cAMP-dependent axon guidance is distinctly regulated by Epac and protein kinase A

Abstract

cAMP is a key mediator of a number of molecules that induce growth cone chemotaxis, including netrin-1 and myelin-associated glycoprotein (MAG). Endogenous neuronal cAMP levels decline during development, and concomitantly axonal growth cones switch their response to cAMP-dependent guidance cues from attraction to repulsion. The mechanisms by which cAMP regulates these polarized growth cone responses are unknown. We report that embryonic growth cone attraction to gradients of cAMP, netrin-1, or MAG is mediated by Epac. Conversely, the repulsion conferred by MAG or netrin-1 on adult growth cones is mediated by protein kinase A (PKA). Furthermore, fluorescence resonance energy transfer reveals that netrin-1 distinctly activates Epac in embryonic growth cones but PKA in postnatal neurons. Our results suggest that cAMP mediates growth cone attraction or repulsion by distinctly activating Epac or PKA, respectively. Moreover, we propose that the developmental switch in growth cone response to gradients of cAMP-dependent guidance cues from attraction to repulsion is the result of a switch from Epac- to PKA-mediated signaling pathways.

Figure 1.

Epac, and not PKA, mediates growth cone attraction to a gradient of a cAMP agonist, but PKA activation induces growth cone repulsion. A–C, Superimposed neurite trajectories in embryonic growth cone turning assays. When transfected with control siRNA, growth cones are attracted to an Sp-cAMPS gradient (A). Attraction is not altered when neurons are transfected with siRNA against PKAc (B) but is switched to repulsion with siRNA-mediated knockdown of Epac (C). D, Cumulative frequency curves showing that the majority of control and PKAc siRNA-transfected neurons exhibit growth cone attraction to the Sp-cAMPS gradient, whereas most growth cones of Epac siRNA-transfected neurons are repelled. Transfection of human Epac1 cDNA along with Epac siRNA switches repulsion back to attraction. The turning angle of each growth cone is plotted on the abscissa against the percentage (ordinate axis) of growth cones turning to that angle or less. The mean turning angle ± SE of each group is represented by a single symbol above the abscissa. E, F, Superimposed traces of neurite trajectories in neonatal DRG growth cone turning assays. Growth cones of untransfected neurons show a neutral turning response to a gradient of a vehicle control (E) but a marked repulsion from a gradient of the PKA agonist 6-Phe-cAMP (F). G, A growth cone turning assay showing the response of a neonatal DRG growth cone at t ₀ (top) and t ₃₀ min (bottom) in a gradient of 6-Phe-cAMP. Average turning angles are indicated by dashed lines in A–C, E, and F. Scale bars: A, G, 10 μm. *p < 0.05.

Figure 2.

Epac, but not PKA, is required for embryonic DRG growth cone attraction to netrin-1. A–C, Superimposed traces of neurite trajectories in growth cone turning assays. Neurons transfected with control siRNA show growth cone attraction to a gradient of netrin-1 (A). Attraction is not altered with knockdown of PKAc (B) but is switched to repulsion with knockdown of Epac (C). D, Cumulative frequency curves (described in Fig. 1 D) show that most growth cones of control transfected neurons are attracted to a netrin-1 gradient. Attraction is unaffected by knockdown of PKAc, but the majority of growth cones are repelled by netrin-1 when Epac is knocked down. Average turning angles are indicated by dashed lines in A–C. Scale bar: A, 10 μm. *p < 0.05.

Figure 3.

Epac, but not PKA, signaling mediates embryonic growth cone attraction to MAG. A–C, Superimposed neurite trajectories in turning assays show that, when transfected with control siRNA, growth cones are attracted to a MAG gradient (A) and that attraction to MAG is not altered in cells transfected with PKAc siRNA (B) but is switched to repulsion when Epac is knocked down (C). D, Cumulative frequency curves (described in Fig. 1 D) show that the majority of growth cones of neurons transfected with control or PKAc siRNA are attracted to the MAG gradient, but most growth cones of neurons transfected with Epac siRNA are repelled by MAG. Average turning angles are indicated by dashed lines in A–C. Scale bar: A, 10 μm. *p < 0.05.

Figure 4.

PKA mediates adult DRG growth cone repulsion from a netrin-1 gradient, and Epac is required for cAMP activation to switch repulsion to attraction. A, Bar chart of mean growth cone turning angles shows that adult growth cones are repelled by a gradient of netrin-1, but the repulsion is switched to attraction with bath application of either Sp-cAMPS or the Epac agonist 8-Me-cAMP. Application of 6-Phe-cAMP has no effect on the netrin-1-induced repulsion of adult growth cones. B, Cumulative frequency curves (described in Fig. 1 D) confirm that the majority of adult DRG growth cones are repelled by netrin-1 even after application of 6-Phe-cAMP, but application of either Sp-cAMPS or 8-Me-cAMP shifts the curve to the right, indicating a switch to attraction. C–G, Superimposed neurite trajectories show that adult DRG neurons transfected with control siRNA are repelled by the netrin-1 gradient (C), as are those of neurons transfected with Epac siRNA (D). Knockdown of PKAc switches off the repulsive response (E). With knockdown of Epac, Sp-cAMPS is no longer able to switch netrin-1-induced repulsion to attraction (F), but neurites transfected with PKAc siRNA and treated with Sp-cAMPS are attracted to netrin-1 (G). H, I, Representative images of growth cones in turning assays in a netrin-1 gradient after transfection with control siRNA (H) or PKAc siRNA (I). Top panels show positions at t ₀ and bottom panels at t ₃₀ min. J, Cumulative frequency curves show that the majority of growth cones transfected with control or Epac siRNA are repelled by a netrin-1 gradient, even after Sp-cAMPS application, but most growth cones of neurons transfected with PKAc siRNA are attracted to netrin-1. Attraction is switched back to repulsion with concurrent transfection of human PKA cDNA and PKAc siRNA. Dashed lines in C–G represent mean turning angles. Scale bars: C, H, 10 μm. *p < 0.05.

