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. 2015 Aug 12;35(32):11315-29.
doi: 10.1523/JNEUROSCI.3645-14.2015.

Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B

Pablo Muñoz-Llancao  1 , Daniel R Henríquez  2 , Carlos Wilson  2 , Felipe Bodaleo  2 , Erik W Boddeke  3 , Frank Lezoualc'h  4 , Martina Schmidt  5 , Christian González-Billault  6
Affiliations

Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B

Pablo Muñoz-Llancao et al. J Neurosci. .

Abstract

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination.

Significance statement: This study identifies the guanine exchange factor responsible for Rap1b activation during neuronal polarization and provides an alternate explanation for cAMP-dependent acquisition of neuronal polarity.

Keywords: EPAC signaling; Rap1b signaling; axon; axon initial segment; cytoskeleton; neuronal polarity.

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Figures

Figure 1.
Figure 1.
EPAC 1 and EPAC2 are differentially expressed in cultured hippocampal neurons. a, EPAC1 immunoblot of lysates from cultured neurons at stages II and III normalized against actin (stage II, 0.86 ± 0.13; stage III, 0.98 ± 0.1; Student's t test, three independent experiments.) b, EPAC1 distribution in cultured hippocampal neurons at stages II and III. Tuj1 antibody against β-III-tubulin was used as neuron-specific marker. c, Quantitative analysis of EPAC1 distribution in stage II and III neurons (n = 20 neurons, one-way ANOVA with Tukey's post hoc test, three independent experiments). d, EPAC2 immunoblot analysis performed as in a (stage II, 0.10 ± 0.01; stage III, 0.07 ± 0.03; Student's t test, three independent experiments.) e, EPAC2 distribution in cultured hippocampal neurons at stages II and III. Cells were immunostained as in b. f, Quantitative analysis of EPAC2 distribution in stage II and III neurons (n = 30 neurons, one-way ANOVA with Tukey's post hoc test, three independent experiments). Data represent the mean ± SEM; n.s., not significant; *p < 0.05; ****p < 0.0001. Scale bars: B and E, top, 10 μm; B and E, bottom, 20 μm.
Figure 2.
Figure 2.
EPAC pharmacological activation results in multiaxonic neurons. a, 4 DIV cortical neurons were incubated with 8-pCPT, ESI-09, or ESI-05 for 30 minutes and then activated Rap1B was evaluated by pull down. Rap1B-GTP was normalized against Rap1B input and corrected against tubulin. b, Quantitative analyses of pull-down data as in a (Student's t test; three independent experiments). c, d Neurons cultured for 3 DIV with DMSO or 8-pCPT were immunostained for Tau-1 (c; axon, arrows) or phosphorylated MAP1B (d; SMI-31; axon, arrows) and MAP2 (somatodendritic compartment). e, Quantitative analysis of neurons stained as in c (n = 76 DMSO and n = 70 8-pCPT neurons, left; n = 70 DMSO and 63 8-pCPT neurons, right; Student's t tests; four independent experiments). f, Hippocampal neurons were transfected with the Ral-GDS-GFP construct, treated with DMSO or 8-pCPT, and stained for Tau-1 (arrows). g, Quantitative analysis of GFP fluorescence in axons (i.e., Tau-1-positive neurites) as in f (n = 36 neurons for DMSO and 55 neurons for 8-pCPT; Student's t tests, three independent experiments). Data represent the mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bars: c, d, f, 50 μm.
Figure 3.
Figure 3.
8-pCPT-treated multiaxonic neurons in long-term (10 DIV) culture display markers of mature axons. a, DMSO- and 8-pCPT-treated neurons were cultured for 10 DIV and stained for AnkG, MAP2, and Tau-1. Arrows indicate axons that are positive for AnkG and Tau-1 staining and negative for MAP2 staining. b, Quantitative analysis of neurons treated as in a (n = 60 neurons; ***p < 0.001; ****p < 0.0001; Student's t tests; four independent experiments). c, Neurons were transfected with vGLUT1-Venus (green) and then treated with DMSO or 8-pCPT for a total of 8 DIV. Fixed neurons were stained for Tau-1 (axons; white, arrows) and MAP2 (dendrites, red). d, Higher magnification of axons in DMSO- and 8-pCPT-treated neurons was used for z-stack reconstruction and semiautomated analyses of Venus-vGLUT1 puncta. Top, Raw images of neurons cultured for 10 DIV. Middle, Higher magnification of area indicated in white rectangle on top. Bottom, Filaments (Tau-1 staining in axons) and dots (Venus-vGLUT1 punctate staining). e, Quantification of data from neurons analyzed as in d (n = 12 neurons per treatment; n.s., not significant; Student's t tests; three independent experiments). f, Neurons were transfected with RFP (white), DMSO, or 8-pCPT and immunolabeled for endogenous synaptophysin (green) and MAP2 (red). Arrows indicate positive synapthophysin spots in axons. Data represent the mean ± SEM. Scale bars: a, 30 μm; c, top, 20 μm; c, bottom, 50 μm; d, top, 40 μm; d, bottom, 10 μm; f, bottom, 50 μm.
Figure 4.
Figure 4.
