Our research is focussed on the question how axons (the long extensions sent out by developing and regenerating nerve cells) are able to protrude, extend, and navigate towards their targets where they will ultimately form synapses. At the tip of each axon is a motile, sensory structure, the growth cone, which is able to sense growth permissive and non-permissive cues in its environment and to react accordingly. Important molecules in the environment as well as in the growth cone membrane are cell adhesion molecules (CAMs) which hence act both as external guidance cues and as axonal receptors by interacting with each other. The external signals are transformed into an adequate growth cone reaction (turn towards or turn away; stop, retraction or advance) by intracellular signalling cascades (triggered among others by CAMs) which ultimately act on the local stability of the cytoskeleton. The growth cone cytoskeleton thus is the structure which defines the future growth direction of the axon: The cytoskeleton is stabilized on one side of the growth cone and destabilized on the other which leads to a local collapse and protrusion formation, respectively, so that the axon in the following turns to the side where the growth cone is net stabilized. This differential stabilisation is mainly modulated by microtubule-associated proteins (MAPs) which are capable of enhancing microtubule growth and stability. Moreover, we study the modulators of the MAPs which activate or inactivate their microtubule stabilizing properties and thereby mediate between plasma membrane receptors and cytoskeleton. Our model system are the retina and spinal ganglia of mouse and chicken (embryo to adult), since are readily accessible parts of the nervous system as they are not covered by bone. For these model systems, we developed a variety of culture systems which allow for the study of navigating axons in their natural environment, e. g. organ culture or whole-mount culture. We concentrate on growth and regeneration of axons and are aiming at the improvement / development of paradigms enhancing axonal regeneration (patents).
Roles of the CAMs for axonal growth
The spatio-temporal expression analyses which we performed for ALCAM and NrCAM in the embryonic visual system revealed that both CAMs are exclusively present on extending, fasciculating RGC axons which form the innermost optic fibre layer in the retina and regional subsets in the optic nerve, chiasm, optic tract, and optic tectum, indicating their function for early axon growth and navigation. Using an in vitro assay system mimicking this restricted presence of ALCAM and NrCAM in the growth cone environment by narrow substrate lanes coated on glass cover slips, we could show that both CAMs are able to guide RGC axons (Fig. 1a). Moreover, both CAMs enhance axon growth on laminin substrate, indicating that the presence of these CAMs could speed up RGC axons in vivo. Both CAMs could be demonstrated to contribute crucial to the correct routing of RGC axons into the optic nerve head by inhibition experiments in the intact retina (Fig. 1b). Also the focussing of the RGC growth cones towards the optic fissure and the limitation of their probing activities away from the correct growth direction depends on the presence of the CAMs as visualized by laser scanning microscopy 3D reconstructions (Fig. 1c). Time lapse analysis in retina flat-mount cultures moreover revealed that the RGC axons grow slower and meander more on their way to the optic nerve head if ALCAM or NrCAM is blocked. Together these findings show for the first time that these two CAMs have an important impact on axonal growth and navigation. We are currently studying the impact of CAM density on axonal functions such as initial outgrowth and regeneration using micro- and nano-patterned substrates.
Fig.1. Impact of CAMs on axons.
(a) NrCAM offered as substrate lanes (N) is preferred over laminin (L) by RGC axons.
(b) Blocking of ALCAM results in RGC axons crossing the optic fissure (OF) and growing into the opposite retina side.
(c) In contrast to the slim growth cones focussed towards the OF (upper panel), blocking of NrCAM (lower panel) leads to perpendicularly probing complex growth cones.
Roles of MAPs for axon growth
Growth cone behaviour such as advance, pause, turn, and retraction and thereby the growth direction of the axon is largely determined by the microtubule system which is modulated in its dynamics by MAPs. In growth cones induced to turn at a substrate border, we could show that a special phosphorylated form of the axon-specific MAP microtubule-associated protein 1B (MAP1B) is only found in the stable region of the growth cone, i. e. in the side of the future growth direction. Kinase Cdk5, which performs this type of MAP1B phosphorylation is present in the entire growth cone. The activator of Cdk5, P35, however, is only found in the stable part of the growth cone; thereby a local activation of MAP1B phosphorylation can lead to a differential stabilisation/ collapse of growth cone regions. We could thus demonstrate a crucial role of MAP1B and its modulators Cdk5/P35 for growth cone turning responses and thereby axonal navigation. We also investigated how APC, another MAP present in the growth cone (Fig. 2a), affects the dynamics of microtubules and thus the behaviour of the growth cone. We neutralized domains of the large, multifunctional protein APC by chromophore-assisted laser inactivation (micro-CALI) in one half of the growth cone. Inactivation of the N-terminal domain (which is crucial for APC’s dimerisation and hence conceivably for microtubule bundling/stabilisation) results in growth cone collapse and turn away of the entire axon (Fig. 2b). In contrast, neutralisation of the 20 amino acid repeat domains in the middle region (which are necessary for APC’s integration into a degradation complex) leads to the formation of protrusions and turn of the axons towards this side. This shows for the first time the role of APC for a local and domain specific modulation of microtubule dynamics in growth cones, thus affecting axonal steering. We are currently investigating a MAP k.o.-mouse during development and regeneration with respect to axonal dysfunctions in vivo and in vitro.
Fig. 2. Role of the microtubule system in growth cones.
(a) Laser scanning microscopy visualizes APC and microtubules in the growth cone.
(b) Local laser inactivation of the N-terminal region of APC in one growth cone half leads to a turn of the axon away from this side whereas inactivation of the middle region of APC results in a turn towards this side as seen by the growth cone trajectories depicted for the first 10 min after laser treatment (10 axons each).