Identifying intracellular signalling pathways that regulate neuronal activity

Lead Research Organisation: MRC Cell Biology Unit

Abstract

The activity of brain cells is controlled in many different ways. The ability to change brain cell activity is thought to be important for many brain functions including mood, memory, and learning. One mechanism is for brain cells to release naturally occurring chemicals that alter the activity of other brain cells. Examples include the chemicals serotonin and dopamine, which control mood and behaviour. In humans, serotonin has been implicated in several aspects of mood and behaviour, including depression, eating disorders, alcohol consumption, and aggression. Dopamine has been implicated in movement control, motivation and reward, Parkinsons disease, and schizophrenia. Both serotonin and dopamine signalling are targets for drugs: cocaine and amphetamines target dopamine pathways, and MDMA (ecstasy) targets serotonin pathways. The Nurrish lab is researching how serotonin and dopamine control brain cell activity. The eventual aim is to understand the pathways involved. Two possible outcomes of this work will be a better understanding of why some people are more susceptible to mood disorders such as depression and to identify targets for drugs to treat disorders such as depression.

Technical Summary

Extracellular neuromodulators alter levels of neurotransmitter release at many, if not all, synapses. Examples include the monoamines serotonin and dopamine, which control global behavioural states of the brain. In humans, serotonin has been implicated in several aspects of mood and behaviour, including depression, eating disorders, alcohol consumption, and aggression. Dopamine has been implicated in movement control, motivation and reward, Parkinsons disease, and schizophrenia. Both serotonin and dopamine signalling are targets for drugs: cocaine and amphetamines target dopamine pathways, and MDMA (ecstasy) targets serotonin pathways. Although many of the molecular components that underlie neurotransmitter release have been characterized, less is known about how they are regulated by neuromodulators and their associated intracellular signalling pathways. Our goal is to understand the signalling pathways that lead from neuromodulator receptors on the cell surface to the control of neurotransmitter release.||Our research uses the model organism C.elegans, in particular the C.elegans neuromuscular junction (NMJ). We and others have shown that C.elegans uses both serotonin and dopamine to modulate NMJ activity using pathways conserved in human neurons. Many of the genes that regulate activity at the NMJ participate in a network of heterotrimeric G-protein signalling pathways controlling the release of synaptic vesicles and/or dense core vesicles (DCVs). At least four heterotrimeric G-proteins (G-alpha-q, G-alpha-12, G-alpha-o and G-alpha-s) act within the motorneurons to control the activity of the NMJ. The G-alpha-q, G-alpha-12, and G-alpha-o pathways converge to control production and destruction of the lipid bound 2nd messenger diacylglycerol (DAG) at sites of neurotransmitter release. DAG acts via at least two effectors, MUNC13 and PKC, to control the release of both neurotransmitters and neuropeptides from motorneurons. The G-alpha-s pathway converges with the other three heterotrimeric G-protein pathways downstream of DAG to regulate neuropeptide release. Released neurotransmitters and neuropeptides then act to control contraction of the bodywall muscles to control locomotion.||Current work focuses on a novel role for RhoA GTPases in the control of neuronal activity in adult neurons. Due to a requirement for RhoA in neuronal development the role of RhoA in adult neurons has been previously missed. Interestingly, many RhoA regulators and effectors have been implicated in human mental retardation and our work suggests this could be due to defects in neuronal activity rather than development. If true this makes it more likely that some forms of human mental retardation could be corrected in adults. RhoA increases neuronal activity in part by inhibiting the DAG kinase DGK-1. This leads to an increase in DAG and thus an increase in neurotransmitter release. However, we have also shown that other pathways exist downstream of RhoA to increase neuronal activity. A genetic screen for RhoA suppressors has identified at least 12 mutations and a combination of genetic mapping and whole genome sequencing is being used to clone these mutations. One mutation is in the nca-1 gene, a homolog of the human NALCN channel. We continue to work on how RhoA regulates the NALCN channel and the other suppressors.||The lipids and proteins involved in these networks are conserved between C.elegans and mammals. Thus, the C.elegans NMJ acts as a model synapse to understand how neuronal activity in the human brain is regulated.
 
