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Comparative Study
. 2006 Oct;9(10):1257-64.
doi: 10.1038/nn1767. Epub 2006 Sep 10.

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Karen Y Wu  1 , Jonathan H Zippin, David R Huron, Margarita Kamenetsky, Ulrich Hengst, Jochen Buck, Lonny R Levin, Samie R Jaffrey
Affiliations
Comparative Study

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Karen Y Wu et al. Nat Neurosci. 2006 Oct.

Abstract

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.

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Figures

Figure 1
Figure 1
Expression of sAC in rat neurons. (a,b) Immunofluorescence localization of sAC in cultured DRG explant axons. E15 dissociated rat DRG neuronal cultures (DIV 3) were labeled with an anti-sAC antibody (R21) to aa 203–217 of human sAC, and an anti-actin antibody. sAC immunoreactivity (green) was detected throughout the axon and the central portion of the growth cone (a), but was less abundant in filopodia and in the actin-rich growth cone periphery (red) (b). (c) Overlay of a and b. Scale bar, 20 μm. (d,e) Immunofluorescence localization of sAC in cultured DSC explant axons. sAC (green) was detected using anti-sAC (R21), and axons were labeled with an antibody to Tuj1 (red). (f) Overlay of d and e. Scale bar, 20 μm. (g,h) sAC activity is present in the brain. Brain lysates were applied to control Ig or sAC Ig-Affigel 10 resin, and bound sAC was eluted and detected in a cAMP accumulation assay. Adenylyl cyclase activity was detected in the eluates from the sAC Ig-resin and was identified as sAC due to its sensitivity to KH7 (g), and its resistance to forskolin (h). Because forskolin stimulation of adenylyl cyclase activity requires MgCl2, forskolin-stimulation assays were performed in the presence of 5 mM MgCl2. Results presented in g and h are representative of experiments repeated at least five times. Error bars represent s.e.m.
Figure 2
Figure 2
sAC overexpression induces axonal outgrowth and growth cone elaboration. (a) Characterization of sAC- and LacZ-expressing lentiviruses. HEK293T cells were infected with an identical titer of each virus, and the recombinant V5-tagged proteins were detected by western blotting using an anti-V5 antibody. (b,c) Lentivirus-mediated expression of sAC resulted in increased growth cone elaboration compared to that observed in LacZ-infected DRG neuronal cultures. Dissociated E15 DRG neurons were plated as pseudoexplants and infected at DIV 1. The percent of growth cones with a filopodial morphology and the average growth cone size were measured 48 h later. Representative phase contrast images of growth cones expressing LacZ and sAC lentiviruses are shown. Scale bar, 10 μm.
Figure 3
Figure 3
Netrin-1 induction of cAMP is blocked by sAC inhibitors. Netrin-1 treatment elevated cAMP levels in a sAC-dependent manner. Dissociated DRG neurons were preincubated with KH7, KH7.15, 2-hydroxy-estradiol (OH-E, a catechol estrogen) or vehicle in the presence of IBMX, a nonselective phosphodiesterase inhibitor. cAMP levels were measured by enzyme-linked immunosorbent assay (ELISA) after treatment with vehicle or netrin-1 (300 ng ml−1) for 15 min. Netrin-1 increased cAMP levels; this increase was blocked by KH7 and OH-E but not KH7.15. Results shown are the average of three separate experiments. Error bars represent s.e.m.
Figure 4
Figure 4
Netrin-1–induced growth cone elaboration is blocked by sAC inhibitors. (a–h) Netrin-1 mediates growth cone elaboration through sAC. E15 DSC explant cultures (DIV 4) were pretreated with the indicated drug for 30 min, and the morphology of individual growth cones were measured at 0 min and 60 min after treatment with netrin-1 (75 ng ml−1) or vehicle. The percent of growth cones that showed an increase in filopodia number and the relative increase in growth cone size were quantified. (a,b) Phase-contrast images of DSC growth cones at 0 min and 60 min after the addition of netrin-1. (c,d) Growth cone elaboration after 60 min of netrin-1 treatment was blocked by pretreatment with KH7 (3 μM), but not by KH7.15 (3 μM), a structurally similar control compound. (e,f) Growth cone elaboration after 60 min of netrin-1 treatment was also blocked by pretreatment with OH-E (5 μM), a structurally unrelated sAC inhibitor, and with a DCC-blocking antibody (1 μg ml−1). Scale bar, 10 μm. (g) Quantification of percent of growth cones with increased filopodia after 60 min for the conditions in a–f. n = 36 growth cones from three different experiments. (h) Quantification for of fold increase in growth cone size after 60 min for the conditions in a–f. n = 36 growth cones from three different experiments. *P < 0.01, **P < 0.001. Error bars represent s.e.m.