Figure 5.

PKA mediates adult DRG growth cone repulsion from a MAG gradient, and Epac is required for cAMP activation to switch repulsion to attraction. A–D, Superimposed neurite trajectories in turning assays show that adult growth cones are repelled by a MAG gradient (A). Repulsion is switched to attraction on application of Sp-cAMPS (B). Application of 8-Me-cAMP also overcomes the repulsion (C), but growth cones are still repelled by MAG on application of 6-Phe-cAMP (D). E, Cumulative frequency curves (described in Fig. 1 D) show that the majority of growth cones are repelled by MAG under control conditions or on application of 6-Phe-cAMP, but a clear shift to the right (attraction) occurs when cAMP or Epac is activated. F, Bar chart shows mean growth cone turning angles of siRNA-transfected adult DRG neurons in a gradient of MAG. Transfection with a control siRNA does not alter growth cone repulsion from the MAG gradient, and siRNA-meditated knockdown of Epac is similarly ineffective. Growth cone repulsion is blocked with knockdown of PKAc. Sp-cAMPS cannot switch repulsion to attraction when Epac is knocked down but can induce attraction when PKAc is knocked down. G, Cumulative frequency curves show that most growth cones of control and Epac siRNA-transfected neurons are repelled by MAG, even when Sp-cAMPS is applied. When PKAc is knocked down, the majority of growth cones are attracted to MAG, and all but three are attracted. Average turning angles are indicated by dashed lines in A–D. Scale bar: A, 10 μm. *p < 0.05.

Figure 6.

FRET analysis confirms the specificity of selective Epac and PKA agonists and shows that netrin-1 activates Epac in embryonic neurons but PKA in postnatal neurons. A, B, Embryonic DRG neurons transfected with an Epac FRET sensor show an increase in growth cone Epac activation on application of 2 μm 8-Me-cAMP but no change in Epac activity after application of 5 μm 6-Phe-cAMP or a vehicle control (A). C, D, Embryonic DRG neurons transfected with PKA FRET constructs show an increase in growth cone PKA activation on application of 6-Phe-cAMP but no change in PKA activity after application of 8-Me-cAMP or a vehicle control (C). E–H, Epac, but not PKA, activation is detected in embryonic DRG growth cones on application of 5 μg/ml netrin-1 (E, F), but PKA and not Epac is activated when netrin-1 is applied to neonatal growth cones (G, H). B, D, F, H, Increased protein activity is represented by darker blue/purple colors within the growth cone (right panels; 5 min after bath application of selective agonists or netrin-1) compared with brighter blue/green colors before application (left panels). Scale bars, 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Growth cone calcium imaging and FRET analysis of B-Raf protein activity. A, C, D, Bath application of 8-Me-cAMP raises calcium in embryonic DRG growth cones (A, C) but not in neurons transfected with Epac siRNA (A, D). B, E, F, Application of netrin-1 also increases calcium in embryonic growth cones (B, E), but this is significantly reduced when Epac is knocked down (B, F). Number of growth cones analyzed in each condition shown in parentheses in legends of A and B. C–F, Representative examples of fluorescence ratio images of fura-2-loaded growth cones with application of 8-Me-cAMP (C, D) or netrin-1 (E, F) in neurons transfected with control (C, E) or Epac siRNA (D, F). G, H, FRET analysis of B-Raf protein activity in embryonic (G) and neonatal (H) DRG growth cones. G, 8-Me-cAMP or netrin-1 application increases B-Raf activity in embryonic growth cones, but 6-Phe-cAMP decreases B-Raf activity. H, B-Raf activity is also increased by 8-Me-cAMP and decreased by 6-Phe-cAMP in neonatal growth cones, but netrin-1 no longer affects B-Raf activity. Scale bars: C–F, 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8.

Schematic diagram proposing a model for distinct cAMP-dependent activation of Epac or PKA signaling to evoke growth cone attraction or repulsion, respectively. cAMP levels in growth cones are controlled by the activities of adenylyl cyclases (which catalyze cAMP formation) and phosphodiesterases (which break cAMP down to 5′-AMP). When an extracellular guidance cue binds to its corresponding receptor on the growth cone surface, an intracellular signaling cascade impinges on cAMP. When cAMP levels are high, Epac is activated and promotes growth cone attraction. When cAMP levels are low, Epac is not activated and thus PKA mediates the cAMP signal, inducing growth cone repulsion.