PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons

Abstract

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

Keywords: d-amphetamine; dopamine; protein synthesis; ribosomal protein S6; striatum.

Figure 1.

Acute d-amph increases rpS6 phosphorylation in whole and synaptoneurosomal fractions of the striatum. A, B, Quantified (top) and representative (bottom) Western blot data of phosphorylated rpS6 at S235/236 sites in the striatum of mice treated acutely (A) or chronically (once daily for 5 d) (B) with d-amph (10 mg/kg) and killed at different time points after injection. Data are expressed as a percentage of untreated control (n = 6 mice/group). Phosphorylated rpS6 levels were normalized to rpS6. Results are represented as mean ± SEM. *p < 0.05, **p < 0.01, by one-way ANOVA followed by Newman–Keuls test (d-amph vs saline). C, Double immunostaining for pS235/236-rpS6 (red) and TH (cyan) in coronal rostral (bregma 1.1 mm) and caudal (bregma −1.7 mm) sections of the striatum from mice receiving a single injection of saline or d-amph (10 mg/kg) and perfused 15 min after injection. Scale bar, 400 μm. D, High-magnification image of pS235/236-rpS6 immunostaining in striatal coronal sections from mice treated with d-amph (10 mg/kg) and killed 15 min after injection. Black arrowheads indicate pS236/236-rpS6 in dendrites. Note that, to detect dendritic pS235/236-rpS6, the staining of the cell body was saturated. Scale bar, 10 μm. E, Representative Western blot of the glial (GFAP), nuclear (H3), presynaptic (TH and synaptophysin, Syp), and postsynaptic markers (PSD95 and mGluR5) in input (I) and synaptoneurosome (S) fractions of the striatum. F, Western blot of pS235/236-rpS6 (normalized to rpS6) of the striatal synaptoneurosome preparation from mice that received a single injection of saline or d-amph (10 mg/kg) and were killed at 15 min after injection. Results are represented as mean ± SEM. **p < 0.01 by Student's t test (n = 6 mice/group). DStr, Dorsal striatum; Acb, nucleus accumbens; Cx, cortex; GPe, external globus pallidus; cc: corpus callosum; ac, anterior commissure.

Figure 2.

d-amph-induced rpS6 phosphorylation in D1R-containing MSNs. A, Triple immunostaining for pS235/236-rpS6 (red), Calb-D28k (magenta), and MOR (cyan) in coronal sections of the striatum from mice treated with d-amph (10 mg/kg) and perfused 15 min after injection. Scale bar, 400 μm. Higher-magnification images of each single staining are shown. Scale bar, 150 μm. B, Double immunostaining for pS235/236-rpS6 and GFP in coronal sections of the striatum from Drd1a- (i) and Drd2-EGFP (ii) mice treated with d-amph (10 mg/kg) and killed 15 min after injection. Yellow arrowheads indicate GFP/pS235/236-rpS6-positive MSNs. C, Double immunostaining for pS235/236-rpS6 and ChAT (i), Parvalbumin (ParV) (ii), nNOS (iii), and calretinin (CalR) (iv) in coronal sections of the striatum from mice treated with d-amph (10 mg/kg) and perfused 15 min after injection. Scale bar in B and C, 35 μm. DStr, Dorsal striatum; Acb, nucleus accumbens; cc, corpus callosum; ac, anterior commissure; v, ventricle.

Figure 3.

d-amph-induced rpS6 phosphorylation depends on D1R but not NMDAR and mGluR1/5-R stimulation. A, pS235/236-rpS6 immunostaining of striatal coronal slices from mice perfused 15 min after d-amph (10 mg/kg) and pretreated 30 min before with saline (left) or the D1/D5-R antagonist SCH23390 (0.15 mg/kg) (right). Scale bar, 800 μm. B–F, Quantified (top) and representative (bottom) Western blot analyses of pS235/236-rpS6 (normalized to rpS6) from mice killed 15 min after d-amph (10 mg/kg) treatment and pretreated 30 min before with SCH23390 (0.15 mg/kg; B), the NMDAR antagonist MK801 (0.1 mg/kg; C), the NR2B subunit of NMDAR antagonist Ro 25–6981 (10 mg/kg; D), the mGluR5 antagonist MPEP (15 mg/kg; E), or the mGluR1 antagonist YM298198 (YM, 15 mg/kg; F). Results are represented as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 (d-amph vs saline), °°p < 0.01 (antagonist pretreatment vs d-amph). DStr, Dorsal striatum; Acb, nucleus accumbens; Cx, cortex; cc, corpus callosum; ac, anterior commissure.

