Identification of the molecular substrate for L-lactate-mediated catecholamine signalling in the brain

Lead Research Organisation: University of Bristol
Department Name: Physiology and Pharmacology

Abstract

It is becoming evident that the function of the brain under normal and pathological conditions cannot be understood without taking into account the role of non-neuronal elements of this complex organ, numerous star-shaped cells known as astrocytes. The roles which astrocytes play in the brain are only currently emerging. It is established that these cells have a unique ability to store glucose in the form of glycogen and convert it into lactic acid (lactate) which they then release. Lactate for many years was thought to be used as an intermediate in the energy generating machinery of the body. However we have evidence that it has also another role in the brain where it acts as a transmitter or messenger between astrocytes and neurones.
Using cutting edge technologies we have discovered that lactate can act on specific subsets of brain neurones which release a critical neurotransmitter and modulator, called noradrenaline. Action of lactate on these neurones excites them and makes them release more noradrenaline. This mechanism could be involved in many functions of the brain, including regulation of sleep-wake cycle, appetite, emotions, blood pressure, attention and others. This needs to be further investigated. Our research implies that there is a dedicated excitatory receptor for lactate in the brain and we aim to discover it in this project. We are therefore proposing a programme of research which will look for the candidate genes which could encode for this new receptor, verify its sensitivity to lactate and prove that it is able to confer sensitivity to lactate to neurones which are naturally not sensitive to lactate. These steps will form the basis for more in depth studies aimed at detailed characterisation of this novel mechanism and ultimately to the understanding of its role in health and disease. Without knowing the molecular identity of the lactate receptor further progress in this direction is essentially impossible. However, when we find the protein responsible for lactate effects we will be in a position to design drugs which act on this receptor and via this route potentially develop new therapies.

Technical Summary

We have recently found that L-lactate (LL) acts as a powerful excitatory stimulus on central noradrenergic neurones, depolarising them and triggering release of noradrenaline. Interestingly, LL is not excitatory to non-noradrenergic neurones in the hippocampus. Our observations allow us to propose the existence of a yet unknown G-protein coupled receptor for LL which is coupled via Gs proteins to cAMP production. Importantly, the EC50 for this new receptor is ~0.5 mM which is within the physiological range of LL concentrations in the brain. Given the potency of this pathway, ubiquitous presence and prominent activity-dependent fluctuations of LL on the one hand, and the paramount role of noradrenergic transmission for the normal functioning of the brain on the other, this new signalling mechanism could be of major biological importance. We have performed an initial transcriptomic analysis of an LL-sensitive area and found a substantial number of currently orphan G-protein coupled receptors expressed. We hypothesise that the new LL receptor is one of them. The project involves a detailed and selective transcriptomic analysis of cells which are sensitive to LL in comparison with cells which are not; wide-scale screens of orphan GPCRs using a beta-arrestin assay; identification of the LL-sensitive receptors using a cAMP assay in cell lines; detailed characterisation of the pharmacological profile of the responses in the areas which we know to be LL sensitive in search of initial lead agonist and antagonist compounds; pharmacological analysis of any candidate receptors; and finally, an experiment where we will attempt to confer LL sensitivity to naturally LL insensitive neurones by expressing in them the newly identified putative target receptor. These studies will form the basis for further analysis of the physiology and pathophysiology associated with this signalling mechanism and for design of small molecule modulators which could have novel therapeutic properties.

Planned Impact

A novel receptor involved in fundamental adaptation of brain functions towards active states and autonomic regulation is likely to incite considerable interest in the public, research communities and pharmaceutical industry. In order to ensure the maximum impact of our findings, we will ensure dissemination of information to a range of relevant audiences:

Scientific audiences
A new receptor with intriguing functional properties will answer some and open up a host of new scientific questions relevant to various fields of neuroscience and neuro-degeneration. We will address researchers by the well-established pathways of
- Presentation of our work at conferences such as IUPS, Society of Neuroscience, Physiological Society, FENS, Gordon Conferences
- Publication in high profile scientific media
- Engagement with young research audiences, including under- and postgraduate students in UK and internationally, for example Young Physiologists (UK) and Neuronus (international scientific youth conference).
- Training in multidisciplinary research skills such as use of viral vectors and optogenetics, in combination with confocal imaging and physiological experiments.

Public audiences
Since lactate is a generally well-known metabolite, findings that explain its central roles will attract interest of the public, creating opportunities to publically highlight issues surrounding healthy brain functions and age-dependent decline. In conjunction with scientific publication, we will
- Prepare press releases in collaboration with the University of Bristol press office
- Publicize findings through websites such as the Bristol Neuroscience platform lectures

Pharmaceutical industry
The receptor which we are seeking is a potentially promising drug target to address health problems associated with a number of diseases with high potential for drug therapy. Drug industry will be informed of our work through
- Our current collaborations with industrial partners at Novartis and GlaxoSmithKline with whom we communicate regularly
- Development of links with clinical research once the relevant protein has been identified
- Pathways that impact on the scientific community (see above) which may attract further interest from pharmaceutical industry. New contacts established in this way will be expanded and any potential patentable issues negotiated via the University of Bristol Research and Development department.