EPAC1 genetic activation results in multiaxonic neurons. a, A Rap1B pull-down activation assay was used to evaluate the effect of WT, DN, and CA constructs of EPAC1 in COS7 cells. In addition, COS7 cells were transfected with a RAP1-GAP construct as a negative control. Only EPAC1 constructs have a HA-tag. b, Quantitative analysis of data shown in a (all compared with normalized control). c, Hippocampal neurons were transfected with the constructs described in a, with RFP as a volume marker. After 3 DIV, neurons were immunostained for Tau-1 (green, arrows) and MAP2 (red). d, e, Quantitative analysis of the percentage of neurons with multiple axons (d) and axonal length (e) from data shown in c. Data represent the mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars: c, 50 μm.
Figure 5.
Figure 5.
EPAC inhibition results in decreased axonal elongation. a, Neurons were treated with control vehicle, EPAC agonist (8-pCPT), or antagonists of EPAC1 and EPAC2 (ESI-09) and then immunostained for Tau-1 (green, arrows) and MAP2 (red). b, c, Quantitative analysis of neurons treated as in a for the percentage of polarized (1 axon), no polarized (0 axon), and mutliaxonic neurons (b) and total axonal length (c; n = 50 neurons per treatment). Data represent the mean ± SEM; n.s., not significant compared with control; *p < 0.05; ****p < 0.001, one-way ANOVA with Dunnett's post hoc test; n = 4 (b) and n = 3 (c) independent experiments. Scale bars: a, 50 μm.
Figure 6.
Figure 6.
EPAC-dependent axonal elongation is independent of PKA signaling. a, Whole protein extracts from 2 DIV cultured hippocampal neurons treated for 6 h with the vehicle (first lane), PKI (a PKA inhibitor; middle lane), and PKI in the presence of 8-pCPT (right lane) were analyzed by immunoblotting with an antibody that recognizes PKA-specific phosphorylation epitopes. Asterisks indicate epitopes that are less abundant in the presence of PKI. b, Quantitative analysis of data shown in a (****p < 0.0001; Student's t tests; three independent experiments). c, Neurons were treated with DMSO, PKI, or PKI + 8-pCPT and assessed for neuronal polarization. Neurons were immunolabeled for Tau-1 (green) and MAP2 (red). d, e, Quantitative analysis of neurons treated in c for the percentage of neuronal phenotypes (d) and axonal length (e; n = 50 neurons per analysis; **p < 0.01 and ***p < 0.001 versus control;#p < 0.0001 versus PKI; n.s., not significant; one-way ANOVA with Dunnett's (d) or Tukey's (e) post hoc test; three independent experiments). f, PKA signaling involved in neuronal polarization was evaluated in control and PKI + forskolin-treated, forskolin-treated, and 8-pCPT-treated N2A neuroblastoma cells as described previously (Cheng et al., 2011a). g, Quantitative analysis of cells treated in f (*p < 0.05, **p < 0.01; n.s., not significant; Student's t tests; three independent experiments). h-i, Cdc42-GTP levels were evaluated in neurons treated with 8-pCPT, 8-pCPT + PKI, and 8-pCPT + ESI-09, no significant differences was observed (three independent studies). A representative blot is presented. Data represent the mean ± SEM. Scale bars: c, 50 μm.
Figure 7.
Figure 7.
Knock-down of EPAC1 delays the polarization and decreases the length in hippocampal neurons. a, Neurons were transfected with ShRNA Scramble-GFP (top) and ShRNA EPAC1-GFP (bottom). After 36 h of culture, neurons were immunostained for EPAC1 (red). Transfected neurons were visualized with GFP marker (arrowhead). a, Right, Close-up view of the boxed areas. b, Neurons were transfected with shRNA as in a and assessed for neuronal polarization. Neurons were immunolabeled for Tau-1 (green, arrow) and MAP2 (red). Quantitative analysis of the percentage of polarized neurons (c, n = 31–40 cells, Student's t tests; *p < 0.05, three independent experiments) and axonal length (d, n = 31–40 cells, Student's t tests; p < 0.0001****, three independent experiments) from data shown in b. Data represent the mean ± SEM; n.s., not significant; *p < 0.05; ***p < 0.0001. Scale bars: a, 50 μm; a, close-up and b, 25 μm.
Figure 8.
Figure 8.
EPAC1 KO hippocampal neurons show delayed polarization. a, Cerebral extracts (40 μg) from WT and EPAC1 KO E18 mice were analyzed for EPAC1 [a, WT brain = 1.4 ± 0.4 arbitrary units (a.u.)] and EPAC2 expression (b, WT brain = 0.37 ± 0.1 a.u.; EPAC1 KO brain = 0.35 ± 0.1 a.u, Student's t tests n.s.; n = 3 samples/genotype). c, Representative images from primary hippocampal neurons isolated from E18 WT and EPAC1 KO mice. Changes in neuronal differentiation at stages II and III were analyzed with anti β III tubulin, phalloidin (F-actin, gray), anti-Tau-1 (green, arrow), or anti-MAP2 (red) antibodies, respectively. d, Percentage of polarized neurons (blue) and nonpolarized neurons without an axon (white) after 3 DIV is shown. Neurons from EPAC KO mice show a significant reduction in the proportion of polarized neurons (n = 47–134 neurons per treatment). e, WT and EPAC1 KO neurons were cultured for 3 DIV and treated either with DMSO or 8-pCPT and immunostained for Tau-1 (green, arrows), MAP2 (red), phalloidin (F-actin, gray), respectively. Representative images show the formation of multiaxonic neurons only in WT neurons treated with 8-pCPT. EPAC1 KO neurons display polarized (white arrow) and no-polarized neurons. Data represent the mean ± SEM; n.s., not significant; *p < 0.05; **p < 0.001. Scale bars: c, 30 μm; e, 50 μm.
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