Figure 4.

d-amph-induced rpS6 phosphorylation is independent of mTORC1/p70S6K1/2, PKC and ERK signaling. A, Representative Western blots (left) and quantified signals (right) of whole striatal lysates from mice killed 15 min after saline or d-amph (10 mg/kg) treatment. The level of each phospho-protein was normalized to the respective total protein. Results are represented as mean ± SEM (n = 6 mice/group). B, Quantified (top) and representative (bottom) Western blot analyses of pS235/236-rpS6 (normalized to rpS6) from mice killed 15 min after d-amph (10 mg/kg) treatment and pretreated with the mTORC1 inhibitor rapamycin (2 injections of 10 mg/kg, 3 h and 30 min prior d-amph). Results are represented as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 6 mice/group). *p < 0.05 (d-amph vs saline), #p < 0.05 (rapamycin vs vehicle). C, Quantified (top) and representative Western blots (bottom) for pS235/236-rpS6 (normalized to rpS6) of whole striatal lysates from p70S6K1/2 double-knock-out (KO) mice that received saline or d-amph (10 mg/kg) and were killed 15 min after injection. Data are expressed as a percentage of saline-treated mice. Results are represented as mean ± SEM. *p < 0.05 by Student's t test (n = 4 mice/group). D, Quantified (top) and representative (bottom) Western blot analyses of pS235/236-rpS6 (normalized to rpS6) from mice killed 15 min after d-amph (10 mg/kg) treatment and pretreated with the PKC inhibitor NPC 15437 (NPC, 8 mg/kg, 30 min prior d-amph). E–H, Quantified (top) and representative (bottom) Western blot analyses of pT185/Y187-ERK2 (E), pS235/236-rpS6 (F), pS209-eIF4E (G), and pS10–H3 (H) (all normalized to the respective total form) from mice killed 15 min after d-amph (10 mg/kg) treatment and pretreated with the MEK inhibitor SL327 (50 mg/kg, 60 min before d-amph). Results are represented as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 (d-amph vs control), °p < 0.05, °°p < 0.01, °°°p < 0.001 (drug + d-amph vs vehicle + d-amph).

Figure 5.

Gαolf/cAMP/PKA signaling regulates striatal rpS6 phosphorylation. A, pS235/236-rpS6 immunostaining of striatal parahorizontal slices from wild-type or Gαolf ^+/− mice perfused 15 min after saline or d-amph (10 mg/kg) treatment. Scale bar, 450 μm. Insets, High magnification of the areas delineated by the yellow stippled rectangle. Yellow arrowheads indicate ChAT interneurons. Scale bar, 30 μm. DStr, Dorsal striatum; Cx, cortex; GPe, external globus pallidus; R, rostral; C, caudal; M, medial; L, lateral. B, Quantification of pS235/236-rpS6-positive cells in an equal-sized region of interest of the striatal parahorizontal slices from wild-type or Gαolf^+/− mice perfused 15 min after saline or d-amph (10 mg/kg) treatment. Results are represented in as a mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 3–4 mice/group). ***p < 0.001 (d-amph vs control), °°°p < 0.001 (wild-type vs Gαolf^+/− mice).

Figure 6.

Inhibition of cAMP degradation by papaverine increases striatal rpS6 phosphorylation independently of mTORC1/p70S6K1/2 signaling. A, Representative (top) and quantified (bottom) Western blot data of pS235/236-rpS6 (normalized to rpS6) in whole striatal lysates from mice that received a single injection of saline or papaverine (30 mg/kg) and were killed at 15 min after injection. Results are represented as mean ± SEM. *p < 0.05 by Student's t test (n = 5 mice/group). B, Triple immunostaining for pS235/236-rpS6 (red), GFP (cyan) and nNOS (magenta) in striatal coronal sections from Drd2-EGFP mice treated with papaverine (30 mg/kg) and perfused 15 min after injection. Scale bar, 400 μm. Higher-magnification images of each single staining are shown. Scale bar, 150 μm. Note the lack of costaining between pS235/236-rpS6 and ChAT-positive interneurons (yellow arrowhead). C, Representative Western blots (left) and quantified signals (right) of whole striatal lysates from mice killed 15 min after saline or papaverine (30 mg/kg) treatment. The level of each phosphoprotein was normalized to the respective total protein. Results are represented as mean ± SEM (n = 5 mice/group). D, Representative (top) and quantified (bottom) Western blot data of pS235/236-rpS6 (normalized to rpS6) of whole striatal lysates from p70S6K1/2 double knock-out (KO) mice that received saline or papaverine (30 mg/kg) and were killed at 15 min after injection. Data are expressed as a percentage of saline-treated mice. Results are represented as mean ± SEM. **p < 0.01 by Student's t test (n = 4 mice/group). E, Representative Western blots of pT185/Y187-ERK2, ERK2, pS235/236-rpS6, and rpS6 from mice killed 15 min after papaverine (30 mg/kg) treatment and pretreated with vehicle or the MEK inhibitor SL327 (50 mg/kg, 60 min before papaverine). F, G, Quantified Western blot analyses of pT185/Y187-ERK2 (F) and pS235/236-rpS6 (G) (all normalized to the respective total form) from mice killed 15 min after papaverine (30 mg/kg) treatment and pretreated with the MEK inhibitor SL327 (50 mg/kg, 60 min prior papaverine). Results are represented as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 6 mice/group). ##p < 0.01 < 0.01 (SL327 vs vehicle) ***p < 0.001 (papaverine vs control), °°°p < 0.001 (vehicle + papaverine vs SL327 + papaverine). DStr, Dorsal striatum; Acb, nucleus accumbens; ac, anterior commissure.

