AMPc microdomains and structural signaling platforms for the development of retinal projections – MicrocAMP

Understanding the spatial organization of intracellular signals requires technologies that allow local monitoring and manipulation of these signals. We use cAMP-sensitive probes that are targeted to individual candidate microdomains to follow local cAMP concentration, and a cAMP buffer to prevent activation of downstream signaling pathways of cAMP. Targeting this buffer to cellular microdomains will lead to identification of cAMP domains involved in the development of neuronal networks in the visual system.

Tools for monitoring and manipulation of local cAMP signals and the techniques for their insertion in retinal ganglion cells are currently in development. Insertion of tools for monitoring global cAMP concentration is now routinely used in our group. We are currently testing the insertion of the tools for local monitoring and manipulation of cAMP concentration.

cAMP participates to a large range of cellular processes and is a potentially interesting therapeutic target. To use it for therapy, specific manipulation of cAMP-regulated signaling pathways is required. Understanding the spatial organization of cAMP signals might be a starting point to achieve this goal, and might unravel the therapeutic potential of this cellular messenger.

This project is currently in its early stage and finalized scientific production has not yet started. Nevertheless, we consider preparing a publication describing our technical methods that would allow an early diffusion of the tools we are developing.

The mature nervous system is an intricate network in which neurons are connected to specific partners. The choice of these partners is crucial for the correct behavior of the network and is determined at very early stages of development. In the visual system, retinal axons have two levels of organization in their main primary targets, the dorso lateral geniculate nucleus (dLGN) and the superior colliculus (SC). In the dLGN the retinal axons coming from both eyes segregate in two non-overlapping territories. In the dLGN and in the SC, retinal axons are topographically organized in their targets: the temporo-nasal axis of the retina projects on the rostro-caudal axis of the SC, in an ephrin-A-dependent manner.
Both level of organization are dependent on cAMP signaling. Among the nine transmembrane adenylyl cyclases – the cAMP synthesizing enzymes – only adenylyl cyclase 1 (AC1) is crucial for the appropriate development of retinal maps, although other ACs are expressed in the retinal ganglion cells (RGCs) and their targets. cAMP regulates a large range of cellular processes and axonal growth cone behaviors, from emergence, outgrowth, and stabilization or elimination of individual filopodia, to turning and retraction. However the mechanisms by which this ubiquitous messenger regulates specifically such spatially and temporally restricted behaviors are still poorly understood. Compartmentalization of cAMP is thought to explain the activation of distinct cAMP-dependant signaling pathways. Microdomains of cAMP have been imaged in cardiac myocytes. In neurons, indirect evidence suggests their presence, but the components of these microdomains are unknown.
Our project aims to determine the composition of microdomains structuring cAMP signaling and to find out if they are critical for the formation of appropriate neuronal networks. Additionally, we will investigate the potential interactions between these microdomains. We identified three candidates with characteristics that make them potential structural organizers of cAMP signals: lipid rafts, focal adhesions, and the anchoring proteins AKAPs (A kinase anchoring proteins).
The project will follow the subsequent plan:
1. Identification of cAMP microdomains required for the development of the visual system (test of the 3 candidates: lipid rafts, focal adhesions, and AKAPs)
2. Importance of adenylyl cyclase 1 targeting to microdomains
3. Interactions between the identified microdomains
The expected results will increase our knowledge of the mechanisms required for the development of neuronal networks, and together with other studies will in time participate to a better understanding of developmental disorders of neuronal networks, and of the absence of axonal regeneration after lesion in the central nervous system. Many aspects of our study involve molecules affecting axonal regeneration. cAMP is an enhancer of axonal regeneration in the optic nerve and the spinal cord. A subset of axon guidance cues including ephrins are potent inhibitors of axonal regeneration after lesion. The lipid rafts structuring proteins flotillins are overexpressed after lesion of the optic nerve in goldfish, an organism in which axons of the central nervous system regenerate. Our project will provide important insights into the contribution of each of these molecules to axonal regeneration (or lack of axonal regeneration).