The mechanistic basis and potential disease relevance of microtubule disorganisation in axons

Here we will study the properties of the microtubule (MT) cytoskeleton of neuronal axons to gain a better understanding of the important roles that MTs play during the formation, maintenance and degeneration of neurons.

Axons are the slender, cable-like, up to several meter long protrusions of neurons which form the nerves that electrically wire our bodies. They can usually not be replaced, hence need to be maintained for up to a century in humans. Unsurprisingly, we gradually lose ~50% of our axons towards old age - and far more in neurodegenerative diseases (ND). In spite of their enormous importance, we know far too little about the mechanisms that maintain these delicate structures long-term or lead to their premature decay in neurodegeneration.

Axon formation and maintenance essentially depends on the microtubule (MT) cytoskeleton. MTs consist of filamentous protein polymers arranged into 25nm thick tubules. In axons, MTs form continuous parallel bundles, serving as structural backbones and highways for life sustaining cargo/organelle transport. In ageing and ND, axons often form swellings where MT bundles become disorganised into criss-crossing curls, which trap organelles and are believed to trigger axonal loss.

For the study of MT disorganisation, we developed the working model of axonal homeostasis based on our experimental data obtained in Drosophila neurons; this model involves three steps (details in CfS pt. 1): (1) MTs have to undergo constant polymerisation/depolymerisation to self-renew. (2) Each MT polymerisation event poses a risk of MT disorganisation, particularly in axons where high densities of MTs and molecular motors generate shear forces which can induce MT curling. (3) Order is imposed by a range of different mechanisms mediated by MT-binding proteins (e.g. by guiding MTs into parallel bundles, or eliminating MTs that have gone off-track). We propose that loss of single (or multiple) of these order-imposing mechanisms increases the risk of MT disorganisation leading to axon swellings - thereby providing potential explanations for late-onset axon degeneration linked genetically to various MT regulators.

So far we have tested and refined this model primarily through experimental work in cultured fly and mouse neurons, by focusing on mechanisms regarding step 3 of our model (i.e. order-imposing MT regulators). Here we will focus on the mechanisms involved in step 2 (i.e. causing the curling of MTs), and compare our knowledge in cultured neurons to the situation in the nervous system in vivo. This work is important for several reasons:

First, data obtained here will reveal the degree to which observations made in the highly efficient model of cultured neurons, reflect mechanisms underlying axon swellings in vivo. This will give important direction for experimental work aiming to unravel how axon swellings form and can be prevented.

Second, we will generate important data concerning MT dynamics and their spatial arrangements in axons. These data will provide important information for the mathematical models of MT behaviours (see support letters) which we are developing in parallel projects - aiming to eventually perform long-term in silico experiments that can test pathological roles of MTs in late-onset neurodegeneration.

Third, our data will provide important understanding, descriptions and concepts of axonal MTs that will aid worldwide research into axonal transport, organelle dynamics and MT regulation, thus promoting general advances in our understanding of axon biology during development, ageing, regeneration and degeneration.

Axons are extremely long, cable-like protrusions of neurons wiring the nervous system. The structural backbones and highways for life sustaining transport in axons are formed by continuous bundles of the microtubule (MT) cytoskeleton. Accordingly, defects in MT regulation can dramatically impact on neuronal development, maintenance, and regeneration, thus causing neuro-developmental or -degenerative disorders. For example, areas of MT disorganisation in pathological swellings of axons, observed during ageing and in neurodegenerative diseases, are associated with axon decay. From work in cultured neurons of fruit flies, we developed a working model explaining the formation of axonal MT disorganisation: polymerising MTs in axons do not automatically arrange into straight bundles but are predisposed to curl up into disorganised arrays. However, a variety of mechanisms mediated by MT-binding proteins are in place to prevent MT disorganisation. We propose that (genetic) defects of these order-imposing proteins render axons more vulnerable to the formation of MT disorganisation/axon swellings, potentially explaining late-onset axon loss, such as in motorneuron disease or spastic paraplegias.
Here we will test the role of MTs in this model. We will use advanced electron microscopy techniques to gain reliable data about MT numbers and spatial arrangements in axons (Obj. 1). We will apply live imaging of cultured neurons to study the processes leading to MT disorganisation in axons (Obj. 2). We will use MT bending assays in vitro, to address how curled MT confirmations are maintained and whether this requires stabilisation through axonal proteins (Obj. 3). We will use three different techniques to study MT disorganisation in axon swellings in vivo and compare it to our observations in cultured neurons (Obj. 4). Data obtained will also be used for our parallel projects developing mathematical models of axonal MT dynamics to test long-term roles of MTs in axon maintenance.

Our project uses the fruitfly Drosophila to unravel fundamental regulatory mechanisms of the 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 be achieved through 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) axons: requiring an understanding of their anatomy, their physiology, their delicate structure, their enormous longevity, and their vulnerability during injury, ageing, developmental and neurodegenerative disease.

B) cytoskeleton: appreciating the existence of different cytoskeletal networks, the requirement of cytoskeleton for virtually all cell functions, the different classes of proteins which associate with and regulate cytoskeleton, the disease relevance of these regulators, the cytoskeleton as a promising drug target.

C) the invertebrate model organism Drosophila: the fundamental concept of using model organisms, understanding why flies came into research, the many experimental advantages of the fly, the translational value rooted in evolutionary conservation, the enormous breadth of topics researched in flies

For several years, I have been proactive in communicating our research at all levels, including fellow scientists, industry, schools and other lay audiences, and our essential strategies and key resources have been successfully implemented. The key task now is to drive these approaches to higher momentum. During this project, we will therefore carry on with our activities, constantly improving their quality and breadth, with the essential goal of driving our engagement to true impact.
The concrete action points/deliverables for this grant period are:

1. Scientific publications (Y2 and Y3; PI, PDRA, RA, SP)
2. Presentation at conferences (~ month 3, 4, 11, 15, 17, 21, 23, 27, 30, 35; PI, PDRA, SP)
3. Conceptual and comprehensive review about axon growth (month 9; PI)
4. Press releases (Y2 and Y3; PI)
5. Further improve web resource on axons and the cytoskeleton (month 18, PI, PDRA, SP)
6. Science fairs and school visits (ca. 4 p.a. , PI, RA, PDRA, SP)
7. Publish an article in a science journal about droso4schools (Y1, PI, SP)
8. Present at a national teacher conference (Y2, PI, SP)
9. Placing candidates at schools and refine/develop teaching resources (Y1-3, PI, SP)