Description Small Arts Grant
Amount £13,133 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2008 
End 03/2009
 
Title Inducible rho-1(gf) C.elegans strain 
Description C.elegans expressing constitutively active RHO-1 from either a cholinergic or heatshock specific promoter. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact These animals have been used in publications from other labs, for example Pub Med paper 17537791 
URL http://europepmc.org/abstract/MED/17537791
 
Title inducible lines for expressing C3 transferase 
Description A line able to express the specific inhibitor of RhoA C3 transferase 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact These lines have been used by other labs (Labouesse, Ahringer, Bargmann) to inhibit RHO-1 in C.elegans 
 
Description Analyzing regulation of protein localization at neurotransmitter release sites. 
Organisation King's College London
Department MRC Centre for Developmental Neurobiology
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a new collaboration. Our collaborator has extensive expertise at quantifying changes in protein localization at neurotransmitter release sites. We are collaborating to test whether changes in RhoA signalling pathways change proteins levels at neurotransmitter release sites.
Collaborator Contribution Our collaborator at Kings College is providing DNA constructs, computer programs and expertise for us to study changes in protein localization at neurotransmitter release sites.
Impact This collaboration has already led to acquiring of data useful to our study of regulation of neirotransmitter release.
Start Year 2009
 
Description Investigating the mechanism of action of the human mood stabilizer valproate 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab performed all the experiments with the exception of analysing the phosphoinositols, which was performed by Adolfo Saiardi at the MRC LMCB.
Collaborator Contribution Adolfo Saiardi provided expertise for analysis of phosphoinositols and valuable advice for the project.
Impact This resulted in a paper, Pub Med 18287529
 
Description Investigating the role of NCA-1 in neuronal function 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We identified nca-1 as required for RHO-1 mediated effects on neuronal activity
Collaborator Contribution They investigated the effects of rho-1 and nca-1 on neuronal calcium signalling and tested locomotion effects using an automated worm tracker
Impact We have identified that nca-1 and other functionally similar genes block the locomotion defects caused by excessive rho-1 signalling. These results are being written up
Start Year 2011
 
Description Regulation of syntaxin by an atypical PKC 
Organisation Medical Research Council (MRC)
Department MRC Cell Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborated with Yasu Fujita to demonstrate that a syntaxin phosphorylation identified by him has an important regulatory role in the release of neurotransmitter
Collaborator Contribution We have identified a new syntaxin mutant that strongly influences neurotransmitter release
Impact A graduate student is continuing to work on this project and we hope to publish our data in 2011
Start Year 2007
 
Description The role of KIN-4 in control of DGK-1 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We identified KIN-4 (a conserved human neuronal protein) as a novel interactor with DGK-1. We collaborated with Julie to demonstrate this interaction was direct by co-immunoprecipiattion experiments
Collaborator Contribution Julie was able to demostrate that the KIN-4 protein binds to the DGK-1 protein using co-immunoprecipitation, an important result for our work
Impact A paper is currently being written up and will be submitted in 2010
Start Year 2007
 
Description The role of RHO-1 in C.elegans innate immunity 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a project set up in my lab that has been carried on by an ex post-doc in her own lab at Imperial College. We provided all the reagents and knowledge to start this project. We are currently providing all of the EM studies to support this project
Collaborator Contribution Strains, plasmids and other regents were exchanged and contributed towards papers currently being written up
Impact A paper is currently being written up for submission
Start Year 2010
 
Description Understanding neural networks in C.elegans behaviors 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution A post-doc worked in my lab (funded as an MRC post-doc) and then in continued her project at a lab in New York, to understand the neural networks and signalling pathways that control the complex behaviour of of males searching for their mates. This resulted in a paper in Current Biology
Collaborator Contribution My lab and the lab in New York funded the salary and research of a post-doc in each lab. The New York lab paid the publication charges for the paper.
Impact Publication Pub Med 19062284
 
Description Use of C.elegans to study AP1 interactors in human epthelial cells 
Organisation Medical Research Council (MRC)
Department MRC Cell Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution A joint post-doc between myself and Dan Cutler performed an RNAi screen to identify new AP1 interactors
Collaborator Contribution The collaboration resulted in a paper together and a position for my ex-post-doc Gregoire Michaux
Impact A paper has just been accepted in the Journal of Thrombosis and Haemostasis
Start Year 2007
 
Description role of nz90 on neuronal activity 
Organisation École Normale Supérieure, Paris
Department Institute of Biology
Country France 
Sector Academic/University 
PI Contribution We have identified a mutation, nz90, with defective neurotransmission
Collaborator Contribution They are using antibodies and genetic constructs to test for changes in muscle neurotransmitter receptor expression and/or colocalization
Impact These results are being written up in a paper to be published
Start Year 2011
 
