Alterations of molecular and behavioral responses to cocaine by selective inhibition of Elk-1 phosphorylation

Abstract

Activation of the extracellular signal-regulated kinase (ERK) signaling pathway in the striatum is crucial for molecular adaptations and long-term behavioral alterations induced by cocaine. In response to cocaine, ERK controls the phosphorylation levels of both mitogen and stress-activated protein kinase 1 (MSK-1), a nuclear kinase involved in histone H3 (Ser10) and cAMP response element binding protein phosphorylation, and Elk-1, a transcription factor involved in serum response element (SRE)-driven gene regulations. We recently characterized the phenotype of msk-1 knock-out mice in response to cocaine. Herein, we wanted to address the role of Elk-1 phosphorylation in cocaine-induced molecular, morphological, and behavioral responses. We used a cell-penetrating peptide, named TAT-DEF-Elk-1 (TDE), which corresponds to the DEF docking domain of Elk-1 toward ERK and inhibits Elk-1 phosphorylation induced by ERKs without modifying ERK or MSK-1 in vitro. The peptide was injected in vivo before cocaine administration in mice. Immunocytochemical, molecular, morphological, and behavioral studies were performed. The TDE inhibited Elk-1 and H3 (Ser10) phosphorylation induced by cocaine, sparing ERK and MSK-1 activation. Consequently, TDE altered cocaine-induced regulation of genes bearing SRE site(s) in their promoters, including c-fos, zif268, ΔFosB, and arc/arg3.1 (activity-regulated cytoskeleton-associated protein). In a chronic cocaine administration paradigm, TDE reversed cocaine-induced increase in dendritic spine density. Finally, the TDE delayed the establishment of cocaine-induced psychomotor sensitization and conditioned-place preference. We conclude that Elk-1 phosphorylation downstream from ERK is a key molecular event involved in long-term neuronal and behavioral adaptations to cocaine.

Figure 1.

Patterning Elk-1 phosphorylation in response to cocaine in the striatum. A–D, Cocaine activates Elk-1 and MSK-1 in the same subset of striatal neurons. P-Elk-1 (A, C, green) was detected along with P-ERKs (A, red) or P-MSK-1 (C, red) by immunohistochemical detection on the same DM striatal section, 10 min after an acute treatment with saline (top rows) or cocaine (20 mg/kg, i.p.; bottom rows). The nuclei were labeled by Hoechst staining (blue). Images are single confocal sections. Scale bar, 25 μm. Note the strong colocalization of P-Elk-1 and P-ERK1/2 (A, merge) as well as P-Elk-1 and P-MSK-1 (C, merge) immunofluorescence occurring in the same subset of striatal cells (Merge + Hoescht). B–D, Quantifications of P-ERK1/2 and P-Elk-1 (B) or P-ERK1/2 and P-MSK-1 (D) immunoreactive cells. E, Schematic representation of striatal regions analyzed for quantifications in the DM and the NACC shell. Cocaine-induced P-Elk-1 immunoreactivity in the DM (F) or NAcc shell (G) is abolished by pharmacological inhibition of NMDA (MK801; 0.1 mg/kg) or DA D₁ (SCH23390; 0.1 mg/kg) receptors or MEK inhibition (SL327; 50 mg/kg). The inhibitors were administered intraperitoneally 30 min before cocaine. B–D, Data (means ± SEM; n = 4 mice per group) were analyzed using unpaired t test for each marker. **p < 0.01, cocaine group versus saline group. F–G, Data (means ± SEM; n = 3–6 mice per group) were analyzed using one-way ANOVA (between subjects) for the DM (F_(4,14) = 34.38, p < 0.001) and NAcc shell (F_(4,14) = 18.81, p < 0.001), followed by post hoc comparisons (Newman–Keuls test). ***p < 0.001, cocaine group versus saline group; °°°p < 0.001, cocaine group versus cocaine groups pretreated with antagonists or MEK inhibitor.

Figure 2.

