Understanding microtubule regulation during the making and maintenance of axons

Axons are slender processes of neurons extending up to meters across the body, serving as information highways that wire the nervous system. Failure to grow axons during development is either fatal or causes developmental brain disorders. Failure to re-grow axons after injury or stroke is an essential cause for lifelong disabilities. Failure to maintain axons in the ageing brain is considered an important cause of neurodegeneration. Pharmacological studies have demonstrated that axon growth and maintenance are essentially mediated by the highly dynamic microtubule (MT) cytoskeleton. However, how MTs are genetically regulated to promote axon growth and maintenance is not understood. The overarching aim of this project is to deliver such understanding, thus bridging an important gap in our knowledge about brain development, regeneration and ageing in both health and disease.
MTs are filamentous, highly dynamic tubulin polymers that form the backbone of axons. MTs provide structural support to axons as well as highways of intracellular transport from and to the cell body. The directed extension of MTs drives axon growth, whereas their destabilisation correlates with axon retraction or degeneration. MT dynamics continue throughout an axon's life (i.e. up to decades) suggesting that axonal maintenance involves steady-state turn-over of MTs. MTs extend/retract through polymerisation/depolymerisation at their plus ends, and their plus ends interact with the intracellular environment to determine the direction and extend of MT elongation. Various proteins have been reported to regulate MT plus end dynamics, and these include EBs (end binding proteins), +TIPs (proteins binding to EBs), XMAP215 (polymerising MTs), DOUBLECORTIN (stabilising MT plus ends), STATHMIN (sequestering free tubulin), and proteins of cell cortex and organelles that can interact with MT plus ends. The principal molecular functions of most of these MT plus end regulators are known in vitro, and various have been linked to brain disorders clearly illustrating their importance in the nervous system. However, functional studies of these proteins in different neuron systems have produced only mild axon phenotypes (if any), falling short of demonstrating the essential roles that MT plus end dynamics are expected to play during axon growth and maintenance. I hypothesise that the different MT plus end regulators contribute to one common MT plus end machinery and that their functions overlap within this machinery. Deciphering this machinery and identifying the key set of components that drive axon growth and maintenance is therefore an important challenge and the overarching objective of this project.
This challenge requires novel approaches. We use a simple genetic model organism, the fruit fly Drosophila. Research in Drosophila is fast, cheap and capitalises on efficient genetic strategies. It has been a powerhouse for the discovery of mechanisms and concepts underpinning brain development and function, many of which are evolutionary well conserved and have laid important foundations for research in higher animals. We have 8 years of experience with work on cytoskeletal regulation during axon growth in Drosophila and have provided substantial proof of principle that novel understanding can be generated and applied to higher animals. Our pilot studies of MT plus end regulators reveal characteristic axon aberrations and allow us to formulate detailed working models. On this basis, we will study cellular mechanisms of MT plus end regulators and functional links between them. Our work will prove the importance of MT plus end machinery during axon growth and maintenance and deliver a step change in understanding of how this machinery works. This will have important implications for research on developmental brain disorders, neuroregeneration, neurodegenerative diseases and ageing.

Axon growth and maintenance are essential for brain development, regeneration and ageing. Dynamic microtubules (MT) form the backbone of axons, and pharmacological studies have shown that they essentially mediate axon growth and maintenance. However, how MTs are genetically regulated to this end is not understood. Here we will generate such understanding.
MTs are highly dynamic filamentous tubulin polymers that extend and shrink at their plus ends. Known regulators of MT plus ends include EBs (end binding), +TIPs (EB binders), XMAP215 (polymeriser), DCX (stabiliser) and MT plus end interactors of cell cortex and organelles. Functional data reported for these proteins fall short of explaining how MTs are regulated to implement axon growth and maintenance. I argue that true understanding will arise when these factors are studied in combination as part of one common MT plus end machinery. For this, we have unique expertise in the fruitfly Drosophila, applying genetic manipulations of cytoskeletal regulators, alone or in combination, and studying their effects on MTs and axons, both in vivo and in cultured primary neurons. Our pilot studies deleting the above mentioned MT plus end regulators have revealed characteristic axon aberrations to be used on this project. Using genetics in combination with imaging approaches, biochemical assays and EM analyses we will assess our working model. We predict that EB1 and XMAP215 display mutual functional dependency in promoting MT polymerisation, whereas DCX and CLASP can maintain basic levels of MT polymerisation in the absence of EB1. Furthermore, EB1 interacts with the actin-MT linker Shot to guide MTs and with the novel cortical collapse factor Efa6 to eliminate MTs that have gone off-track, thus maintaining MT organisation required for proper axon function. Experimental validation of this model will lead to a step change in our understanding of how MT plus end regulation implements axon growth and maintenance.

Costs caused by brain disorders and brain damage are increasing at a dramatic rate, whilst efforts to develop remedial strategies are diminishing. A recent comprehensive landmark study, commissioned by the European Brain Council estimated the total cost of disorders of the brain to account for 25% of the direct healthcare costs in Europe (Gustavsson et al., 2011, Eur Neuropsychopharmacol 21, 718ff.). Tragically, most pharmaceutical companies have withdrawn from brain research due to the enormously high failure rates in drug development (Abbott, 2011, Nature 480, 161ff.). More fundamental brain research is required to enhance our understanding of the nervous system, and we need new ideas and strategies for drug development.

The cytoskeletal machinery of neurons provides a promising path to be explored to this end. This statement can be deduced from the increasing number of brain disorders linking to cytoskeletal machinery. It is supported by precedent cases, such as the use of the microtubule stabiliser taxol in treatment of nerve regeneration, neurodegeneration as well as cancer. It is also suggested by the mere logic that cytoskeleton regulation underlies virtually all cellular functions, yet involves a relatively limited number of genes. The cytoskeleton is therefore likely to establish common pathomechanisms, comparing to oxidative stress or unfolded protein responses. Our work actively explores this enormous potential of cytoskeletal machinery, with our current focus being on fundamental brain science, but with a clear view to future application.

As the FIRST PATHWAY TO IMPACT for our research, I have established a communicational route to Theo Meert (head of the neuroscience division of Johnson & Johnson in Europe) with the aim of establishing joint projects (CASE studentship, The University of Manchester / Johnson & Johnson Co-Managed Fund). BBSRC funding of this project proposal would allow us to continue on this path by providing further proof-of-principle. A future industrial application will be to capitalise on our expertise and cellular systems to generate models for cytoskeleton-related brain disorders which can then be used for drug screens. We already generated promising pilot data showing that Drosophila primary neurons can be cultured and imaged on automated systems.

The SECOND PATHWAY TO IMPACT regards public awareness. Thus, AP is the initiator of thoroughly planned and elaborate outreach activity that explains the importance of fruit flies in research, with most examples and displays referring to research into brain function and disease. This exhibition has featured with great success at a number of public science events. The underlying concepts and materials are currently being prepared for public dissemination through the web page of the Manchester Fly Facility, to stimulate broader use of these kinds of activities.

As a THIRD PATHWAY TO IMPACT, concepts for the outreach activities are being further developed into teaching packages for schools, using Drosophila to enhance the learning experience of genetics teaching. Recent workshops on extracurricular experience days at two secondary schools were most successful and have started to focus discussions on designing Drosophila-based teaching materials that match curricular requirements. Tools and ideas for this have been generated already and have been made available to the wider Drosophila community (Roote & Prokop, 2013, G3/Bethesda, in press).