The fundamental roles of axonal actin during neuronal growth and longevity

The actin cytoskeleton within axon shafts has long been neglected, but supra-resolution microscopy has now made it amenable to investigation. Here we will use Drosophila genetics to dissect the roles of axonal actin, building on promising pilot data which suggest important functions in axon growth and in maintaining axonal MTs.

Axons are the cable-like protrusions of neurons which electrically wire the nervous system and are indispensable for its function. In spite of their importance, the fundamental mechanisms which underpin the formation and maintenance of axons remain poorly understood. Important understanding will come from work on the actin and microtubule (MT) cytoskeleton which is absolutely required for the growth and maintenance of axons. Actin and MTs are filamentous protein polymers which arrange into intracellular scaffolds maintaining cell architecture and mediating cell dynamics. So far, research has primarily been focussed on the prominent cytoskeletal networks of neurons: firstly, abundant actin networks in motile growth cones (GCs) which guide axon elongation during development; secondly, bundles of MTs which form the structural backbones of axons and are required to establish and then maintain axons for an organism's lifetime (i.e. decades in humans). Further important roles are likely to come from the actin cytoskeleton in axon shafts, but this actin has been difficult to visualise and has been widely neglected.

Recently, supra-resolution microscopy of mouse neurons delivered a precise template for studies of axonal actin. These studies revealed bundles of short actin filaments arranged into periodically patterned rings which surround the axonal MT bundles, ideal to regulate their dynamics. Notably, we find similar repetitive patterns when using supra-resolution microscopy on neurons of the fruitfly Drosophila, suggesting that these structures are evolutionary conserved and functionally relevant. Such relevance is further supported by our experiments with two classes of genetic and drug manipulations: one class is expected to affect actin in axon shafts, the other to maintain actin rings, but both clearly remove actin from GCs. These treatments have differential effects on axon extension which strongly support a model in which axonal actin has growth-promoting roles. Such a role of axonal actin would introduce novel mechanistic concepts into models of axon growth, thus providing new opportunities to unravel this still unresolved, fundamental problem in neurobiology.

Furthermore, our experiments with the two classes of actin manipulations suggest that axonal actin has a second role, which is to maintain axonal MTs. Thus, when MTs are destabilised through specific genetic manipulation, additional removal of axonal actin eliminates their proliferation and axons retract and eventually vanish. This surprising and novel finding likewise opens up new opportunities, and we believe that it will have potential implications not only for axon growth but also for axon degeneration and branching.

To turn our pilot data on axonal actin into substantial understanding of axon biology, we will capitalise on the unique genetic and experimental opportunities provided by fly neurons, for which we have 10 years of experience. Thus, we have already investigated ~40 actin and MT regulators of Drosophila, alone or in combinations, during axon growth and GC regulation. This provides us with a solid and unique knowledge base for the research on this project. Here, we will 1) use supra-resolution microscopy in combination with our actin manipulations to functionally validate the model of axonal actin rings, 2) proof the growth-promoting roles using micro-fluid chambers, refined live imaging and traction fore microscopy, 3) unravel the underlying mechanisms by focussing on actin-dependent MT polymerisation and forward sliding of MTs, and 4) demonstrate the relevance of axonal actin in vivo.

This project explores newly described actin structures of axons as a novel research direction to study the development and maintenance of axons. This topic is important, timely and promising since axon biology closely links to central aspects of brain development, function, regeneration and decay, but how axons are regulated during these processes remains little understood.

Axons are slender cable-like protrusions of neurons which wire the brain, and are indispensable elements of nervous systems. Their structural backbone is formed by bundles of microtubules (MTs) which are the key drivers of axon growth and essential for axon longevity. Supra-resolution microscopy of mouse axons has now shown that axonal MT bundles are surrounded by evenly spaced rings of bundled actin, ideally positioned to regulate axonal MTs. Notably, they are evolutionary conserved, since our supra-resolution microscopy of Drosophila neurons reveals similar repetitive axonal actin structures.

We have started to study their function using versatile fly genetics which provides powerful means to dissect the roles and regulation of the neuronal cytoskeleton. Using the ring model as a template we assess its proposed properties with specific actin manipulations and functional readouts. Our data so far support the model. Notably, they suggest important roles of axonal actin in promoting axon growth and in maintaining the polymerisation of axonal MTs.

Here we will test these proposed roles. 1) We will use supra-resolution microscopy to assess whether our manipulations affect actin rings as predicted, thus functionally validating the ring model. 2) Assessing actin manipulations with micro-fluid chambers, traction force microscopy, live imaging and stretch-biosensors we will test roles of axonal actin in growth promotion. 3) We will assess MT polymerisation and MT sliding as likely candidate mechanisms downstream of axonal actin, and 4) test the functional relevance of axonal actin in vivo.

We use the fruitfly Drosophila to unravel fundamental roles of the actin and microtubule cytoskeleton in axons, during nervous system development and maintenance. The key pathway for achieving impact on this project is to improve the wider appreciation and understanding of our research, and this will primarily involve communication with various target audiences, including other researchers (cell, developmental and neurobiologists), students, representatives from industry and members of the general public. This task is challenging because full appreciation of our research and its enormous potentials requires the integrated understanding of three very different topics, each loaded with specific ideas and concepts:

a) the function, organisation, growth, maintenance and decay of axons; this topic touches on anatomy, neurophysiology, developmental and cell biology, and has important implications for brain disorders or events of spinal cord injury.

b) the key roles of the cytoskeleton during all biological processes of axons, the cytoskeleton as a major lesion site in the ageing brain, the prevalence of "cytoskeletal genes" in human brain disorders and, consequently, the cytoskeleton as a promising but little explored drug target.

c) the strategy of using the invertebrate model organism Drosophila, in particular its powerful genetics, as effective means to understand fundamental biology, the impressive track-record of Drosophila in delivering significant scientific advance, and the translational value of such work in flies.

I am already proactive in communicating our research at all levels, including scientific audiences (several conceptual reviews and a specialist blog), industry (interaction with the Manchester robotics unit, connections to J&J and Boehringer) and the wider public (press releases, an online layman's guide, public engagement in museums and schools). During this project, we will keep momentum through carrying on with these activities, we will qualitatively improve them to increase their impact, and will engage in two new and complementary activities designed to reach even wider audiences.

Impact activities will involve (1) publication of our research in peer-reviewed journals, (2) presentation on conferences three times a year, (3) the writing of a conceptual review about axon growth, (4) active contributions to the nEUROskeleton blog, (5) collaboration with our Media Relations Officer Kath Paddison to turn out press releases, (6) the reactivation of links to industrial representatives (J&J, Boehringer Ingelheim) to discuss the application of our Drosophila models for drug screens, (7) build a web resource informing about axons and the neuronal cytoskeleton, (8) generate an animation film and filmed interviews for this resource, (9) continue with school and museums events, (10) collaborate with North West Schools, the Directorate for the Student Experience, and the Manchester Institute of Education to develop resources which will help to establish Drosophila as a modern teaching tool in schools. The last activity will include an application for a Wellcome Trust People Award. Finally, I will take on one Nuffield student each year.