Projects
A cGMP signaling cascade is implicated in axonal branching of sensory axons
For an integrated processing of information perceived from different places of the body or for the divergent distribution of information it is essential that individual neurons establish contacts to multiple neurons that might be located in different parts of the nervous system. To generate this complex circuitry axons are able to branch at specific places during extension and the resulting daughter axons grow to different target regions. Branching that is common to almost every neuron therefore contributes to the enormous complexity of circuits within the nervous system.
A relatively simple and accessible model system for the molecular analysis of axonal branching is the projection of sensory axons into the spinal cord. When entering the spinal cord the growth cone of a sensory axon splits into two arms (bifurcation) after which one of the resulting daughter axons grows in rostral whereas the other extends in caudal direction. After a waiting period collaterals are generated from these stem axons (interstitial branching) that grow to their termination zones where further arborization occurs (terminal branching) and synapses are finally established. Collaterals of proprioceptive neurons terminate in the ventral cord, whereas nociceptive and mechanoreceptive collaterals are confined to the dorsal horn (Figure 1).
Figure1:
Schematic drawing of the trajectories of sensory axon projections within the spinal cord. (adapted from Schmidt et al. J. Cell Biol. 179: 331-340, 2007)
Our detailed analysis of cGMP signaling in mutant mice using axon tracing methods with fluorescent dyes or genetic markers revealed that a cGMP signaling cascade is essential for the bifurcation of sensory axons at the entry zone of the spinal cord (Figure 1). Three components of this signal pathway are currently known: the ligand natriuretic peptide C (CNP), the receptor guanylyl cyclase Npr2 (natriuretic peptide receptor 2) and the cGKIα (cGMP-dependent kinase Iα). In the absence of one of these components, sensory axons are unable to bifurcate; instead, all axons simply turn in either rostral or caudal direction. The ligand is released by neurons and precursor cells in the dorsal horn and its binding to Npr2 on the surface of sensory growth cones leads to the activation of the intracellular guanyly cyclase domain of Npr2 that synthesizes cGMP from GTP. cGMP then activates the kinase cGKIα which in turn phosphorylates so far unknown intracellular proteins. Candidates for phosphorylation might be cytoskeletal elements (actin or tubulin associated proteins) that provide the machinery for bifurcation or other intracellular signaling components.
Figure2:
A signaling cascade composed of the ligand CNP (natriuretic peptide C), the receptor guanylyl cyclase Npr2 and the kinase cGKIα (cGMP-dependent kinase Iα) is implicated in the branching of sensory axons when these enter the spinal cord. The ligand CNP binds to Npr2 which generates cGMP from GTP on the intracellular side. cGMP in turn activates the kinase cGKIα which phosphorylates so far unknown components (scheme). In the absence of one of these components sensory axons do not bifurcate. CNP is generated by dorsal horn neurons and precursor cells, whereas Npr2 and cGKIα are expressed by sensory axons. Microscopic images of DiI labeled sensory axons: Left – wildtype, middle – mutant of CNP. The scheme on the right also illustrates ongoing projects: activation of the ligand by proteolytical processing (PC – protein convertase), search for downstream phosphorylation targets of cGKIα and termination of the cGMP signalling cascade (by phospodiesterases, the scavenger receptor or by phosphorylation of Npr2 in its kinase homology domain).
Electric activity and modulation of neuronal circuits
Neuronal activity appears to be essential for the correct development of the nervous system. As functional circuits form, spontaneous activity becomes correlated contributing to the refinement of circuits. For example in the visual system spontaneous activity is important for the generation of eye specific layers in the LGN and ocular dominance columns in the visual cortex. Neuronal activity appears to enhance the precision and strength of specific circuit connections. However, the links between neuronal activity and molecules contributing to the structural remodelling of circuits are less understood. We have therefore undertaken several approaches to identify cell surface proteins that are linked to neuronal activity and which might have a function in structural modelling during development. These approaches led to the identification of (a) the Ig cell adhesion protein CAR and (b) the EGF- and chondroitinsulfate-containing protein CALEB.
CAMs are considered as candidates to link electric activity to structural changes and synaptic plasticity. CAMs establish initial cell-cell contacts by bringing apposed cell membranes into contact via trans homophilic or heterophilic molecular interactions. They are required to expand initial contact sites and therefore might provide a platform for contact mediated intercellular signalling. Four major structural groups of CAMs are found within the developing nervous system: cadherins/protocadherins, IgCAMs, integrins and neurexins/neuroligin.
In past years Dr Rathjen´s laboratory has discovered a number of cell adhesion molecules belonging to the Ig superfamily. The group has therefore investigated a possible direct link between electric activity and cell adhesion molecules of the Ig superfamily at developmental stages. Several IgCAM deficient neurons (NCAM, L1, neurofascin, NrCAM, CHL1, TAG-1, neurotractin or CAR) were screened whether their absence affects action potential generation and membrane input resistance. The studies showed that the homophilic/heterophilic IgCAM CAR - in contrast to other tested IgCAMs - drives the electric properties of excitable cells.
CALEB is implicated in presynaptic differentiation
CALEB is a transmembrane protein composed of an N-terminal segment that contains chondroitinsulfate chains followed by an acidic stretch, an EGF-like domain, a transmembrane and a cytoplasmic segment. CALEB expression is restricted to the central nervous system and appears to be generated as a precursor protein that becomes converted in a truncated transmembrane form with an exposed EGF domain and in a secreted form. This conversion is facilitated by membrane depolarization, calcium influx through voltage gated calcium channels and occurs at the surface of neurons. CALEB is implicated in synaptic maturation, i.e. CALEB-deficient synapses displayed higher paired-pulse facilitation, reduced depression during prolonged repetitive activation, a lower rate of spontaneous postsynaptic currents as a result of a lower neurotransmitter release probability at early but interestingly not at mature postnatal stages. All measured electrophysiological differences are confined to very young ages. These findings indicated that CALEB might be essential for aspects of synapse maturation.