A Systems Biological Approach to Elucidate Local Protein Synthesis Code in Plasticity and Memory

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Informatics

Abstract

Diseases which affect the nervous system such as Alzheimer's, Parkinson's, depression and schizophrenia account for the single largest cost to the healthcare system of the UK and are often associated with long-term disability, and distress for patients and their families. A common clinical feature of many of these and other disorders is a cognitive (often learning/memory) deficit. However learning and memory is a very complex biological process that is only partly understood. Of particular interest to us are the changes that occur after learning (i.e. during memory establishment) in the synapses - the critical structures that join two neurons together and mediate information flow and processing in all brains. In this study we aim to identify the biochemical changes (at the protein level) that are associated with forming memories. In particular, we aim to test the involvement of several candidate molecules that have been implicated but whose importance has not yet been proven. This work will also allow us to try and bridge the gap between what we see at the biochemical level in terms of molecules and their abundance with the actual behaviour of the brain (and subsequently of the animal). The observed biochemical changes will be used to refine and extend computational models of neuronal synapses. These computational models will provide a unique method to visualise these complex biochemical networks which involve more than 1000 proteins. Mathematical methods will then be applied that allow us to predict which molecules are more likely to be involved in the memory and a selection of the best candidates will be tested in the laboratory. These new insights will help us understand how memory is formed in the brain. Unravelling these core biological processes is vital to our understanding of animal behaviour in the first instance. In the longer term our research will have relevance to human cognition ultimately aiding the search for new drug therapies for cognitive illness.

Technical Summary

This project fits within the overall envelope of a systems biology approach to understanding the regulation of mRNA translation in neurological systems in response to synaptic signalling. Specifically, we will undertake the following studies: 1. we will use the newly-developed pulsed SILAC (pSILAC; stable isotope labelling with amino acids in culture) method to identify and quantitate changes in protein synthesis that occur in response to synaptic stimulation. In pSILAC, newly made proteins are 'tagged' with heavy isotope versions of arginine and lysine. Mass spectrometric analyses reveal both the identities of proteins whose rates of synthesis have changed and allow accurate quantitation of the changes. 2. well-established methods (e.g., using phosphospecific antisera and assays for translation factor function) will be employed to investigate the nature and temporal sequence of changes to the translational machinery induced by specific neural stimuli. Inhibitors, e.g., of specific signalling pathways and/or protein kinases will be employed to dissect the upstream pathways that elicit these changes. 3. data from transgenic mouse models (from collaborators) will be used to determine the role of individual regulatory inputs to the control of the synthesis of specific proteins: already available are knockouts of the initiation factor eIF4E kinases Mnk1 and Mnk2, and a kinase-dead knockin for elongation factor eEF2 kinase. 4. for selected proteins identified in #1, we will create reporter constructs containing potential regulatory features from their 5'- or 3'-untranslated regions to explore which elements of the mRNA confer control in response to specific stimuli and/or signaling events/translation factors. 5. integrate data from above with prior studies to describe and model the translation machinery within the post-synaptic proteome.

Planned Impact

1. Who will benefit from this research? The beneficiaries from this research include: (i) basic neurobiologists, especially those interested in the fundamental cellular and molecular mechanisms of learning and memory; (ii) investigators studying the control of gene expression, especially those examining the molecular mechanisms by which the synthesis of specific proteins can be controlled at a post-transcriptional level; (iii) researchers studying cellular signalling pathways and their physiological functions, in particular those studying pathways such as the mammalian target of rapamycin and protein kinases that impinge on the translational machinery; (iv) neuroscience researchers aiming to understand the basis of learning and memory why these processes may become defective or can be enhanced. A major strength of this programme of work is that it encompasses studies ranging from in vitro experiments in cell culture to work using established paradigms of learning and memory in animal models; (v) systems biologists, through the development of models to relate experimental data at the molecular levels to findings obtained in parallel relating to behavioural outputs, i.e., measurable outcomes in learning and memory. 2. How will they benefit from this research? For basic scientists in all the areas mentioned above, a major strength of this proposal is its multidisciplinary approach, which will allow relationships between molecular and behavioural data to be examined, developed into hypotheses and tested. This will provide key new data on the changes in the protein synthesis machinery and in protein synthesis itself to be related to quantifiable behavioural outcomes. It will yield insights that are important to all these researchers about the importance of specific changes in the translational machinery to alterations in the synthesis of specific proteins, and the relevance of both to learning and memory. For experimental neuroscientists, the findings will provide a coherent and testable set of models relating to the changes in gene expression linked to paradigms of learning and memory. This will help to explain the molecular basis of these key neurological processes. In the longer term, these findings, together with others in the field, will potentially provide a basis for understanding clinical conditions in which learning or memory are impaired. Although there are no immediate commercial impacts, there may be long term potential for engaging with, e.g., pharmaceutical companies with interests in targeting the signalling pathways we identify as being important in learning. This work has the potential to benefit patients with disorders of learning and memory. 3. What will be done to ensure that they have the opportunity to benefit from this research? Communication: The findings of our work will be communicated to the biological/biomedical research community through the peer-reviewed scientific literature and via presentations at meetings across a broad area of biosciences, e.g., in the areas of neuroscience, signal transduction, gene expression and system biology, to ensure that our findings reach a wide audience. They will also be published on the consortium's website, to maximise exposure. Collaboration: strong collaborations exist between members of the consortium, and discussion meetings will take place at regular intervals. Staff exchange visits also form an integral element of the programme. Plans for exploitation, where appropriate - there are no immediate plans for commercialisation. CGP & JDA have experience of commercial explotation of research (patents, spin-outs) where appropriate. Relevant experience /record: CGP and JDA have extensive experience of communicating science in specialist scientific presentations, as plenary talks to broader scientific audiences, speaking to health care professional professionals (e.g., CME) and working with the public media (radio, the press)