Figure 7.

Stimulation of cAMP production by forskolin increases striatal rpS6 phosphorylation through PKA signaling. A, B, Representative (top) and quantified (bottom) Western blot data of the effect of the PKA inhibitor Rp-cAMP on forskolin (Fsk) (0.1 or 1 μM) induced pS235/236-rpS6 (normalized to rpS6) (A) and pT34-DARPP-32 (normalized to DARPP-32) compared with control (Ct) group in mouse striatal slices. Results are represented as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 3). **p < 0.01, ***p < 0.001 (Fsk vs Ct), °p < 0.05, °°p < 0.01 (Fsk vs Fsk + Rp-cAMP). C, Representative Western blot analyses of the effect of the MEK inhibitor U0126 on forskolin (Fsk) (0.1 or 1 μM)-induced pT34-DARPP-32, pT202/Y204/T185/Y187-ERK1/2, and pS235/236-rpS6 (normalized to their respective total forms) compared with Ct group in mouse striatal slices.

Figure 8.

d-amph-induced rpS6 phosphorylation in MSNs requires inhibition of PP-1. A, pS235/236-rpS6 immunostaining in striatal parahorizontal slices from wild-type or DARPP-32 T34A mice perfused 15 min after saline or d-amph (10 mg/kg) treatment. Scale bar, 450 μm. Insets, High magnification of the areas delineated by the yellow stippled rectangle. Yellow arrowheads indicate ChAT-positive interneurons. Scale bar, 30 μm. DStr, Dorsal striatum; Cx, cortex; GPe, external globus pallidus; R, rostral; C, caudal; M, medial; L, lateral. B, Quantification of pS235/236-rpS6-positive cells in an equal-sized region of interest of the striatal parahorizontal slices from wild-type or DARPP-32 T34A knock-in mice (DARPP-32 T34A ki) perfused 15 min after saline or d-amph (10 mg/kg) treatment. Results are represented in as mean ± SEM and by one-way ANOVA followed by Newman–Keuls test (n = 3–4 mice/group). ***p < 0.001 (d-amph vs control), °°°p < 0.001 (wild-type vs DARPP-32 T34A ki). C, Diagram depicting the signaling pathway involved in rpS6 phosphorylation in the striatum in response to acute d-amph administration. d-amph through the stimulation of D1R, but not NMDAR or mGluR1/5Rs, induces rpS6 phosphorylation in D1R-expressing MSNs. D1R coupled G-protein Gαolf triggers adenylyl cyclase (AC) activity and thus stimulates cAMP signaling. Stimulation of cAMP production by forskolin or inhibition of cAMP degradation by papaverine (a PDE10a inhibitor) is sufficient to increase rpS6 phosphorylation in the striatum. Increased cAMP levels lead to the activation of PKA, which phosphorylates DARPP-32 at T34 site. In turn, DARPP-32 inhibits PP-1 and promotes rpS6 phosphorylation via the suppression of its dephosphorylation. The studied phenomenon is independent of mTORC1/p70S6K1/2, ERK/p90RSK, and PKC signaling.

