various routes including the University of Newcastle, local schools, and through our involvement in patient outreach programs. These have mainly focussed on our specialist research interests of epilepsy (AT: through the annual North East Epilepsy Research Meetings, Epilepsy Interest Group, which is part of the Northern England Stategic Clinical Network, Epilepsy Research UK and International League Against Epilepsy meetings, local and national patient groups, including through Epilepsy Action (contributions to Epilepsy Professional magazine)) and mitochondrial disease (RL, through his involvement as founding member and PI of the Mitochondrial Research Group - see numerous local and national level work, leading ultimately to the recent change in the law regarding so-called "three parent families"). These events also stimulate interest and understanding of science in children and young adults, providing academic role models and roadmaps for their own career ambitions.

Medical charities, such as Epilepsy Research UK and Epilepsy Action who have both funded topics related to this project, will benefit by communicating to the public about recent scientific discoveries and new breakthrough technologies. This boosts fundraising, by providing tangible evidence of the effective use of the benefactions, which in turn supports further research and services for people suffering with these conditions.

Through these various pathways, these research efforts will help raise the profile of the North East and the UK generally, as leaders of biomedical and health care innovation.