Dose–response curves and kinetics of TDE effects on cocaine-induced phosphorylation of ERK, MSK-1, Elk-1, and H3 (Ser10). A–D, Immunohistochemical detection of cocaine-induced P-ERK1/2 (A), P-MSK-1 (B), P-Elk-1 (C), and P-H3 (Ser10) (D) was performed on striatal slices (illustrated here is the DM) 10 min after an acute treatment with saline or cocaine (20 mg/kg, i.p.) in mice. When indicated, the mice were pretreated with the TDE peptide (8 mg/kg), 60 min before cocaine treatment. Note the increased immunoreactivity for all markers in the cocaine group compared with saline control group. Note also the decreased P-Elk-1 and P-H3 (Ser10) immunoreactivities in the TDE-treated cocaine group when compared with cocaine alone. Scale bars, 50 μm. E–H, Quantifications of P-ERK1/2 (E), P-MSK-1 (F), P-Elk-1 (G), and P-H3 (Ser10) (H) immunoreactive cells in the DM, 10 min after an acute treatment with saline, cocaine alone (20 mg/kg), or cocaine and TDE (0–12 mg/kg) 60 min before cocaine. I, Illustration of the TDE effects on cocaine-induced phosphorylation of Elk-1. TDE (8 mg/kg) administered from 0.5 to 8 h before cocaine (20 mg/kg); mice were killed 10 min after cocaine administration. J, Quantification of P-Elk-1 immunoreactive cells in the DM in mice pretreated with TDE at indicated times (0–8 h) before a cocaine treatment occurring 10 min before being killed. Note the limited time window of TDE inhibitory effect between 30 and 120 min before cocaine administration. E–H, Data (means ± SEM; n = 8–16 mice per group) were analyzed using one-way ANOVA (between subjects) for P-ERK1/2 (F_(5,62) = 22.29, p < 0.001), P-MSK-1 (F_(5,62) = 31.76, p < 0.001), P-Elk-1 (F_(5,62) = 17.05, p < 0.001), and P-H3 (Ser10) (F_(5,62) = 6.665, p < 0.001), followed by post hoc comparisons (Newman–Keuls test). *p < 0.05, **p < 0.01, ***p < 0.001, cocaine groups versus saline group; °p < 0.05, °°p < 0.01, °°°p < 0.001, TDE pretreated cocaine groups versus saline pretreated cocaine group. J, Data (means ± SEM; n = 7–20 mice per group) were analyzed using one-way ANOVA (between subjects) (F_(6,65) = 2.254, p < 0.05), followed by post hoc comparisons (Dunnett's multiple test). *p < 0.05, 0.5–8 h TDE pretreated groups versus immediately TDE pretreated group.

Figure 3.

Mutations of Elk-1 DEF or DEJL domain alter glutamate-induced H3 (Ser10) phosphorylation but preserve MSK-1 activation in vitro. A, Schematic representation of the Elk-1 mutants encoded by the plasmids used for transfections. Striatal neurons were transfected at DIV6 with cDNAs encoding HA-tagged versions of WT Elk-1 (Elk-1 Wild-Type), Elk-1 carrying a deletion of the DEJL domain (Elk-1 DEJL deletion), or a mutation of the DEF domain (Elk-1 DEF mutation). Twenty-four hours later, neurons were incubated in the absence (Control) or presence of glutamate (Glutamate; 10 μm) for 20 min, with or without U0126 (10 μm) applied 30 min before and during glutamate (Glutamate + U0126) application. B–D, Representative pictures of striatal neurons transfected with the WT or HA-tagged DEF Elk-1 mutants treated or not with glutamate in the absence or presence of U0126. Transfected cells were detected with anti-HA antibody (red) or P-Elk-1 with a phospho-specific antibody (green). White arrows indicate transfected striatal neurons that are P-Elk-1 positive. E–H, Quantifications of P-ERK1/2 (E), P-MSK-1 (F), P-Elk-1 (G), and P-H3 (Ser10) (H) immunoreactive cells, in HA-transfected cells. Note the strong decrease in P-Elk-1 and P-H3 (Ser10) signals in cells transfected with the DEF mutant, whereas the DEJL mutant only affects P-H3 (Ser10). Note also the total inhibition of all markers in the case of a pretreatment with U0126. E–H, Data (means ± SEM; n = 3–4 independent experiments per group) were analyzed using two-way ANOVA: for P-ERK1/2, effect of treatment, F_(2,18) = 95.28, p < 0.001; effect of mutation, F_(2,18) = 0.059, NS; for P-MSK-1: effect of treatment, F_(2,18) = 53.71, p < 0.001; effect of mutation, F_(2,18) = 0.39, NS; for P-Elk-1: effect of treatment, F_(2,27) = 120.4, p < 0.001; effect of mutation, F_(2,27) = 29.54, p < 0.001; for P-H3 (Ser10): effect of treatment, F_(2,18) = 19.05, p < 0.001; effect of mutation, F_(2,18) = 7.36, p < 0.01, followed by post hoc comparisons (Bonferroni's test). **p < 0.01, ***p < 0.001, control (Ctrl) versus glutamate (Glu); ⁺p < 0.05, ⁺⁺⁺p < 0.001, glutamate versus glutamate + U0126; °p < 0.05, °°°p < 0.001, wild-type Elk-1 versus Elk-1 construct (DEF mutation or DEJL deletion).

Figure 4.

