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Spatial and Temporal Second Messenger Codes for Growth Cone Turning

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Spatial and Temporal Second Messenger Codes for Growth Cone Turning Spatial and temporal second messenger codes for growth cone turning Xavier Nicol1,2, Kwan Pyo Hong, and Nicholas C. Spitzer Neurobiology Section, Division of Biological Sciences, Kavli Institute for Brain and Mind, University of California at San Diego, La Jolla, CA 92093 Edited* by Lynn T. Landmesser, Case Western Reserve University, Cleveland, OH, and approved July 5, 2011 (received for review January 6, 2011) Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent se- generated in growth cones in response to Netrin-1, the principal cond messengers that regulate a wide range of cellular processes. axon guidance molecule required for attraction of commissural During development of neuronal networks they are critical for the axons by the spinal floor plate. We report a major difference in first step of circuit formation, transducing signals required for axon cAMP/calcium interplay between filopodia and the growth cone fi pathfinding. Surprisingly, the spatial and temporal cAMP and cal- center. Optogenetic elevation of cAMP in lopodia but not in fi cium codes used by axon guidance molecules are unknown. Here, growth cone centers drives growth cone attraction. Both lopo- we identify characteristics of cAMP and calcium transients gener- dial and growth cone center signaling pathways are mediated by ated in growth cones during Netrin-1–dependent axon guidance. In activation of the same Netrin-1 receptor, Deleted in Colorectal Cancer (DCC). We show that cAMP dynamics are also essential filopodia, Netrin-1–dependent Deleted in Colorectal Cancer (DCC) for midline crossing of commissural axons in vivo. receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activa- Results tion of DCC in growth cone centers leads to a transient calcium- We identified spatial and temporal second-messenger codes that dependent cAMP increase and a sustained increase in frequency of fi regulate axon guidance using dissociated cell cultures of Xenopus calcium transients. We show that lopodial cAMP transients regu- spinal neurons. Application of a local Netrin-1 gradient to growth fi late spinal axon guidance in vitro and commissural axon path nd- cones of these cultured neurons attracts axons in a cAMP- and ing in vivo. These growth cone codes provide a basis for selective calcium-dependent manner that requires the DCC Netrin-1 re- activation of specific downstream effectors. ceptor (8, 16) mediating both attraction and repulsion in Xenopus spinal neurons (22). In contrast to previous studies (16, 23), we cyclic AMP dynamics | calcium dynamics | compartmentalization | did not include serum in the culture medium because it blocked photoactivated adenylyl cyclase alpha | cyclic AMP oscillations some of the growth cone calcium signals observed in vivo (Fig. S1 A and B). The response to Netrin-1 and its dependence on cAMP yclic AMP (cAMP) is a major cellular second messenger that and calcium were robust: Netrin-1 is an attractant in the control Cactivates and integrates multiple intracellular signaling path- condition, converted into a repellent when adenylyl cyclase is ways. Microdomains of cAMP are generated in cardiac myocytes inhibited, and does not affect the direction of axon growth in the (1), regional domains of cAMP are present in neurons (2, 3), and absence of extracellular calcium (Fig. S1C). cAMP diffusion is restricted by rapid degradation by phospho- We used a plasma membrane-targeted Epac2-camps (pmEpac2- fl diesterases (4) that limit the duration of cAMP signaling (5, 6). camps) FRET probe (24) to monitor uctuations in cAMP con- However, little is known about spatial compartmentalization and centration. Growth cones expressing pmEpac2-camps responded temporal dynamics of cAMP in neuronal growth cones, despite to bath application of forskolin, an activator of transmembrane the importance of cAMP for axon pathfinding in response to adenylyl cyclases, with an increase in the CFP:YFP ratio (Fig. S2 a wide range of molecular guidance cues, including Netrin-1 (7– and Movie S1). Although we did not calibrate the sensor, we 11), semaphorins (12), Slits, and ephrins (13). demonstrated that saturation was not reached with the resting Calcium is another ubiquitous second messenger involved in concentration of cAMP in growth cones (Fig S2). Targeting axon pathfinding, mediating responses to axon guidance mole- pmEpac2-camps to the plasma membrane reduces differences in fluorescence intensity between the center of the growth cone and cules. In addition to axon steering, calcium modulates axon out- fi growth and retraction (14, 15). A sustained gradient of calcium lopodia, and allows monitoring of cAMP in both compartments across the growth cone is thought to be generated by asymmetric (Fig. S3). However, measurement of cAMP levels with this Epac- activation of axon guidance