Summary of work
We are studying the cellular and molecular processes underlying growth and navigation of axons during development and regeneration of the nervous system. Specifically, we analyse the roles of cell adhesion molecules of the immunoglobulin superfamily (IgSF-CAMs) and the intracellular signalling cascades they elicit. ALCAM is a small and unusual IgSF-CAM present in the plasma membrane of growing axons and their growth cones (i.e. the sensory structure at the axon tip). We could show that ALCAM is synthesized and degraded in the growth cone which fine-regulates its plasma membrane density and is crucial for axonal substrate preference. By use of ALCAM-biofunctionalized nano-patterns, we were could demonstrate that ALCAM per se is able to promote cell adhesion and axon growth (initial axon outgrowth as well as axon elongation). This triggered a study aiming at the application of nano-patterned ALCAM as a biomimetic implant-coat to enhance axonal regeneration. We could also show that ALCAM clustering activates the kinase Erk and now want to identify the triggers. Downstream targets of the intracellular signaling are microtubules and microtubule-associated protein (MAPs), and we could show that kinase Cdk5, its activator P35, and phosphatase PP2B play crucial roles in regulation of the phosphorylation state and thereby the MAP functions. Moreover, by analysing a MAP k.o. mouse, we could show the importance of MAPs for axonal navigation. Recently, we developed a technique to long-term image microtubules inside growth cones and are aiming at the quantitative analysis of microtubule dynamics during growth cone explorative behaviour and steering reactions.
During development and regeneration of the nervous system, neurons send out axons with a motile sensory structure at their tip, the growth cone. Interactions of proteins in the growth cone's plasma membrane with molecules in the environment trigger intracellular signal cascades that act on the cytoskeleton; this ensures appropriate steering reactions of the growth cone and ultimately the correct target-finding of the axon during nervous system development and regeneration. We study a subgroup of integral plasma membrane proteins of the growth cone, the cell adhesion molecules of the immunoglobulin superfamiliy (IgSF-CAMs), in particular ALCAM (previously also termed DM-GRASP). We could show that ALCAM plays an important role for the path-finding of axons, for example they fail to enter the optic nerve if ALCAM is blocked. We moreover demonstrated - for the first time for a CAM - that ALCAM is translated in growth cones (Thelen et al., 2012a). This local translation is controlled by regulatory elements (CPEs) in ALCAM's mRNA untranslated region (3'UTR), depends on the kinase Erk; it is moreover a prerequisite for the ALCAM-induced enhancement of axon elongation as well as for the axonal preference of ALCAM-containing microlanes. We also showed that ALCAM's synthesis in the growth cone is counterbalanced by its endocytosis; both processes together fine-regulate the density of ALCAM in the plasma membrane and are moreover crucial for the capability of axons to prefer to grow on ALCAM.
To further elucidate the role of ALCAM spacing, we made use of nano-patterned substrates produced by the group of Prof. J. Spatz (PCI, Heidelberg) which allow for the presentation of ALCAM in highly defined distances (varying from 30-140 nm) as substrate for growing axons (Jährling et al., 2009; Thelen et al., 2012b). These studies revealed for the first time that ALCAM is able to promote cell attachment and axon growth correlating to the substrate ALCAM density in a roughly dose-dependent manner and without the assistance of other molecules. The 70 nm spacing of substrate ALCAM, however, unexpectedly turned out to be incompatible with efficient axon growth which induced us to model the spectrin-based cortical cytoskeleton structure/dimensions. This gave rise of the hypothesis that ALCAM is anchored to the cortical cytoskeleton which we could indeed prove by a variety of approaches recently. In addition, we also performed screens for novel intracellular (Y2H approach) and extracellular (cellular display) binding partners of ALCAM (Pollerberg et al., 2013). Among others, we could identify a guidance molecule as well as a cytokine-like molecule and are currently studying the functional role of these molecular interactions for axon growth. The findings obtained by using the nano-patterned ALCAM substrates also induced translational studies aiming at the development of a biomimetic implant presenting ALCAM in an optimized nano-spacing to enhance axonal regeneration; upon positive external evaluation, the University of Heidelberg supported an international patent application for such an implant and a second one is currently in preparation.
We also analyzed the CAM signaling elicited by inside the growth cone as well as the down-stream signaling targets, in particular microtubules and microtubule-associated proteins (MAPs) which regulate the dynamics of the microtubules. We could show that activation of ALCAM - by clustering this CAM in the plasma membrane - activates the kinase Erk (a series of other signaling components could be excluded). Moreover, MAP1B is regulated in its phosphorylation levels by kinase Cdk5, its activator P35, and phosphatase PP2B which together control the association of MAP1B to the microtubules and thereby their dynamic stability. We are currently imaging microtubule dymanics using fluorescent fusion proteins which binding to their plus tips. We could to optimize various parameters so that high frequency time-lapse videos up to two hours can be obtained; thereby we are now able to long-term image microtubules in navigating axons of warm-blooded vertebrates for the first time worldwide. We are at the moment employing this optimized technique for a comprehensive quantitative study how microtubules inside the growth cone react to external stimuli and which signaling components are involved. To gain deeper insight into the role of MAPs for axonal navigation in vivo, we are also analysing k.o. mice and could find abnormalities with respect to several aspects of axonal growth, including axon elongation and navigation; the findings reveal for the first time a crucial role of the studied MAPs for these important axonal features underlying development and regeneration of the nervous system.
Neuron sending out an axon in vitro, with its growth cone contacting another axon.
Green: CAM-labelling visualizing the plasma membrane, red: microtubules, blue: nucleus
Spinal ganglion with its regenerating axons extending preferentially on ALCAM lanes.
Green: microtubules, red: ALCAM coated as micro-stripes on Laminin substrate