Regulation of glutamate transporter EAAT2 activity lateral diffusion and membrane trafficking by palmitoylation and interacting proteins

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

In our brains information is encoded as changes in the voltage across the membrane of nerve cells. Nerve cells send signals to each other by releasing chemicals called neurotransmitters at special sites called synapses. One key neurotransmitter called glutamate acts on special proteins (receptors), to generate excitation (a more positive voltage). Controlling the levels of the neurotransmitter glutamate available to activate the receptor proteins is a key way to regulate communication in the brain. Controlling the levels of glutamate in the brain is also important because if glutamate levels get too high this causes too much excitation which is toxic and can lead to neuronal death (as occurs in stroke). Glutamate levels in the nervous system are mainly controlled by another type of protein on the surface membrane of brain cells, called a transporter protein. The transporters have a specialised role to bind glutamate released at synapses and pump it back inside cells so that it can be packaged up and recycled which stops it building up to toxic levels. My application is on a key aspect of brain function: how do brain cells regulate the number, activity and location of the key glutamate regulating transporter proteins in the plasma membrane of brain cells to control the duration and strength of neurotransmitter signaling at synapses. In particular we want to investigate how the regulation of the glutamate transporter proteins is performed, characterising the molecular properties of the proteins which are crucial for regulating transporter function and movement in the membrane. Studying the molecular mechanisms that underlie these regulatory processes will allow us to better understand how the brain works under healthy conditions. In addition because glutamate signalling is implicated in many neurodegenerative and neuropsychiatric diseases, our proposed work may also lead to an improved understanding of diseases where the levels of the neurotransmitter glutamate are altered such as epilepsy, stroke, Alzheimer's disease, motor neuron disease, Huntington's disease and schizophrenia.

Technical Summary

Glutamate is the major excitatory neurotransmitter in the central nervous system. It affects neuronal and glial function by acting on ionotropic and metabotropic glutamate receptors. Controlling the extracellular concentration of glutamate in the brain is crucial, both for normal information processing to occur and to prevent neuronal death that occurs when extracellular glutamate levels get too high. In most brain areas, the glutamate transporter EAAT2 is the major mechanism regulating the basal glutamate concentration, yet little is known of how its function is controlled. Using a combination of biochemistry, electrophysiology and state of the art imaging approaches such as Quantum Dot tracking we will investigate the molecular mechanisms that regulate the activity, lateral diffusion in the surface membrane and trafficking (exo/endocytosis) of EAAT2. We will focus in particular on the role of palmitoylation of EAAT2 and it's interaction with the trafficking protein Nischarin which we recently found to be potentially important modulators of EAAT2 activity and trafficking. This work will lead to a better understanding of the mechanisms that regulate the critical glutamate uptake systems in the brain.

Planned Impact

Who will benefit from this research? Academic beneficiaries UK science base. Pharmaceutical industry. General public. How will they benefit from this research? Training the next generation of scientists: The academic community and the UK Science base will directly benefit from this programme of research through the scientific and professional training of the post doctoral scientists on the project. The high level of training given within the Department of Neuroscience, Physiology and Pharmacology at UCL also has the potential to directly benefit the pharmaceutical and biotech industries as research skills learned during the project would be readily transferable to research and development projects in the pharma and biotech sectors. Transferable skills gained by the postdoctoral researchers during the project are also relevant to the wider UK economy, therefore potentially benefiting the wider UK commercial sector and general public. Pharma and Public Health: Neurological diseases representing a very significant disease burden to the UK include a component of disrupted glutamatergic signalling. Research on diseases such as stroke, epilepsy, motor neuron, Alzheimer's and Huntington's disease and schizophrenia will directly benefit from a detailed understanding of the molecular mechanisms that control extracellular glutamate levels and glutamate clearance in the brain. The knowledge generated from the proposed work will thus benefit pharmaceutical and biotech sectors targeting these disease and will also benefit the public health. While we do not anticipate our findings will directly impact on patient health within the timecourse of the project, by contributing towards an improved understanding of the mechanisms that underlie diseases where glutamate signalling is altered the proposed research will benefit in the long term the pharmaceutical industry competitiveness and UK quality of life. What will be done to ensure that they benefit from this research? Our main aim is to publish our work in high impact international journals as an excellent means of contributing to UK research competitiveness and disseminating our findings and data to the widest academic and pharmaceutical audiences. Previous published work from my lab has received scientific commentary such as 'News and Views' articles (e.g. Qi and Sheng, Neuron 2009; http://www.cell.com/neuron/abstract/S0896-6273(09)00125-1) and Faculty of 1000 'Must Read' (http://f1000biology.com/article/id/1157424). We will also disseminate our data at earlier stages prior to publication in the form of presentations of the data at invited lectures at universities and pharmaceutical companies and at national and international meetings and symposia. I am active in organising symposia both in the UK and abroad (e.g. FENS 2008; BNA 2007; Physoc 2006) and will aim to organise a more focused symposia on this area of research which will allow discussion and exchange of ideas and data with others in the field of glutamate signalling, transporter biology and membrane dynamics. We will also engage the public regarding our key findings. 'UCL Neuroscience' (www.ucl.ac.uk/neuroscience) which brings together neuroscience research at UCL has a very active public engagement policy. UCL was recently named one of the six 'Beacons of Public Engagement' nationwide. My lab is actively engaged in communicating with UCL Public Engagement to disseminate our research to a wider public audience. For example, as part of UCL Neurosciences promotion of brain awareness week 2009, UCL Neuroscience showcased work from several labs including our work 'Brain power where it is needed' as part of the 'latest breakthroughs' for UCL Neuroscience (see http://www.ucl.ac.uk/news/news-articles/0903/09031604). Work from my lab has also been previously showcased in the news section of the MRC website (see http://www.ucl.ac.uk/news/news-articles/MRC005717).
 
