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

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.

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.

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)