Figure 9.

d-amph-induced rpS6 phosphorylation is uncorrelated with global translation. A, Polysome profiles of whole striatal lysates from mice treated with saline and d-amph (10 mg/kg) and killed at 1 h after injection (n = 6 mice/group). B, Representative Western blot analyses of puromycin staining (pmy) of whole striatal lysates incubated 10 min with puromycin or not. C, Representative and quantified Western blot analyses of puromycin staining (normalized to β-actin) of whole striatal lysates incubated 10 min with puromycin from mice killed 1 h after intrastriatal infusion of saline or harringtonin (harr, 10 μg; n = 8 mice/group). D, Representative (left) and quantified (right) Western blot analyses of puromycin staining (pmy, normalized to β-actin) of whole striatal lysates incubated 10 min with puromycin from mice killed 1 or 2 h after saline or d-amph (10 mg/kg) treatment (n = 7 mice/group). Results are represented as mean ± SEM. E, Quantified Western blot signals were expressed as a correlation between the percentage of increase in pmy normalized to β-actin and the percentage of increase in pS235/236-rpS6 normalized to rpS6. F, Representative (left) and quantified (right) Western blot analyses of striatal synaptoneurosomal preparations incubated 10 min with puromycin of mice killed 1 h after saline or d-amph (10 mg/kg) administration. Results are represented as mean ± SEM (n = 8 mice/group). G, Validation of anti-puromycin antibody specificity. Immunofluorescence for GFP (cyan) and pmy (magenta) of Drd2-EGFP mice perfused 5 min after vehicle (upper) or pmy infusion (27 μg, i.c.v.) (bottom). Scale bar, 100 μm. H, Triple immunostaining for GFP (cyan), pmy (magenta) and pS235/236-rpS6 (red) in coronal sections of the striatum from Drd2-EGFP mice that were pretreated with saline or d-amph 15 min before pmy infusion (27 μg, i.c.v., n = 4 mice/treatment) and killed 5 min later. Note the presence of some GFP-expressing MSNs, which are also positive (red arrowhead) or negative (green arrowhead) for pmy and the presence of some pS235/236-rpS6-stained MSNs, which are also positive (white arrowhead) or negative (yellow arrowhead) for pmy. Scale bar, 20 μm. I, Triple immunostaining for GFP (cyan), pmy (magenta), and DARPP-32 (red) in coronal sections of the striatum from Drd2-EGFP mice that were pretreated with saline or d-amph 15 min before pmy infusion (27 μg, i.c.v., n = 4 mice/treatment) and killed 5 min later. Note that pmy staining was observed in D2- and D1-MSNs, in ChAT-positive interneurons (yellow arrowhead), and most likely in other classes of interneurons (white arrowheads). Scale bar, 10 μm. J, Quantification of pmy-positive cells (pmy +, filled bars) in DARPP-32-positive/GFP-negative (D1-MSNs) and DARPP-32-positive/GFP-positive (D2-MSNs) cells counted in coronal sections of Drd2-EGFP mice pretreated with saline or d-amph.

Figure 10.

d-amph-induced rpS6 phosphorylation is not correlated with TOP mRNA translation. A, B, Triple immunostaining for GFP (cyan), DARPP-32 (red), and rpS6 (blue) (A) or eEF1A (blue) (B) in coronal sections of the striatum from Drd2-EGFP mice that were pretreated with saline or d-amph 15 min before perfusion. Yellow arrowheads indicate ChAT-positive interneurons and white arrowheads most likely correspond to GABAergic interneurons. Scale bar, 30 μm. C, D, Quantified Western blot analyses of eEF1A and rpS6 in crude homogenate 15, 30, or 60 min after acute saline or d-amph (10 mg/kg) administration (C) or 15 and 30 after chronic saline or d-amph (10 mg/kg) administration (D) (n = 6 mice/group). E, Quantified Western blot analyses of eEF1A and rpS6 in synaptoneurosomes 60 min after saline or d-amph (10 mg/kg) administration (n = 6 mice/group).

Figure 11.

Haloperidol- and papaverine-induced rpS6 phosphorylation is not correlated with global and TOP mRNA translation. A, Representative (left) and quantified (right) Western blot analyses of puromycin staining (pmy, normalized to β-actin) of whole striatal lysates incubated 10 min with puromycin from mice killed 1 h after vehicle or haloperidol (0.5 mg/kg) treatment (n = 6 mice/group). B, Quantified Western blot analyses of eEF1A and rpS6 1 h after vehicle or haloperidol (0.5 mg/kg) administration (n = 5–6 mice/group). C, Representative (left) and quantified (right) Western blot analyses of puromycin staining (pmy, normalized to β-actin) of whole striatal lysates incubated 10 min with puromycin from mice killed 1 h after saline or papaverine (30 mg/kg) treatment (n = 5–6 mice/group). D, Quantified Western blot analyses of eEF1A and rpS6, 15 min after saline or papaverine (30 mg/kg) administration (n = 5–6 mice/group). Results are represented as mean ± SEM and were analyzed by Student's t test.