Support for synapses: the role of cell adhesion molecules in glial morphogenesis

The central nervous system (CNS) consists of the brain, spinal cord and retina (eye). It controls most functions of the body and mind. Despite the importance of these tissues are made up of only two major cell types: neurons and glia. Most researchers focus on neurons because they are the electrical wires passing signals to perform daily functions. However, glial cells outnumber neurons in the CNS and they support neurons to make sure they are healthy and function properly. To make up the CNS, neurons and glia need to meet during development and make specific partnerships that last a lifetime. Glial cells have special shapes so that they can connect to the neurons. Changes in glial shape can make neurons sick and potentially lead to disease. So it is important to understand how glial cells get their shape in the first place so we can make sure they keep it and support the neurons throughout the lifespan. We don't know how glial cells get their shape and meet their neuronal partners. We also don't know exactly what happens to neurons if glial cells don't make these connections in the first place. I want to explore these really important fundamental questions.

In order to really understand how glia get their shapes and support neurons the best way is to watch it happen in a living animal during their development. I am an expert in studying glial cells in the retina of the zebrafish using genetics and microscopy techniques. The retina is a really simple CNS tissue, if compared to the brain. I will use the zebrafish to study this very interesting problem as we can see inside it during early development, its retina has neurons and glia just like humans, and we can follow individual cells using fluorescent proteins. Thus, using microscopy I can watch how neurons and glia behave to meet and make their connections in a living fish. I have found that glia are active and change their shapes very quickly to find and contact specific neurons. We don't know what molecules are controlling the glia to find their neuronal partners. To identify molecules I carried out a genetic screen looking for glial cells with shape defects. This identified the cell adhesion molecules, which are important molecules for cell connections in many different tissues, including the retina and brain. However, we don't know how these particular ones work in the glia to control their shapes during development. To find out why these genes are important and how they help the glia find their partners I will delete them in zebrafish and observe how retina development goes wrong in animals without these genes. To achieve this I will use microscopy to watch glia and neurons in retinas that grow abnormally. Finally, I have shown before that if you don't have any glia in the zebrafish retina then it doesn't function properly. So I will test the vision of fish that still have glia but only their shapes, and connections to neurons, are affected. I will test this by using visual behavioral tests and stimulation with specific light patterns, these are experiments that can easily be done and something myself and other experts will work on together to accomplish.

My research programme will tell us how glia find their partner neurons, which cell adhesion molecules are important for glia to get their shapes and how they make sure our CNS function normally. These answers will be very important for understanding how our retinas and brains are built in the first place. If we understand how glia shape is set up then maybe it will be the same molecules to maintain the connections, so this will also be very important for keeping each part of the CNS healthy as we age. Finally, these behaviors and molecules might help with discovering drugs and treatments to change glial shape and make sure they connect to neurons and support them again. This will be very important for patients with neurodegenerative diseases, like Alzheimers, or retina degeneration (major cause of blindness).

Glial morphogenesis is critical for establishing close contacts between glia and neuronal synapses. These close contacts are necessary for support functions and to ensure the proper function and maintenance of the nervous system. In this fellowship I will define the dynamic cell interactions leading to glial contacts with neurons in the synaptic layer of the retina, identify the role of cell adhesion molecules in guiding these contacts and determine the consequences on synapses if glial morphology is not properly established. Using time-lapse confocal microscopy on transgenic zebrafish I have characterised dynamic cell behaviors by Muller glia (MG), the principal glia in the retina, during development. I have carried out transcriptomics on these MG and through a reverse genetic screen identified five cell adhesion molecules (CAMs) that may regulate these morphogenic behaviours and neuronal contacts resulting in properly formed and functioning synaptic circuits. I will use cell specific reporters and antibody markers to define the precise contacts between mature MG in the retina with synapses, including visualising their dynamic sub-cellular interactions with precise neuronal circuits in the developing retina in real time in vivo. To determine the role of CAMs on these cell behaviours and synaptic contacts I will use established mutants, clonal analysis and the novel technique of CRISPRi to knockdown each CAM specifically in the MG. I will characterise the cell behaviors leading to morphogenesis defecrts and improper synaptic contacts, building a defined genetic pathway regulating morphogenesis. I will use mutants and/or CRISPRi to induce defined MG morphology defects and assay neuronal degeneration and synaptic function in the retina. Collectively this will allow me to define the importance of CAMs in the establishment of glial morphology to contact and support synapses, and determine their necessity for proper neuronal survival and function.

Who will benefit from this research?
My proposed research programme will make an immediate impact on the fields of developmental neurobiology and glial biology. It falls directly in line with the BBSRCs remit of "healthy ageing across the lifecourse" as it will determine critical cellular and molecular mechanisms important for the establishment of glial morphology and synapse support. These mechanisms will have clear relevance for the maintenance of glial-synapse contacts potentially enhancing healthy ageing of the nervous system. The primary goal of my research programme is to produce novel knowledge that will benefit academics and clinicians in many fields, but it also has potential implications for patients. This research has direct relevance for people with retinal degenerations or visual impairments, as Muller glia are central to pathologies in most disease. However, the broader implications are for patients with neurodevelopmental disorders (Epilepsy) and neurodegenerative disease (Alzheimers) throughout the CNS. The majority of research focuses on neurons, ignoring an obvious and potentially central player: the glial cells. Glial cells are the support cells of the nervous system, as such the loss of glia contact with neurons would likely result in the loss of support and dysfunction. Thus, it is of the utmost importance to begin to understand the cellular and molecular mechanisms establishing the vital partnership between glia and neurons during nervous system development. As my proposal addresses several unexplored fundamental issues for proper nervous system development and function I feel that this is timely and given the ageing populations all over the world, including the UK, understanding fundamental mechanisms of brain health is imperative.

How will they benefit from this research?
The aim of this research is to tackle fundamental questions for proper nervous system development, with relevance to the maintenance of the CNS in ageing using the zebrafish retina as a model. The final aim is to determine the cellular and molecular mechanisms guiding glia cells to make contacts and support synapses, which may identify pathways for pharmacological or genetic intervention to treat or even reverse the devastating effects of neurodegenerative disease. As glia are modified and react in almost all neurological insults it has been proposed that glial cells are potential key targets for pharmacological intervention. For example, alterations in glial cell morphology and organisation have been noted in the epileptic brain (Oberheim et al. 2008, J Neurosci). Intriguingly, after drug treatment the astrocyte morphology domains recede and the seizures are reduced. This research may also provide genetic targets to screen for genetic counseling in neurodegenerative disorders and rare disease. To this affect I am a member of the Genomics England Clinical Interpretation Partnership. As such I have access to their large genomic datasets to identify human mutations that might implicate disruption of genetic pathways and glial morphology, such as cell adhesion molecules, and lead to the identification of currently unknown cellular processes resulting in disease or susceptibility to particular insults. An amazing property of Muller glia in the retina is they are a source of neuron regeneration after injury in lower vertebrates, and potentially mammals and humans. This is directly relevant to patients with many age-related eye diseases, such as macular degeneration, the leading cause of adult blindness. The datasets and mechanistic insights from this fellowship will therefore be of interest to researchers who wish to understand the mechanisms by which the retina regenerates, which embryonic programs are reactivated during the regenerative process, and how they might promote the integration of neurons and glia into damaged synaptic circuits to result in the regeneration of vision.