Periodic Report Summary - MESSENGER CODES (Dynamics of second messengers in axon guidance: decrypting the codes)
Project context and objectives
The mature nervous system is an intricate network in which neurons are connected to specific partners. The choice of partners is crucial for the correct behaviour of the network and is determined during the early stages of development. As soon as axons begin to grow out, they must navigate through a complex environment to reach their appropriate targets. Cellular second messengers, and particularly cyclic nucleotides - cAMP and cGMP - and calcium are critical for axonal pathfinding. They regulate a large range of axonal growth cone behaviours, from emergence and outgrowth, to turning and retraction. These behaviours enable axons to respond to guidance cues that attract or repel growth cones. However the mechanisms by which these ubiquitous messengers regulate such spatially and temporally restricted behaviours are still poorly understood.
Work performed
We used spinal Xenopus laevis axons as a model. They are attracted by Netrin-1, one of the main axon guidance cues. We exposed spinal axons in culture to Netrin-1, while monitoring two second messengers required for Netrin-1-dependent attraction, cAMP and calcium, using fluorescent sensors. We identified two distinct compartments in the growth cone, with different second messenger signals and interactions. In filopodia, the Netrin-1 application generates a transient cAMP signal followed by a brief increase in calcium transient frequency. In the centre of the growth cone, the cAMP signal is transient but delayed compared to filopodia. In contrast, the calcium transient frequency increases in a sustained manner.
We investigated the interactions between calcium and cAMP in both compartments. In filopodia, removing calcium from the extracellular medium does not affect Netrin-1-dependent cAMP signals. Reducing cAMP concentration blocks the spontaneous filopodial calcium transients, and their frequency is increased when the cAMP synthesis is stimulated. Both treatments block the Netrin-1-dependent increase of calcium transient frequency. Thus, cAMP is upstream of calcium in filopodia. Using similar experiments, we showed that calcium is upstream of cAMP in the centre of the growth cone.
Main results
To determine which pathway is required for axon pathfinding, we used a light sensitive adenylyl cyclase to elevate cAMP concentration locally. cAMP transients that were generated in filopodia on one side of growth cones were able to attract axons. In contrast, elevating cAMP on one side of the growth cone centre did not change the direction of axon outgrowth. To confirm this observation in vivo, we imaged spinal commissural axons and manipulated cAMP concentration, with pharmacological agents or using the photoactivatable adenylyl cyclase. The sustained blockade of cAMP synthesis leads to aberrant axon trajectories: they fail to cross the ventral midline of the spinal cord, unlike untreated axons. The sustained increase in cAMP concentration leads to similar defects. These defects are similar to those observed when Netrin-1 signalling is blocked. We showed that localised pulses of cAMP are able to rescue the defects of Netrin-1 signalling deficient axons, confirming that the signalling pathways we identified in vitro are likely to occur in vivo.
We expect to go further and identify the biochemical components of the neurons responsible for the compartmentalisation of cAMP during axon guidance. We started evaluating the involvement of cellular microdomains in the formation of neuronal network in mice. As most of the adenylyl cyclases are targeted to the plasma membrane, our first candidates are microdomains of the plasma membranes. Understanding the structure of second messenger signalling for axon pathfinding could lead to the development of new recovering therapeutic strategies for a damaged nervous system, after injury or neurodegenerative diseases.
The mature nervous system is an intricate network in which neurons are connected to specific partners. The choice of partners is crucial for the correct behaviour of the network and is determined during the early stages of development. As soon as axons begin to grow out, they must navigate through a complex environment to reach their appropriate targets. Cellular second messengers, and particularly cyclic nucleotides - cAMP and cGMP - and calcium are critical for axonal pathfinding. They regulate a large range of axonal growth cone behaviours, from emergence and outgrowth, to turning and retraction. These behaviours enable axons to respond to guidance cues that attract or repel growth cones. However the mechanisms by which these ubiquitous messengers regulate such spatially and temporally restricted behaviours are still poorly understood.
Work performed
We used spinal Xenopus laevis axons as a model. They are attracted by Netrin-1, one of the main axon guidance cues. We exposed spinal axons in culture to Netrin-1, while monitoring two second messengers required for Netrin-1-dependent attraction, cAMP and calcium, using fluorescent sensors. We identified two distinct compartments in the growth cone, with different second messenger signals and interactions. In filopodia, the Netrin-1 application generates a transient cAMP signal followed by a brief increase in calcium transient frequency. In the centre of the growth cone, the cAMP signal is transient but delayed compared to filopodia. In contrast, the calcium transient frequency increases in a sustained manner.
We investigated the interactions between calcium and cAMP in both compartments. In filopodia, removing calcium from the extracellular medium does not affect Netrin-1-dependent cAMP signals. Reducing cAMP concentration blocks the spontaneous filopodial calcium transients, and their frequency is increased when the cAMP synthesis is stimulated. Both treatments block the Netrin-1-dependent increase of calcium transient frequency. Thus, cAMP is upstream of calcium in filopodia. Using similar experiments, we showed that calcium is upstream of cAMP in the centre of the growth cone.
Main results
To determine which pathway is required for axon pathfinding, we used a light sensitive adenylyl cyclase to elevate cAMP concentration locally. cAMP transients that were generated in filopodia on one side of growth cones were able to attract axons. In contrast, elevating cAMP on one side of the growth cone centre did not change the direction of axon outgrowth. To confirm this observation in vivo, we imaged spinal commissural axons and manipulated cAMP concentration, with pharmacological agents or using the photoactivatable adenylyl cyclase. The sustained blockade of cAMP synthesis leads to aberrant axon trajectories: they fail to cross the ventral midline of the spinal cord, unlike untreated axons. The sustained increase in cAMP concentration leads to similar defects. These defects are similar to those observed when Netrin-1 signalling is blocked. We showed that localised pulses of cAMP are able to rescue the defects of Netrin-1 signalling deficient axons, confirming that the signalling pathways we identified in vitro are likely to occur in vivo.
We expect to go further and identify the biochemical components of the neurons responsible for the compartmentalisation of cAMP during axon guidance. We started evaluating the involvement of cellular microdomains in the formation of neuronal network in mice. As most of the adenylyl cyclases are targeted to the plasma membrane, our first candidates are microdomains of the plasma membranes. Understanding the structure of second messenger signalling for axon pathfinding could lead to the development of new recovering therapeutic strategies for a damaged nervous system, after injury or neurodegenerative diseases.