Regulation of cellular interactions and synapse development in the CNS.

Brains are networks of interconnected cells. Their functions depend on the selective formation of connections, so that they behave as appropriately configured information processing machines. The process of forming connections, though vital, is not well understood, and we are particularly ignorant of what happens in living networks as they form, make connections and begin to function. As nervous systems develop, nerve cell terminals grow. The growth of these terminals is intimately linked to the formation of connections on them. During normal development, nerve terminal growth is therefore tightly regulated, so as to ensure that normal function can emerge. Conversely, mutations in genes that regulate nerve terminal growth or the formation of connections between them have been linked to mental retardation, cognitive disorders and neurodegeneration. In addition to nerve cells, so-called glia cells also contribute to the make up of the nervous system. Glia cells have in recent years become appreciated for their role in regulating the formation and function of connections that nerve cells form between each other. However, our understanding of how glia cells interact with nerve cells so as to regulate nerve terminal growth and connectivity is particularly patchy. One reason for this is that cell-cell interactions are very dynamic and it has been exceedingly challenging to differentially label and image interacting cells in a developing nervous system. We have a simple model network in the fruitfly fly embryo. Importantly, most developmental processes have been conserved during evolution. It is for this reason and because one can do fly genetics with relative ease, that the fruitfly has been instrumental in driving forward our understanding of nervous system development. We have developed new genetic, microscopy and electrophysiological methods that allow us to study and manipulate particular cells in this system. Using genetics, we can differentially label partner nerve and glia cells (with fluorescent dyes). Using our microscope setup we can image how these cells interact as their terminals grow, form connections with one another and mature. We will determine precisely what happens as connections form - we can study it both morphologically and functionally (as a signaling junction) throughout the period of its development. Through a recent genetic screen we have identified a gene called Anaplastic lymphoma kinase (Alk) as an important regulator of nerve terminal growth. Mutations that alter the activity of this gene have been found to cause cancers in humans and this aspect has been studied. However, other functions of Alk, namely during nervous system development are less well understood. Our preliminary work strongly suggests that Alk is important for regulating the communication between nerve cells and glia and that it may thus regulate nerve terminal growth. We will apply our genetic and imaging methods to test this hypothesis. Having established the consequences of what happens to nervous system development when Alk function is altered, we will investigate underlying mechanisms to understand how these changes occur. The results from this work will help us understand the mechanisms and molecules that are required to form a functioning nervous system.

During nervous system development connections between neurons emerge as a result of dynamic cell-cell interactions: outgrowth, pruning and selective stabilisation of neuronal arbors. In this proposal we focus on two fundamental and important issues: i) how interactions between pre- and postsynaptic terminals and associated glia lead to the stabilisation of neuronal branches and the development of synapses in vivo; ii) the role of Jelly Belly (Jeb)-Anaplastic lymphoma kinase (Alk) signalling, as a new pathway regulating these interactions. We work with the locomotor network of the Drosophila embryo as a model. We have developed new genetic tools to genetically label and manipulate identified connecting neurons independently from one another. We have developed methods to image the dynamics of these cells in vivo. We will use custom algorithms to digitally reconstruct these cells for quantitative analysis of complex morphological data. Our specific objectives are: 1) To fully characterise the normal sequence and dynamics of: i) neuron-glia interactions and ii) dendrite growth and synapse formation between identified partner neurons. We will image differentially labelled, interacting cells in vivo. Using patch clamp recording we will correlate anatomical changes with changes in synaptic function. 2) We identified Jeb-Alk signaling as a new regulator of dendritic growth. Building on the detailed characterization of normal development, we will determine how these events are affected by changes in Jeb-Alk signaling. 3) To identify molecular mechanisms by which Jeb-Alk signaling mediates intercellular interactions, dendritic growth and synapse development. We will investigate the role of specific cell adhesion molecules and signaling pathways that have been identified as candidate effectors for Jeb-Alk signaling.

Who will benefit from this research proposal? The development and maintenance of appropriate synaptic connections is essential for all forms of behaviour and cognition, animal and human. Understanding how synaptic connections are formed and maintained lies at the heart of developmental neuroscience and many disabling disorders, notably mental retardation, epilepsy and cognitive disorders, can be directly related to aberrations in these processes. It is increasingly clear that many disorders can be traced to mutations in highly conserved genetic pathways. One focus of this proposal is the role of Anaplastic lymphoma kinase (Alk) in regulating cell-cell interactions and synapse development in the central nervous system (CNS). Alk encodes a conserved receptor tyrosine kinase. In humans, mutations in Alk cause non-Hodgkin's lymphomas and are associated with retinoblastomas. The role of Alk in the developing nervous system is less well understood, though a possible link between mutations in Alk and a predisposition in Schizophrenia has been reported. Another focus of this work is the role of glia-neuron interactions in the regulation of nerve terminal growth and synapse formation and function. The importance of glia in the development and regulation of synapses is illustrated by discoveries that link defects in (CNS) glia to the pathogenesis of conditions such as epilepsy, neuropathic pain and a number of neurodegenerative diseases (e.g. amyotrophic lateral sclerosis, spinocerebellar ataxia, Huntington's disease, Parkinson's disease and multiple system atrophy). The results from this work, on glia-neuron interactions, likely mediated through Alk regulating dendritic growth and synapse development, will form the basis for future studies on neurodevelopmental and degenerative diseases and thus this work will contribute significantly to the fundamental knowledge base on which clinical studies can build. We use the fruitfly, Drosophila melanogaster, as the experimental organism for well considered reasons. First, the cellular and molecular events we investigate will be of relevance to humans, because the high degree of conservation in the cellular, genetic and molecular pathways that underlie nervous system development and function, from neurogenesis to learning and memory. Second, to genetically label, manipulate and image the dynamics of interactions between identified partner neurons and glia in a network as it develops and matures cannot currently be done in any other system, as far as we are aware. Third, the power of Drosophila genetics is second to none. Use of the fruit fly, with a clear track record of driving discoveries in biology, allows for rapid and effective experimentation, such as the testing of genetic interactions and being able to literally watch the effects of manipulations in living, intact animals and their nervous systems. Fourth, working with Drosophila means less use of vertebrate models, saving on housing and husbandry costs as well as ethical considerations. This has long been a goal of the UK Research Councils and of society at large and falls under the aims of the 3Rs programme: replacement, reduction and refinement. How might individuals, organisations or society benefit from this research? M.L. undertakes regular outreach activities with local schools, particularly during Science Week. This proposal has the potential for medically relevant discoveries. This work will also produce evocative experimental paradigms for teaching school children and undergraduates alike the importance of cell-cell interactions during nervous system development (e.g. movies of interacting nerve cells and the effects on larval behaviour when normal interactions are disrupted).