Molecular cross-talks during commissural axon guidance in the developing spinal cord – YADDLE

We make extensive use of various mouse models, and several types of tissue and cell cultures, combined with biochemical approaches. We also use of classical techniques to trace axon tracts. We set up several methods to islate tiny pieces of nervous tissue to culture them and analyze various parameters.

We could identify a novel player implicated in the guidance of neuronal projections and having an original contribution. We also characterized novel and unexpected sets of molecular interactions between several types of proteins, playing roles in axon guidance and neuronal survival.

This is a fundamental research project having perspectives in the design of novel therapeutic targets in the context of cancerogenesis and neurological disease.

The first part of the work produced a publication in the scientific journal Neuron. The ongoing part will be presented this year at several meetings.

The principal goal of our project is to study newly identified molecular cross-talks implicated in the guidance of spinal commissural projections across the ventral midline. Dorsal interneurons establish complex network of connections with neurons of spinal and higher brain centers, and participate in circuits controlling sensorimotor information and left right coordination. Commissural projections interconnect the two halves of the central nervous system, and their elaboration require complex signaling to control axon path-finding across the midline.

Appropriate sensitivity of axon tips, the growth cones, which are responsible for perception and integration of environmental positional information, is a key parameter of axon navigation. Given the rather limited number of guidance cues related to the complexity of neuronal networks to build, modulations of growth cone responsiveness to guidance cues over axon navigation might be instrumental to increase the possibilities of pathway choices. Better understanding of axon guidance requires detail investigation of the mechanisms setting appropriate responsiveness to guidance cues. Over the past years, the midline crossing has proven to be a very powerful model for such investigations. Spinal commissural growth cones cross the midline in a ventral structure, the Floor Plate (FP) and indeed alter their sensitivity to local various guidance cues during crossing. These modulations are instrumental for proper pathway selection.

By using combinations of ex vivo and in vivo approaches we recently uncovered a molecular pathway controlling the gain of response to the midline repellent Sema3B. We found that at pre-crossing stage, commissural neurons synthesize the Sema3B receptor sub-units Nrp2 and Plexin-A1 but Plexin-A1 growth cone expression is prevented by a calpain1-dependent processing, which silences growth cone responsiveness. During FP in-growth, this protease pathway is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone structures and sensitization to Sema3B. In our current investigations, we could identify a signaling emanating from FP cells mediated by a cooperation of two cues, a Neurotrophic Factor, GDNF, and a IgSuperFamily Cell Adhesion Molecule, NrCAM, controlling calpain activity and increase of Plexin-A1 level. The first part of our project plans to investigate in details this cooperative FP signaling.

In a second part, we will concentrate on some molecular aspects of Plexin-A1 processing by calpains.

The last part of our project will be devoted to a second molecular cross-talk, linking Plexin-A1 to a key ligand-receptor signaling for midline crossing, the Slits/Robos. We uncovered a molecular interaction between Plexin-A1 and the midline repellent Slit1. Moreover, our analysis of Plexin-A1 null embryos revealed unexpected midline crossing of some commissural axons. Notably this phenotype has been observed in embryos lacking all Slit1-3 proteins, thus supporting a functional relevance of the Plexin-A1/Slit cross-talk. We will thus characterize the network of interactions between Plexins and Robo Slit proteins and using various in vivo models and ex vivo assays, we will investigate the functions of the cross-talk during commissural axon guidance.