Publications

10 25 50
 
Description Memories can last an entire life-time. A series of complex biological processes thought to underpin the ability to form long-term and, despite their obvious important us they are only partly understood. Evidence gathered by many groups over the past years suggests that the brain needs to synthesise new molecules (proteins) in order to encode new memory. How and when these proteins are made needs to be tightly controlled so that it occurs in the right place at the right time. To try and understand this process better we assembled an international group of researchers each specialising in a related but different piece of the jigsaw puzzle. We combined expertise in the genetic control of memory and its measurement in living brains; expertise in the proteins thought to trigger memory associations; expertise in the biochemical pathways that regulate the formation of new proteins and our own expertise in building mathematical models of molecules in the brain.

As an interdisciplinary group we are able to achieve more during the research programme than we could have as single groups. The specifics of the research are complex but we were able to apply new analytical methods to the data used by the neuroscience groups in order to bring new understanding to the topic. As an example, in collaboration with the Proud group we looked at one of the most important molecular circuits thought to regulate when new proteins are made when neurons need them for memory formation. There were several alternative ways that this circuit could be formed. Together with them we generated a mathematical model for each of the alternative circuit layouts. We showed that all three circuits were theoretically capable of behaving in a way that matched the available data in the laboratory. However we were were able to predict new experiments that would differ depending on the circuit layout. These were then performed and a preferred circuit emerged. This was only possible through the close interaction between the research groups, each taking a very different approach. The new insights and understanding of these mechanisms are general in that they are likely to impact across all animal species. In the longer term we can also now look to see how these mechanisms fit into dysfunction either as a normal effect of cognitive decline with age or in disease.

The interdisciplinary approach we used in the study could be applied to many other areas of biology and of course there are many other groups of researchers coming together to achieve similar goals in other topics. While doing this we identified that some of the computational tools that we produced would be of wider interest for researchers. Therefore we packaged them up and made these freely available to other researchers to use in and even adapt for their own research.
Exploitation Route A key outcome of the research beyond the academic findings are the the software packages and statistical methods used. We have put effort into making these available on a variety of open-source software sites so that they can easily be obtained by research groups in academic and in industry for their own use and further development.

In terms of the 3Rs - replacing, refining and reducing the use of animals in research, it could be argued that the modelling work we have performed addresses aspects of reducing. However we feel at present the immediate impact is in refining the data we obtain from animal studies.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description TI Simpson (lead) and JD Armstrong have established a new collaboration with UCB Pharmaceuticals. In the first instance they have funded a PhD student at Edinburgh to be supervised jointly by UCB, Simpson and Armstrong. This directly builds on the expertise developed in the project and translates the new methodologies and expertise developed by the programme into a new area of direct health relevance.
First Year Of Impact 2012
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description EPSRC Institutional Sponsorship Award 2016
Amount £52,310 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 03/2017
 
Description Future Emerging Technologies, Flagship Programme
Amount € 89,000,000 (EUR)
Funding ID 720270 
Organisation European Union 
Sector Public
Country European Union (EU)
Start 04/2016 
End 03/2018
 
Description The Human Brain Project
Amount € 54,000,000 (EUR)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 10/2013 
End 03/2016
 
Title InterologWalk 
Description InterologWalk is a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. 
Type Of Technology Software 
Year Produced 2011 
Open Source License? Yes  
Impact This has allowed a number of subsequent studies to incorporate evolutionary information into protein-protein interaction studies. The article is rated as Highly Accessed by BiomMedCentral. External research groups have started contributing to the open source code. 
URL http://search.cpan.org/~ggallone/Bio-Homology-InterologWalk/
 
Description Outdoor Science activities for young people 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact This is an ongoing activity under constant development and delivered approximately 2-3 times per year with young people involved in Scouting primarily aged 13-17 although some activities are being extended to younger age groups. A series of tasks look to develop an awareness of scientific method, how to design experiments, measure accurately, identify and handle sources of noise and handle data. This is only indirectly associated with any specific research funding and more generic in its aim to engage young people in science.
Year(s) Of Engagement Activity 2016,2017
 
Description School visit Linlithgow 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact We had a lot of interaction with the young people with a lot interest in the work we are doing.

Many took away information packs on the brain, others took away contacts for a junior programming club.
Year(s) Of Engagement Activity 2014