receptors, and to be the relevant based sensor could miss detection of cAMP levels that would be calcium signal for axon pathfinding (16). Spontaneous fast and revealed with a PKA-based sensor. spatially restricted filopodial calcium transients are also sufficient to steer axons (17, 18). The frequency of slower transients in the Dynamics of cAMP and Calcium Signals in Filopodia. When growth cones were stimulated by Netrin-1, cAMP increased transiently in entire growth cone controls the rate of axon extension in vivo and fi ∼ ∼ in vitro (19). The regulation of these transients by axon guidance lopodia 1 min after stimulation, reaching a maximum by 4 min (Fig. 1A). Although stimulation was maintained, the cAMP cues has not been investigated. ∼ Filopodia are critical for axon pathfinding (20), and clues to level returned to baseline 10 min after the onset of Netrin-1 their operation are provided by subcellular localization of signal- application. This effect was not due to desensitization of the ing components. Filopodial enrichment of regulatory subunit II of probe, because pmEpac2-camps reported a sustained increase in protein kinase A, a major effector of cAMP, is required for growth cAMP concentration (Fig. S2). The cAMP response to Netrin-1 cone attraction mediated by intracellular gradients of cAMP or PACAP (21). Modulation of spontaneous calcium transients in filopodia regulates axon turning (17). Temporal regulation of Author contributions: X.N. and N.C.S. designed research; X.N. and K.P.H. performed re- cAMP synthesis is also important for axon responses to ephrin-As search; X.N. analyzed data; and X.N. and N.C.S. wrote the paper. (14), and the frequency of calcium transients in the growth cone The authors declare no conflict of interest. center that includes the lamellipodium regulates the rate of axon *This Direct Submission article had a prearranged editor. extension (19). However, little is known about regulation of filo- 1Present address: Institut National de la Santé et de la Recherche Médicale, U839, Uni- podial second messenger signals and their temporal dynamics by versité Paris 6, Institut du Fer à Moulin, 75005 Paris, France. diffusible axon guidance cues. 2To whom correspondence should be addressed. E-mail: [email protected]. We have investigated the subcellular localization, temporal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dynamics, and local interactions of cAMP and calcium signals 1073/pnas.1100247108/-/DCSupplemental. 13776–13781 | PNAS | August 16, 2011 | vol. 108 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1100247108 Downloaded by guest on September 28, 2021 ) Netrin-1 was blocked when it was replaced with a vehicle or when cultures -1 A 12.5 C 50% were preincubated in a solution containing a function-blocking ΔF/F 2 antibody against the DCC Netrin-1 receptor (Fig. 1 A and B). 10.5 10 s The frequency of fast filopodial calcium transients (17) followed R/R (%) 1 control the same dynamics as the cAMP transient (Figs. 1 C and D and Δ 8.5 2A, Fig. S4,andMovie S2). Starting from a baseline (6.8 ± 0.7 Netrin-1 −1 0 6.5 min ) before Netrin-1 stimulation, the frequency began rising ∼2 cAMP Netrin-1 Filopodial calcium min after the initiation of stimulation and achieved its maximum SQ 22536 Netrin-1 −1 4.5 transient frequency (min (10.9 ± 1.8 min ) ∼4 min after the onset of Netrin-1 application; -5 0 5 10 15 20 it returned to baseline after ∼14 min (Fig. 2A and Movie S2). time (min) Stimulation with vehicle or exposure to anti-DCC prevented this forskolin fi C D time (min) increase in lopodial calcium transient frequency (Fig. 1 and ). 1% Netrin-1 -0.5 0 +4 +4.5+14 +14.5 Ratiometric calcium imaging that avoids artifacts due to volume B ΔR/R fi – 2+ 5 min changes con rmed the Netrin-1 induced increase in frequency of ) fi -1 before lopodial calcium transients. The increase in frequency was D E Netrin-1 fi ** after greater in proximal than in distal lopodia (Fig. S4). 0 mM Ca Netrin-1 16 ** 2 * 2 mM Ca2+ 1.5 cAMP Drives Generation of Filopodial Calcium Transients. Because 0 mM Ca2+ cAMP and calcium regulate each other and modulate axon turning 1.5 * 12 1 (25), we investigated the interplay between cAMP and calcium 1 8 – fi R/R (%) during Netrin-1 induced signaling in lopodia. To determine Δ fi 0.5 0.5 whether cAMP regulates production of lopodial calcium tran- 4 transient frequency Spontaneous filopodial *** sients, we suppressed its synthesis with SQ22536, a blocker of cAMP 0 transmembrane adenylyl cyclases, and stimulated its synthesis with 0 Normalized filopodial calcium 0 calcium transient frequency (min -0.5 before SQ 22536 - + - SQ 22536 - + - forskolin. SQ22536 reduced and forskolin increased the frequency 2-6 min 10-14 min of spontaneous filopodial calcium transients (Fig. 2 C and D). In stimulation forskolin - - + forskolin --+ – contrast, both treatments precluded the Netrin-1 induced in- Fig. 2. The Netrin-1–induced elevation of cAMP drives generation of filo- crease in frequency (Fig.
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