Figure 5
Figure 5
siRNA-mediated knockdown of sAC reduces netrin-1–dependent growth cone elaboration. (a) sAC-specific siRNAs reduced sAC levels in DRG neurons. Top, E15 dissociated DRG neuronal cultures were transfected with control or sAC-specific siRNAs on DIV 1, and sAC protein levels were detected 48 h later by immunoblotting with a sAC-specific antibody (R21). Bottom, the same blot was probed with anti-actin to confirm equal loading in all lanes. (b,c) DRG neurons were transfected as in a and then assayed for netrin-1–dependent growth cone elaboration 48 h later. We measured the morphology of individual growth cones at 0 min and 60 min after treatment with netrin-1 or vehicle, and quantified the percent of growth cones showing an increase in filopodia number (n = 50 growth cones from three different experiments) and the fold increase in growth cone size (n = 27 growth cones from 3 different experiments) after 60 min. siRNA-mediated knockdown of sAC blocked netrin-1–mediated outgrowth. *P < 0.01. Error bars represent s.e.m.
Figure 6
Figure 6
Netrin-1–mediated growth cone elaboration requires cAMP and PKA. E15 DSC explant cultures (DIV 4) were pretreated with the indicated drug for 30 min, and the morphology of individual growth cones was measured at 0 min and 60 min after treatment with netrin-1 (75 ng ml−1) or vehicle. (a,b) Phase-contrast images of DRG growth cones 60 min after the addition of vehicle and netrin-1. (c,d) Netrin-1–induced growth cone outgrowth at 60 min was blocked by pretreatment with KH7 (3 μM) and was rescued by simultaneous treatment with 8-bromo-cAMP (8Br, 3 μM). (e) Netrin-1–induced growth cone outgrowth at 60 min was also blocked by pretreatment with the PKA inhibitor KT5720 (1 μM). Scale bar, 10 μm. (f,g) Quantification for a–e of percent of growth cones with increased filopodia and fold increase in growth cone size, after 60 min. n = 36 growth cones from three different experiments. *P < 0.01, **P < 0.001. Error bars represent s.e.m.
Figure 7
Figure 7
Differentiation of the roles of tmAC and sAC in growth cone signaling. The tmAC inhibitor 2′,5′-ddAdo did not affect netrin-1–mediated growth cone elaboration, and KH7 did not block PACAP-mediated growth cone elaboration. E15 DSC explant cultures (DIV 4) were pretreated with the indicated drug for 30 min, and the morphology of individual growth cones was measured at 0 min and 60 min after treatment with netrin-1 (75 ng ml−1), PACAP (10 nM) or vehicle. (a,b) Phase-contrast images of DRG growth cones 60 min after the addition of vehicle or PACAP. (c,d) PACAP-induced growth cone outgrowth was blocked by 2′5′-ddAdo (50 μM), but not by pretreatment with KH7 (3 μM). (e,f) Netrin-1–induced growth cone elaboration was not blocked by pretreatment with 2′5′-ddAdo. (g,h) Quantification for a–f of percent of growth cones with increased filopodia and of fold increase in growth cone size, after 60 min. n = 36 growth cones from three different experiments. *P < 0.01, **P < 0.001. Error bars represent s.e.m.
Figure 8
Figure 8
Netrin-1–induced axonal outgrowth is blocked by sAC inhibitors. (a) E15 DSC explants cultured on PDL/fibronectin substratum (DIV 4) were pretreated with the indicated drug for 30 min, and axon outgrowth was measured at 0 h and 3 h after treatment with netrin-1 (75 ng ml−1), PACAP (1 nM) or vehicle. Netrin-1–induced axon outgrowth was blocked by pretreatment with KH7 (3 μM), but not by KH7.15 (3 μM). PACAP-induced axon outgrowth was not blocked by pretreatment with KH7. n = 36 spinal cord axons. *P < 0.01, **P value < 0.001. (b–g) E15 DSC explants were cultured in collagen gels and axonal outgrowth was elicited axons. *P < 0.01, **P value < 0.001. (b–g) E15 DSC explants were cultured in collagen gels and axonal outgrowth was elicited by treatment with netrin-1 (300 ng ml−1). Axon growth was measured at 20 h using anti-Tuj1 immunofluorescence to detect axons. (b,c) Images of DSC explant 20 h after the addition of vehicle and netrin-1. (d,e) Netrin-1–induced axon outgrowth was blocked by treatment with KH7 (30 μM), but not by KH7.15 (30 μM). Scale bar, 200 μm. (f,g) Quantification of axon bundles and axon length, per explant, for the conditions in b–e. n = 6 spinal explants from two different experiments. * P < 0.01. Error bars represent s.e.m.
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