Publications

10 25 50
 
Description We have discovered a potentially important effect of the signalling system under study for release of noradrenalin in the brain. We have established some characteristics of the putative receptor and obtained initial information about the structure-activity profile. We have also tested effect of lactate on a range of recombinant orphan receptors and made observations which should help to understand their function. This may lead to creation of new drugs.
We have discovered a powerful modulatory action of L-lactate on GPR4 - a proton receptor, this is published (Hosford et al 2018). We have screened a wide range of other receptors which were identified in the target area. We have also found that there are olfactory receptors sensitive to L-lactate but they are not responsible for the effects we were seeking to explain.In 2018, after the expiration of the award WE HAVE FINALLY IDENTIFIED A CURRENTLY ORPHAN RECEPTOR WHICH COULD BE A NEW RECEPTOR FOR L-LACTATE. This information has been published by our group in Neuroglia in 2018.
Exploitation Route We have performed a wide range of experiments which led us to identification of one orphan G-protein coupled receptor which, upon stimulation with L-lactate, leads to an increase in production of cAMP. This was our primary target. We have also developed an array of methods related to measuring and manipulating cAMP. We are in the process of publishing a study which describes a modulator action of L-lactate on yet another receptor, GPR4. Finally, we have tested whether Olfactory receptor 78 is the mediator of L-lactate actions but concluded that this is unlikely, this is published (Mosienko et al 2017).
We have data for at least one more publication which is in preparation now. We have applied for support to validate our new L-lactate receptor.
This research may be used as a basis for further analysis of the Lactate-mediated signalling in the brain and potentially lead to the development of new drugs.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title Genetic constructs distribution 
Description Clones of various constructs distributed via AddGene (~40 over this period) 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Dozens of labs have been enabled to generate molecular constructs they require for various experiments. We also have shipped packaged viral vectors to 3 different Universities at no cost. 
 
Description Collaboration with Novartis (Basel) on the effect of L-lactate on GPR4 receptor 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution Collaboration was used to jointly supervise a student with a "year in industry" option. She performed experiments on a topic of mutual interest related to the theme of Lactate signalling.
Collaborator Contribution Supervision of the student and organisation of several training and research trips to Novartis (Basel)
Impact Joint publication has been submitted, still in the stage of publishing.
Start Year 2016
 
Description Collaboration with Novartis (Basel) on the effect of L-lactate on GPR4 receptor 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution Collaboration was used to jointly supervise a student with a "year in industry" option. She performed experiments on a topic of mutual interest related to the theme of Lactate signalling.
Collaborator Contribution Supervision of the student and organisation of several training and research trips to Novartis (Basel)
Impact Joint publication has been submitted, still in the stage of publishing.
Start Year 2016
 
Description Invited plenary lecture at 4th Winter seminar on Dementia (Italy) 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture on technologies we use and our research
Year(s) Of Engagement Activity 2016
 
Description Invited presentation at Gordon Research conference, Ventura, CA 5-10 March 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact GRC is the highest ranked international conference in the year. We presented our data there and this caused multiple discussions and sparked new collaborations.
Year(s) Of Engagement Activity 2017
 
Description Invited talk at the 12 International Conference on Brain Energy and Metabolism, Hong Kong 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Lecture on the research related to our work for scientists, and local scientific media and local politicians.
Year(s) Of Engagement Activity 2016
 
Description Presentation at FENS 2016 meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation on our work which attracted a significant interest and led to development of the future research strategies.
Year(s) Of Engagement Activity 2016
 
Description Talk at Demencia winter conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote speaker at the 4th Winter seminar on Dementia (Italy). Attended mainly by clinical scientists (~80 participants) involved in dementia research. Multiple discussions with participants and speakers.
Year(s) Of Engagement Activity 2016
URL http://www.sindem4juniors.it/
 
Description Talk at Gordon conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture at Gordon Research conference Angiotensin 2016 - Italy. Attended by key lab leaders around the world and a number of PhD students and young clinicians with various interests. Various discussions afterwards. Was also involved in poster ranking.
Year(s) Of Engagement Activity 2016
URL http://www.grc.org/programs.aspx?id=13998
 
Description Volga Neuroscience conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Two invited lectures were made and a poster presentation which was awarded as best presentation. Various collaborative projects were discussed.
Year(s) Of Engagement Activity 2016