Description Nerve cells send signals to each other by releasing chemicals called neurotransmitters at special sites called synapses. One key neurotransmitter in the brain is called glutamate and is released by neurones at synapses to affect neuronal function by acting on proteins called glutamate receptors which can activate the downstream neuron. Controlling the levels of glutamate available to activate the glutamate receptor proteins is a key way to regulate communication in the brain. It is also important because if glutamate levels get too high this can lead to excessive glutamate receptor activation which can be neurotoxic and can lead to neuronal death. Glutamate levels in the nervous system are mainly controlled by proteins called glutamate transporters which are mainly located on a specialised type of brain cell called an astrocyte. The transporters bind the glutamate released at synapses and pump it back inside cells so that it can be packaged up and recycled, stopping its building up to toxic levels. Despite the important role of glutamate transporters we still don't know enough about how their function, expression levels and distribution on astrocyte processes nearby to synapses are controlled.
The project has focused in particular on the mechanisms that regulate the number, activity and location of the glutamate transporter proteins in the astrocyte cell surface membrane to control the duration and strength of neurotransmitter signaling at synapses. As part of this work we firstly established several state of the art approaches using live cell video microscopy to be able to visualise with very high accuracy the spatial distribution of the transporters in the surface membrane of live astrocytes grown alongside a network of neurones. This then allowed us to establish that neuronal activity and glutamate levels can tune the distribution and movement of transporters in the astrocyte membrane on rapid time scales to regulate transporter localisation close to synapses and hence brain glutamate signaling. We were also able to identify protein motifs within the transporters, which can regulate this process via their interaction with intracellular scaffolding proteins providing new mechanistic insight regarding how transporters are stabilised in subdomains of the astrocyte membrane. Finally in related work we also further studied the machinery that regulates the trafficking of transporters between the cell surface and storage compartments inside the cell. This allowed us to establish new proteins that regulate glutamate transporter trafficking and surface expression levels which allows us to better understand how neurones and astrocytes talk to each other to regulate brain glutamate levels. In addition because glutamate signalling is implicated in many neurodegenerative and neuropsychiatric diseases, the identification of new components of the transporter trafficking machinery could be used to regulate glutamate uptake and may eventually provide new therapeutic targets for diseases where the levels of glutamate are altered such as epilepsy, stroke, Alzheimer's disease, motor neuron disease and schizophrenia.
Exploitation Route The identification of new components of the EAAT2 transporter trafficking machinery could be used to regulate glutamate uptake and may eventually provide new therapeutic targets for the pharmaceutical industry working in treatments for diseases where the levels of the neurotransmitter glutamate are altered such as in epilepsy, stroke, Alzheimer's disease, motor neuron disease, Huntington's disease and schizophrenia. As part of our work on transporter trafficking proteins we have also made significant progress in characterising a poorly understood protein called Nischarin. We aim to continue studying the Nischarin protein which is highly expressed in the nervous system and which we think plays a critical role in brain function. We are also characterising several tools to study Nischarin including knockout mice lines which may also be useful to the wider cell biology community. As part of our work imaging the surface diffusion dynamics of the transporters we have also developed new Quatum Dot tracking techniques to study membrane proteins in astrocytes in brain slices which may be useful in the future to other groups wishing to study protein dynamics in situ in intact tissue.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The identification of new components of the EAAT2 transporter trafficking machinery could be used to regulate glutamate uptake and may eventually provide new therapeutic targets for the pharmaceutical industry working in treatments for diseases where the levels of the neurotransmitter glutamate are altered such as in epilepsy, stroke, Alzheimer's disease, motor neuron disease, Huntington's disease and schizophrenia. We are currently in discussion with the pharmaceutical industry regarding these findings and their translational applicability.
First Year Of Impact 2015
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description In2Science UK 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact 1-2 week laboratory placement scheme in the summer for gifted A-level science students from disadvantaged backgrounds (the poorest 10% of our society).

Stimulated thinking of future young scientist
Year(s) Of Engagement Activity 2013