Understanding essential roles of microtubule regulators during synapse formation and maintenance

A key prerequisite for nervous system function is the capability of neurons to communicate with other cells via specialised cell junctions called synapses. Synapses contain complex machinery for rapid transmission of signals to partner cells. Once formed, synapses have to be maintained in a plastic state, and precocious loss of synapses is considered a potential cause of neuronal decay in ageing and in neurodegenerative diseases. However, in spite of this importance, the mechanisms underlying synaptic maintenance are very little understood. The overarching aim of this project is to deliver such understanding, thus bridging an important gap in our knowledge about processes of ageing and degeneration in the brain.
During a neuron's life, its synaptic machinery is constantly recycled. To this end, its building blocks have to be efficiently transported between the neuronal cell body and the very distant synapses (up to a meter away in humans), connected only by a cable-like neuronal protrusion called the axon. Such precise movement of synaptic proteins along the axon is achieved by motor proteins which bind to transport vesicles containing synaptic proteins and trail along highways made out of parallel bundles of microtubules (MTs). MTs are dynamic filamentous polymers which are continuously built and degraded throughout a neuron's life, and these processes have to be regulated to sustain proper axonal transport. The number of MTs needs to be well controlled, they have to bear the right posttranslational modifications (PTMs) to promote the right motor protein interactions, and they have to maintain their bundled organisation - all so that blockage or slowdown of transport is prevented. For this, MTs are regulated through MT-binding proteins (MTBPs) which can control MT de/polymerisation, stabilisation, cross-linkage and PTMs. It seems therefore obvious that MTBPs, through controlling MT networks, can regulate axonal transport and consequently also synaptic maintenance and neuronal survival, and this causative chain could provide important explanations for why a number of MTBPs are associated with neurodegenerative disease. However, MTBP-based mechanisms of synaptic maintenance remain poorly understood.
For example, the MTBP Tau was discovered several decades ago. It has been associated with Alzheimer's Disease and Frontotemporal Dementia and has therefore been intensely researched. However, its function in health and disease remains surprisingly poorly understood. This is due to the complexity and robustness of the regulatory networks underpinning MT regulation which are experimentally difficult to decipher. To tackle this problem I am using a genetic model organism, the fruit fly Drosophila, I have extensive experience with this system and the role and regulation of the neuronal cytoskeleton therein. I have provided substantial proof of principle that regulatory mechanisms can be deciphered and applied to higher animals. Apart from the enormous amenability and speed of experimentation, the fundamental advantage for cytoskeletal research in Drosophila is the efficiency with which genes can be manipulated and investigated in combination. Thus, on this project, I capitalise on my finding that functions of tau become apparent when combined with loss of a second MTBP, called Shot. Only upon combined deletion does a new phenotype occur consisting in dramatic loss of synapses caused by collapse of axonal transport of synaptic proteins. This phenotype provides robust readouts to decipher the underlying mechanisms, which will be one key objective of this project. In addition, I will study the relevance of these mechanisms for neuronal survival and assess their potential conservation in mouse neurons. This work will unlock important new mechanistic understanding that will advance research on brain development, ageing and degeneration.

This project aims to understand the role of two microtubule-binding proteins (MTBPs), Shot and Tau, in synapse formation and maintenance. Synapses require sustained axonal transport of synaptic proteins from the distant soma, which is a prerequisite for brain development, function and longevity, and synapse loss is considered an early step in neurodegeneration. Axonal transport is driven by motor proteins trailing along bundles of microtubules (MT), and changing the properties of these MTs can significantly impact on synaptic transport. MTs are regulated by MTBPs which are therefore in a key position to regulate synapse formation and homeostasis, although the underlying mechanisms remain speculative. Understanding these mechanism would help to explain the cell biology of synapse formation and maintenance and deliver mechanisms which sustain neurons in healthy ageing or are targeted in neurodegeneration.
Mutations in the MTBP Tau result in Alzheimer's and other neurodegenerative diseases, but its functions in neurons remain little understood. We previously suggested that Tau has functional links to the MTBP Shot (homologue of dystonin, linked to neurodegeneration in mouse and humans). Meanwhile, I have tested this in shot tau double-mutants, using efficient Drosophila genetics. I find striking suppression of synaptic transport leading to entire loss of synapses, a phenotype that is absent in shot or tau single mutants. This suggests functional synergy and opens up new research avenues into tau and the role of MTBPs in synapse regulation. Here I will use a range of synaptic proteins, mutations for different motor proteins, and refined imaging to pinpoint the precise properties of transport affected; I will investigate underlying mechanisms focussing on MT properties and the JNK pathway as candidates; I will demonstrate their in vivo relevance and test whether combined loss of the Shot and Tau homologues in mouse neurons causes comparable synaptic phenotypes.

The proposed project will unravel fundamental mechanisms of the formation and maintenance of the nervous system, relevant also for work on ageing and neurodegeneration. To make sure impact is reached we will disseminate our work by a) publishing in high impact journals and contributing book chapters; b) presenting our work on national and international research conferences; c) developing on-line resources such as a lab webpage illustrating the main objectives, strategy and outcomes of the project; d) initiating and contributing to scientific multi-author blogs, discussing and informing about main breakthroughs in the area of our research (including our own work).

Since public opinion has an increasing impact on political funding decisions, we will foster public awareness and understanding of the importance of science and research. For this, our research is appealing to the general public which is fascinated by topics of brain research. To impact on the public we will: a) present our work on open days and in public lectures: the University of Liverpool has over 15 years experience in delivering high quality activities on a local, national and global level, and we will make frequent contributions within this framework; b) we will develop a special section on our webpage to inform and enthuse the public about our research; c) we will use the knowledge and experience we have on brain and cytoskeleton research to provide material and concepts for museum exhibitions whenever opportunities arise (see my contributions to the permanent exhibition "All about us" at the "At Bristol" venue).

This project addresses synapse loss and "dying-back" mechanisms likely to bring better understanding of neurodegenerative diseases, pave the way for future research and potential long-term benefits for social well-being. This will be of interest to the commercial and private sector. We know from the treatment of many cancers that the microtubule cytoskeleton is a promising drug target, and in the context of brain disorders this potential has not been well explored. Therefore, we will establish suitable contacts capitalising on the Research and Business Gateway provided by the University of Liverpool which aims to catalyse communication between researchers and business.