Comparative effects of TDE and TAT–DEJL–Elk-1 peptide on molecular responses to cocaine. Quantification of P-ERK1/2 (A, E), P-MSK-1 (B, F), P-Elk-1 (C, G), and P-H3 (Ser10) (D, H) immunoreactive cells in the DM (A–D) or in the NAcc shell (E–H), 10 min after an acute treatment with saline or cocaine (20 mg/kg). When indicated, the mice were pretreated with the TDE or the TAT–DEJL–Elk-1 (TAT–DEJL) (8 mg/kg, i.p.) 60 min before cocaine treatment. Note the increased immunoreactivity for all of the markers in the cocaine groups compared with saline control groups. Note also the decreased P-Elk-1 and P-H3 (Ser10) immunoreactivities in the cocaine group pretreated with TDE when compared with cocaine alone and the absence of effect of TAT–DEJL–Elk-1 on P-Elk-1 immunoreactivity. A–H, Data (means ± SEM; n = 3–7 mice per group) were analyzed using one-way ANOVA (between subjects) in the DM for P-ERK1/2 (F_(3,14) = 91.30, p < 0.001), P-MSK-1 (F_(3,14) = 103.3, p < 0.001), P-Elk-1 (F_(3,14) = 25.63, p < 0.001), and P-H3 (Ser10) (F_(3,14) = 15.99, p < 0.001) and in the NAcc shell for P-ERK1/2 (F_(3,14) = 5.99, p < 0.01), P-MSK-1 (F_(3,14) = 11.46, p < 0.001), P-Elk-1 (F_(3,14) = 4.52, p < 0.05), and P-H3 (Ser10) (F_(3,14) = 41.20, p < 0.001), followed by post hoc comparisons (Newman–Keuls test). *p < 0.05, **p < 0.01, ***p < 0.001, cocaine groups versus saline group; °p < 0.05, TDE pretreated cocaine group versus saline pretreated cocaine group.

Figure 5.

Inhibition of Elk-1 phosphorylation alters SRE-driven gene regulation by cocaine in vivo. Immunohistochemical detections of c-Fos (A–C) and Zif268 (D–F) were performed in the DM and the NAcc shell 60 min after an acute treatment with saline or cocaine (10 mg/kg) in mice pretreated with TDE or SCR (8 mg/kg) 60 min before cocaine treatment. Note the increased immunoreactivity of c-Fos (A) and Zif268 (D) in mice pretreated with SCR before cocaine administration. Note also the decreased immunoreactivity in TDE pretreated cocaine groups. Scale bars, 50 μm. G, Real-time quantitative PCR was performed to evaluate levels of ΔfosB, arc/arg3.1, homer1a, srf, and actin mRNA 45 min after an acute cocaine injection in mice pretreated for 1 h with either TDE or SCR. B, C, E, F, Data (means ± SEM; n = 8 mice per group) were analyzed using one-way ANOVA (between subjects) in the DM for c-Fos (F_(3,28) = 16.54, p < 0.001) and Zif268 (F_(5,21) = 16.80, p < 0.001) and the NAcc shell for c-Fos (F_(3,28) = 14.51, p < 0.001) and Zif268 (F_(3,28) = 10.86, p < 0.001), followed by post hoc comparisons (Newman–Keuls test). *p < 0.05, **p < 0.01, ***p < 0.001, cocaine groups versus saline group; °p < 0.05, °°p < 0.01, TDE pretreated cocaine group versus SCR pretreated cocaine group. G, Data (means ± SEM; n = 5–6 mice per group) were analyzed using one-way ANOVA (between subjects) for ΔfosB (F_(3,19) = 34.87, p < 0.001), arc/arg3.1 (F_(3,19) = 16.64, p < 0.001), homer1a (F_(3,19) = 5.349, p < 0.01), srf (F_(3,19) = 0.581, NS), and actin (F_(3,19) = 0.163, NS), followed by post hoc comparisons (Newman–Keuls test). *p < 0.05, **p < 0.01, ***p < 0.001, cocaine groups versus saline group; °p < 0.05, °°p < 0.01, TDE pretreated cocaine group versus SCR pretreated cocaine group.

Figure 6.

Inhibition of Elk-1 phosphorylation inhibits cocaine-induced dendritic plasticity. A three-dimensional morphological analysis of DiI-labeled medium spiny neurons was performed in the NAcc shell of mice chronically treated after a chronic treatment with saline or cocaine (10 mg/kg; 5 injections over 3 d) in mice pretreated with TDE or SCR (8 mg/kg) 60 min before cocaine treatment. A, Representative pictures of DiI-stained dendritic sections in each experimental group. B, Quantifications of the number of dendritic spines detected per 10 μm of dendritic sections. Note that pretreatment with TDE totally abrogates cocaine-induced increase in dendritic spine density. C, Quantifications of head-spine diameters for each experimental group. Black dots represent the mean head-spine diameters per animal (n = 7 animals per group), and gray dots represent the median head-spine diameters per neuron (n = 10–14 neurons per animal). The black bar represents the mean head-spine diameters per group. Note the decreased head-spine diameters in chronically TDE pretreated groups independently of cocaine. B, Data (means ± SEM; n = 7 mice per group) were analyzed using two-way ANOVA: effect of pretreatment, F_(1,24) = 5.443, p < 0.05; effect of treatment, F_(1,24) = 5.342, p < 0.05, followed by post hoc comparisons (Bonferroni's test). **p < 0.01, cocaine groups versus saline group; °°p < 0.01, TDE pretreated cocaine group versus SCR pretreated cocaine group. C, Data (n = 72–79 neurons) were analyzed using Kruskal–Wallis test (p < 0.0001), followed by post hoc comparisons (Dunn's tests). **p < 0.01, ***p < 0.001; NS, not significant.

