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Approaches to study protein complexes and signaling systems during neural circuit formation using ES cell-derived neurons.

Reference	BB/I022392/1
Principal Investigator / Supervisor	Professor Britta Eickholt
Co-Investigators / Co-Supervisors	Dr Patricia Kreis, Dr Ivo Lieberam
Institution	King's College London
Department	MRC Ctr for Developmental Neurobiology
Funding type	Research
Value (£)	700,907
Status	Completed
Type	Research Grant
Start date	01/08/2011
End date	17/01/2015
Duration	42 months

Abstract

The PTEN phospatase represents a key node in protein-protein interaction networks, and, through binding and modulation of other signalling component, regulates diverse aspects of cellular morphology and homeostasis. In neurons and their progenitors, PTEN has been shown to specifically regulate cell cycle, neurite outgrowth, synapse formation and synaptic plasticity. We propose here to explore the role of the PTEN pathway at successive stages of neurogenesis and circuit formation, using motor neurons derived directly from embryonic stem cells (ES cells) as our primary experimental system. ES cell-derived motor neurons allow us to rapidly introduce genetic modifications such as fluorescent fusion proteins, cell compartment-specific variants, null mutations, and RNAi knock-downs into undifferentiated ES cell and then systematically assay the interaction of PTEN pathway components in differentiated motor neurons, which closely resemble their primary counterparts and are representative of CNS neurons in general. Previously, we have identified a large panel of factors that physically associate with PTEN. We now plan to dissect the cellular and developmental dynamic of these interactions in neurons using biochemical methods, live cell imaging and in vitro formation of neuromuscular synapses, as well as in vivo approached such as chick in ovo electroporations and xenografting of mouse ES cell-derived motor neurons into embryonic chick spinal cord. We believe that our approach may provide unique insights into the regulation of neural circuit formation through PTEN signalling, as well as identify potential targets for the regenerative therapies of degenerative diseases that affect CNS neurons.

Summary

This research looks at the mechanisms involved in the formation of the complex arrangement of nerve cells within the nervous system. In recent years, it became apparent that a specific factor called PTEN regulates many processes required for nerve cells to proper assemble a functioning nervous system. In addition to this, it was realised that inhibition of PTEN helps the survival of nerve cells under conditions of degeneration, and, also, that it can re-install growth of nerve cells following injury to the nervous system. Therefore, this research proposes to investigate the mechanisms that control PTEN function in the nervous system. We will study (1) how PTEN is regulated by other factors in the nerve cell, (2) how PTEN helps nerve cells to communicate with each other, (3) how deregulation of PTEN by different proteins has an effect on the formation of the nervous system, and (4) how we can translate this information in order to develop novel ways in which we can inhibit the function of PTEN in a very specific way. We will use different experiments, which have novel and complementary advantages. Firstly, we will use stem cells, which we will differentiate into neurons. We will manipulate these cells with DNA sequences known to alter PTEN functions. In this way we generate clones of cells that can be amplified to virtual unlimited quantities. Using these stem cells, we will investigate how alteration in PTEN function affects the nerve cells and which cellular mechanisms are involved in mediating these effects. Secondly, we will study PTEN function in chick embryos as they develop. In this research we will use a set of temporary manipulations of the chick. We will test if our nerve cells produced from stem cells integrate into the nervous system of the chick, and we will also test if direct manipulation of PTEN in the nervous system of the chick affects the establishment of connection between nerve cells. This approach helps us to avoid expensive and time-consuming experiments in commonly used animal models such as the mouse. This work will help us understand how PTEN is regulated in the nervous system, and will help us to develop future treatment strategies that help neurons to re-integrate into neuronal networks following injury or during degenerative conditions.

Impact Summary

PTEN is an important tumor suppressor with established roles in neuronal circuit formation; deregulation of this pathway component increases the risk to develop cancers, but also leads to neurodevelopmental disorders including autism. Interestingly, loss of PTEN in mice has been shown to increase the regenerative growth potential following injury to the nervous system and in degenerative conditions. We believe strongly that our proposed research will help to provide new avenues for the development of more specific approaches and/or validation of pharmacological strategies in the treatment of various disease conditions of the nervous system. In addition to this, the generated embryonic stem cell clones will constitute a universally exploitable system for investigating PTEN regulation (and protein-protein interactions) and will, we are certain, provide powerful tools with which to pursue an avenue of research that will prove of value to the wider neuroscience community and to researchers working in industry. The proposed grouping (PIs and collaborators) have considerable expertise in the molecular, biochemical, as well as cellular or morphological analyses of neuronal tissue and are experts in applying ambitious and modern techniques to our work, which will provide excellent training opportunities for both postdoctoral researchers in a variety of skill sets. Both postdoctoral researchers will be involved in the supervision of lab project that are part of the final undergraduate program here at King's. In this way, we will provide an environment that will contribute to the training of scientists, and which will facilitate the development of skills of postdocs to increase their expertise to manage, to teach, and to direct and supervise lab projects. This commitment will require postdocs to develop expertise in skills that will prove essential in their future career as scientist, but also in other professions. We will offer an MSc thesis projects in Bioinformatics. Thestudent will participate in the project by modeling logical relationships between PTEN pathway components based on emerging experimental data. We believe that this project would offer a unique opportunity for the training of a computational biologist, as his/her theoretical work would be tightly interwoven with the experiments undertaken in the Eickholt and Lieberam groups. The laboratory of B.J.E. has a solid track record of contributing to the public understanding of science and seeking to promote understanding of scientific research in other academic disciplines in a range of ways. For example, in collaboration with Dr. Frances Stracey from the History of Art Department at UCL, she organised a debate on the use of science in art. This event was attended by students and researchers from the sciences and the humanities. It provoked a healthy debate on a range of ethical and technical issues. As a direct result of her visit to the MRC Centre Dr Stracey was invited to publish an article on this topic: 'Bio-art: the ethics behind the aesthetics' (Nature Reviews Molecular Cell Biology, 2009, 10, 496-500). More recently, one of her PhD students also wrote an article on 'visualising firing neuronal synapses', which was published in a non-scientific photography journal. Both PIs regularly host students from different schools in order to provide some hands-on work experiences in the lab environment and to increase the general interest of pupils in the sciences. B.J.E also accepted to accommodate and educate students as part of the 'in2scienceuk' organisation. In2science, UK, offers underprivileged students currently completing Science AS levels in deprived schools the opportunity to work alongside scientists for a 2 week period. We believe these examples demonstrate our commitment to the idea that a considerable engagement in teaching and education at different levels is an important aspect of scientific practice.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Neuroscience and Behaviour, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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