Regulation of neuronal connectivity by second messengers: decrypting the codes – MessengerCodes

We are using combinations of molecular and imaging (FRET, optogenetics) techniques to monitor and manipulate second messengers in subset of retinal ganglion cells and in subcellular compartment of the neuron. These techniques are compatible with in vitro and in vivo experiments, enabling to assay the behavior of developing axons in simplified conditions or in a more complex and physiological environment.

Using an innovative molecular technique developed for this project, we demonstrated that retinal ganglion cells cooperate with their neighbors to form precise connectivity maps in the brain.
To identify the features of calcium and cGMP signals involved in wiring retinal axons into the brain, we developed a molecular toolset enabling to monitor and manipulate second messengers in vivo with subcellular resolution.
We identify phosphorylation sites of proteins modulating cellular adhesion, that are regulated by local cAMP signals required for the development of retinal axon connectivity into the brain.

Globally, our project will provide a better understanding of the cellular and molecular processes requiring second messengers for the development of the neuronal connectivity. Since alteration of second messenger signaling during development perturbs sensory perception in rodents and humans, it might reveal developmental processes affected in a subset of neuro-ophthalmological disorders including amblyopia. More generally, it will help understanding the mechanisms of neuronal connectivity development, providing useful knowledge to apprehend neurodevelopmental pathologies.

At this early stage of the project, scientifique production and patents are still in preparation.

The mature nervous system is an intricate network in which the connectivity between neurons is tightly regulated. Precise connections are crucial for appropriate functioning of the network. Connectivity is organized during development and undergoes limited remodeling in the mature central nervous system (CNS). Consequently, ectopic or imprecise connections during development can lead to major and enduring neurological disorders. Second messengers and especially cyclic nucleotides – cyclic AMP (cAMP) and cyclic GMP (cGMP) – as well as calcium are critical modulators of neuronal network development. They integrate not only extracellular signals guiding axons during navigation to their targets, but also modulate axonal competition in the targets. Therein, these cellular messengers are crucial for refinement of exuberant axonal arbors and pruning of misplaced axonal branches. In addition, these second messengers are involved in a wide range of distinct cellular processes. Despite their early identification as signaling hubs in multiple signaling pathways, we still have partial understanding of how specificity is achieved for numerous downstream effectors. Focusing on subcellular localization, kinetics, and interactions of second messengers, the present project aims to identify and understand the second messenger codes used to shape CNS connectivity, focusing on subcellular localization, kinetics and interactions of cyclic nucleotides and calcium.
The visual system will be used as a model to study the development of neural connectivity. Retinal projections in their main brain targets, the dorsolateral geniculate nucleus (dLGN) and the superior colliculus (SC), are organized in three finely tuned maps. In most mammals, retinal projections target both the same (ipsilateral) and the opposite (contralateral) hemisphere, enabling binocular vision. The first map organizes ipsi- and contralateral projections in segregated regions in the SC and dLGN, requiring a mechanism that involves axonal competition. Physiologically distinct retinal ganglion cells (RGCs) detect different features of light stimuli including motion or border detection. RGC subtypes connect distinct layers of the SC and the dLGN building the second map. The third retinal map is topographic, i.e. axonal arbors of two neighboring RGCs develop in neighboring areas in their targets. This is crucial for an efficient transfer of spatial information to the brain. The development of all three maps requires cyclic nucleotides and calcium signaling, making this system ideal to identify codes of these second messenger signals used during the development of neuronal networks. Using second messengers as an investigative tools, we will investigate developmental processes required for the formation of retinal maps (competition between populations of axons for the binocular map and identification of the yet unknown molecular mechanisms leading to physiologically distinct RGC axon lamination).

We will focus on the following aims, centered on each map of retinal projections:
Aim 1: cAMP-dependent cooperation between populations of axons (binocular map).
Aim 2: Axon guidance and cGMP/Calcium signaling codes for the lamination of axons from physiologically-distinct RGCs.
Aim 3: Topographic map and downstream signaling of cAMP signals restricted to subcellular microdomains.

Globally, our project will provide a better understanding of the cellular and molecular processes requiring second messengers for the development of the neuronal connectivity. Since alteration of second messenger signaling during development perturbs sensory perception in rodents and humans, it might reveal developmental processes affected in a subset of neuro-ophthalmological disorders including amblyopia. More generally, it will help understanding the mechanisms of neuronal connectivity development, providing useful knowledge to apprehend neurodevelopmental pathologies.