Spatial and temporal second messenger codes for growth cone turning

Abstract

Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calciumdependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.cyclic AMP dynamics | calcium dynamics | compartmentalization | photoactivated adenylyl cyclase alpha | cyclic AMP oscillations C yclic AMP (cAMP) is a major cellular second messenger that activates and integrates multiple intracellular signaling pathways. Microdomains of cAMP are generated in cardiac myocytes (1), regional domains of cAMP are present in neurons (2, 3), and cAMP diffusion is restricted by rapid degradation by phosphodiesterases (4) that limit the duration of cAMP signaling (5, 6). However, little is known about spatial compartmentalization and temporal dynamics of cAMP in neuronal growth cones, despite the importance of cAMP for axon pathfinding in response to a wide range of molecular guidance cues, including Netrin-1 (7-11), semaphorins (12), Slits, and ephrins (13).Calcium is another ubiquitous second messenger involved in axon pathfinding, mediating responses to axon guidance molecules. In addition to axon steering, calcium modulates axon outgrowth and retraction (14,15). A sustained gradient of calcium across the growth cone is thought to be generated by asymmetric activation of axon guidance receptors, and to be the relevant calcium signal for axon pathfinding (16). Spontaneous fast and spatially restricted filopodial calcium transients are also sufficient to steer axons (17, 18). The frequency of slower transients in the entire growth cone controls the rate of axon extension in vivo and in vitro (19). The regulation of these transients by axon guidance cues has not been investigated.Filopodia are critical for axon pathfinding (20), and clues to their operation are provided by subcellular localization of signaling components. Filopodial enrichment of regulatory subunit II of protein kinase A, a major effector of cAMP, is required for growth cone attraction mediated by intracellular gradients of cAMP or PACAP (21). Modulation of spontaneous calcium transients in filopodia regulates axon turning (17). Temporal regulation o...

Calcium signaling in axon guidance

“…Surprisingly, calcium transients [63,64] and calcium entry through mechanosensitive channels slow axon growth, whereas calcium release from ER stores enhances growth [41]. The relation between calcium transients and cAMP signaling may be different between the central domain of the growth cone and its filopodia [19]. Further, manipulating growth cone calcium waves is sufficient to affect neurite extension directly [65].…”

“…The spatial distribution and level of intracellular calcium is crucial for axon guidance [19], and regulation of this distribution begins with calcium entry into the growth cone. Methods of entry particularly important for axon guidance include voltage-dependent calcium channels (VDCCs), release of intracellular calcium stores, and store-operated calcium entry (SOCE) from extracellular sources ( Figure 1).…”

Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

“…In Xenopus neurons, Netrin-1 gradient has been shown to induce different signaling mechanisms in growth cone centers and in filopodia. 24 According to a model proposed by Nicol and colleagues, 24 Netrin-1 induces a short-term elevation in cAMP levels in filopodia, which causes a brief increase in the frequency of Ca 2+ transients. In contrast, in the growth cone center, Netrin-1 induces a sustained increase in the frequency of Ca 2+ transients, which causes a transient increase in the cAMP levels.…”

“…Traditionally, the gradient has been generated by locally dispensing a concentrated solution using a micropipette. 9,24 The introduction of microfluidic devices enabled the generation of stable gradients, whose profile can be precisely controlled. [31][32][33] The role of Netrin-1 as a growth factor is rarely the main research objective; it is usually investigated to support turning assay results.…”

Dissection, Culture, and Analysis of <em>Xenopus laevis</em> Embryonic Retinal Tissue

“…For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,[19][20][21][22][24][25][26][27][28][29][30][31][32][33] . Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .…”