Description the effect of the nz90 mutation on neuronal signalling 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution We identified a mutation, nz90, that has defective neuronal signalling
Collaborator Contribution They are using electrophysiology to determine at what stage nz90 mutants have defective neurotransmission
Impact This work is being written up for publication
Start Year 2011
 
Description Fabrics for Life 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Fabrics for Life was set up by Amanda Fisher at the MRC CSC were Nobel Prize winners were paired with senior designers at Central St Martins. I was included in the collaboration between Sir John Sulston and Carole Collet as Sir John no longer actually works with C.elegans. Thus, Carole worked in my lab to understand Sir John's research.

Outpits include a web site, a DVD (in which I appear) and an exhibition of the designs at the ICA in London
Year(s) Of Engagement Activity 2008
 
Description MRC Public Panel meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Approx 30 members of the MRC Public Panel were invited to the MRC LMCB. After the panel meeting they were given a tour of the building and spent 30 minutes in my lab being shown C.elegans as I described how we use them to study diseases relevant to humans.

The MRC received a very enthusiastic feedback about our presentations. In particular-
The scientist working on the worms (Bristol),introduced a totally new concept to all of us, his explanation was at such a fundamental level, that he injected a serious desire to know more and more, his explanation how you inject serotonin/dopamine into the worms brain to evaluate changes in brain activities was mind boggling.
Year(s) Of Engagement Activity 2010
 
Description Music from the worm farm 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact I collaborated with a composer, and we obtained a small arts grant from the Wellcome Trust. Keith spent 6 months in my lab and composed music based on general and specific science concepts in my lab, for example general concepts of genetics and mutations, and specific concepts of control of neurotransmitter release.

The composer and I gave an evening presentation at the DANA centre an the science museum to a general audience. I explained my science to the audience and the composer explained how my research inspired him to create a number of compositions which were played. A CD containing music inspired by my research was made and given out. In addition a website explaining the collaboration was created http://www.wormusic.org/welcome.html
Year(s) Of Engagement Activity 2008,2009
 
Description Nobellni 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact I was involved with an ongoing collaboration between the MRC Clinical Sciences Centre (Amanda Fisher) and the Central Saint Martins College of Arts and Design funded by Epigenome. Various science PhD students interacted with design students who then came up with ideas for designs. This was run at the DANA Centre at the Science Museum. In a session open to the general public 2 science/designer pairings were decided by public vote in a 'Blind-date' style and I was the compere.

Several websites recording the work and collaborations http://www.csc.mrc.ac.uk/PublicScience/FabricsOfLife/Nobelini2/ http://www.nobeltextiles.com/nobeltextiles_/Home.html. The Nobelini involved production of a film in which I appear. The 3 winning collaborations were announced at a joint MRC LMCB/MRC CSC retreat and these three groups were given the money to proceed with their designs and these were presented at a second evening open to the general public at the DANA Centre.
Year(s) Of Engagement Activity 2008,2009
 
Description Participation at meetings 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact A part of a small arts grant from the wellcome trust I collaborated with the composer Keith Johnson. The results of our collaboration were presented at an evening at the DANA centre at the Science museum and are recorded on the website http://www.wormusic.org/welcome.html. I was also interviewed for the UCL monthly podcast for March 2009

Creation of the website http://www.wormusic.org/welcome.html
Year(s) Of Engagement Activity 2008,2009
URL http://www.wormusic.org/welcome.html
 
Description Seminar speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Activities have included participating with the pairing of Nobel Prize Winners with Designers (Fabrics of Life), pairing of science and design students to come up with a design representing the work of the scientist (Nebelini), and being involved with presenting my work followed by a an afternoon session discussing science/design with Design MA students at CSM. The primary organizer for these events has been Amanda Fisher at the MRC CSC.

Fabrics of Life resulted in a DVD and a presentation of work/lectures at the Institute of Contempory Art (ICA). Nebelini involved an evening describing the ideas to the general public at the DNA centre at the Science Museum. These are described at the websites http://www.nobeltextiles.com/nobeltextiles_/Home.html http://www.csc.mrc.ac.uk/PublicScience/FabricsOfLife/Nobelini2/
Year(s) Of Engagement Activity 2008,2009
URL http://www.csc.mrc.ac.uk/PublicScience/FabricsOfLife/Nobelini2/