Figure 7.

Inhibition of Elk-1 phosphorylation and cocaine-induced behavioral adaptations. Spontaneous locomotor activity: TDE compared with SCR injected at the dose of 8 mg/kg 60 min before the exposure to a novel environment (actimeter) does not alter either the spontaneous horizontal (ambulation) or vertical activity (rearing). B, Locomotor habituation. TDE compared with SCR injected at the dose of 8 mg/kg 60 min before the exposure to the same environment (actimeter) for 2 consecutive days does not alter the locomotor habituation. C, After 3 habituation days (days −2 to 0), mice were pretreated daily for 5 consecutive days, with either TDE or SCR at the dose of 8 mg/kg 60 min before saline (left) or cocaine (10 mg/kg; right) injection, and locomotor activity was measured. This procedure was also repeated on day 12 after a 7 d withdrawal. D, E, CPP induced by cocaine (n = 8 mice per group) was performed from mice pretreated during the conditioning phase of the protocol with TDE or its scramble version 1 h before cocaine administration in a one-pairing (D, left; E, F) or three-pairing (D, right; G, H) CPP paradigm. C, Data (means ± SEM; n = 14–15 mice per group) were analyzed using mixed-factor two-way ANOVA (repeated measure over time): saline: effect of time, F_(8,224) = 7.53, p < 0.001; effect of pretreatment, F_(1,224) = 0.35, NS; 10 mg/kg cocaine: effect of time, F_(8,216) = 10.69, p < 0.001; effect of pretreatment, F_(1,216) = 6.55, p < 0.05, followed by post hoc comparisons (Bonferroni's test). *p < 0.05, **p < 0.01, present day versus day 1; °p < 0.05, °°p < 0.01, TDE pretreated cocaine group versus SCR pretreated cocaine group. D, Data (means ± SEM; n = 8 mice per group) were analyzed using mixed-factor two-way ANOVA (repeated measure over conditioning session): one-pairing protocol SCR pretreatment: effect of conditioning, F_(1,14) = 7.37, p < 0.05; effect of treatment, F_(1,14) = 3.12, NS; one-pairing protocol TDE pretreatment: effect of conditioning, F_(1,14) = 2.51, NS; effect of treatment, F_(1,14) = 0.62, NS; three-pairing protocol SCR pretreatment: effect of conditioning, F_(1,14) = 32.68, p < 0.001; effect of treatment, F_(1,14) = 7.73, p < 0.05; three-pairing protocol TDE pretreatment: effect of conditioning, F_(1,14) = 21.0, p < 0.001; effect of treatment, F_(1,14) = 5.35, p < 0.05, followed by post hoc comparisons (Bonferroni's test). **p < 0.01, ***p < 0.001, posttest versus pretest; °p < 0.05, °°°p < 0.001, saline versus cocaine. E–H, Data (means ± SEM; n = 8 mice per group) were analyzed using paired t test for all of the groups before (Pre) and after (Post) conditioning. *p < 0.05, **p < 0.01, ***p < 0.001, drug-paired chamber versus vehicle-paired chamber. Dashed lines represent equal exploration of both chambers.

Figure 8.

Schematic representation of molecular events driven by Elk-1 phosphorylation in response to cocaine. A, During cocaine administration, combined activation of D₁R and NMDAR drives ERK activation via calcium entry (Pascoli et al., 2011). This leads to the phosphorylation and nuclear translocation of Elk-1 along with ERKs. Within the nucleus, Elk-1 binds to the SRE sites of IEGs (including c-fos, zif268, and arc/arg3.1) and recruits ERK1/2 and MSK-1 at the proximity of the nucleosome. In turn, MSK-1 can phosphorylate histone H3 on Ser10 residue, allowing nucleosome repositioning and transcription. These molecular events are critically involved in MSN spine plasticity and long-term behavioral alterations in response to cocaine. B, In the presence of the TDE peptide, Elk-1 phosphorylation and nuclear translocation is impeded. Cocaine-induced ERK and MSK-1 phosphorylation persist, but MSK-1 is not recruited to the nucleosome. SRE-driven gene regulation is inhibited, along with spine plasticity and long-term behavioral responses.