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The role of ventral striatal cAMP signaling in stress-induced behaviors

Nature Neuroscience volume 18, pages 1094–1100 (2015)Cite this article

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Abstract

The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum– or D1 dopamine receptor–specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression.

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Figure 1: Cdk5 regulates cAMP signaling via phosphorylation of PDE4.
Figure 2: Cdk5 cKO mice exhibit elevated cAMP, increased PKA activity, and altered behavioral responses in acute and chronic stress procedures.
Figure 3: Virus-mediated Cdk5 knockout in ventral striatum reduces PDE4 phosphorylation and alters stress-induced behaviors.
Figure 4: Development of a small interfering peptide that raises cAMP levels, increases PKA activity and reduces immobility time in the FST.

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Acknowledgements

We thank N. Heintz (Rockefeller University) and GenSat for D1R-Cre mice, K. Deisseroth (Stanford University) for the Dio-Cre vector, C. Burger (University of Wisconsin-Madison) for AAV vectors, H. Ball and Y. Li (University of Texas Southwestern (UTSW) Protein Technology Center) for peptides, the UTSW Animal Resource Center for help with phosphorylation state–specific antibody generation, D.M. Dietz, M. Lutter, M. Kouser and J. Kumar for help with the social defeat procedure, and T. Singh and G. Mettlach for technical assistance. We thank M. Trivedi and the UTSW Depression Center for support. This work was supported by a Brain and Behavior Research Foundation NARSAD Young Investigator Award (K.H.), a pre-doctoral National Research Service Award from the National Institute on Drug Abuse (D.R.B.), a grant from the Darrell K Royal Research Fund for Alzheimer's Research (F.P.) and the California Metabolic Research Foundation (M.D.H.), as well as by US National Institutes of Health grants to A.C.N. and P.G. (MH090963, DA10044), E.J.N. (MH51399), R.T. (GM084249) and J.A.B. (MH79710, MH083711, DA016672, DA018343, DA033485, NS073855).

Author information

Author notes

  1. Kanehiro Hayashi & David R Benavides

    Present address: Present addresses: Department of Anatomy, Keio University School of Medicine, Tokyo, Japan (K.H.), and Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA (D.R.B.).,

Authors and Affiliations

  1. Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Florian Plattner, Kanehiro Hayashi, Adan Hernández, David R Benavides, Tara C Tassin, Chunfeng Tan, Matthew S Goldberg & James A Bibb

  2. Division of Neuroscience and Molecular Pharmacology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK

    Jonathan Day

  3. Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Maggy W Fina & Ronald Taussig

  4. Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York, USA

    Eunice Y Yuen & Zhen Yan

  5. Department of Neurology and Neurotherapeutics, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Matthew S Goldberg & James A Bibb

  6. Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA

    Angus C Nairn

  7. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, USA

    Angus C Nairn & Paul Greengard

  8. Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Eric J Nestler

  9. Department of Pharmacology, Kurume University School of Medicine, Fukuoka, Japan

    Akinori Nishi

  10. Institute of Pharmaceutical Science, King's College London, London, UK

    Miles D Houslay

  11. Harold C. Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    James A Bibb

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  1. Florian Plattner
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Contributions

F.P., K.H., D.R.B., A.H., T.C.T., C.T., J.D., M.W.F., E.Y.Y., M.S.G. and A.N. collected data and analyzed the experiments. F.P., Z.Y., A.C.N., E.J.N., A.N., P.G., R.T., M.D.H. and J.A.B. contributed to study design, supervision and interpretation of the experiments. F.P. and J.A.B. wrote the manuscript.

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Correspondence to James A Bibb.

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Integrated supplementary information

Supplementary Figure 1 PDE4 phosphorylation state-specific antibody generation, distribution of PDE4 in brain, and cGMP levels in striatal slices.

(a) Identification of the Cdk5-dependent phosphorylation site on PDE4B1 by LC-LC MS analysis. The presence of a single charged peptide (SDSDYDLpSPK) indicates the site of phosphorylation at amino acid residue Ser145 of the rat PDE4B1 sequence, with “pS” denoting the position of the phospho-serine. The spectrum depicts the y-ion series as a vertical line in the peptide sequence and corresponding site of y-ion generation (i.e. y1, y2, etc.). (b) Time-course of in vitro phosphorylation of recombinant PDE4B1 by PKA. Time-dependent 32P incorporation, Coomassie-stained (CBB) PDE4B1, and quantified reaction stoichiometry are shown. (c) PDE4 family member and Cdk5 brain tissue distribution. Immunoblots with pan-specific PDE4A, 4B, and 4D antibodies are shown with individual isoforms indicated. Blots for Cdk5 and GAPDH are also shown. ob, olfactory bulb; ctx, cortex; str, striatum; nac, nucleus accumbens; hip, hippocampus; cer, cerebellum. (d) Validation of phosphorylation state-specific antisera to phospho-Ser133 PDE4B1 [pPDE4 (PKA)] phosphorylated by PKA. Dot blot analysis to confirm phosphorylation state-specificity of antibody. Selective detection of phospho- versus dephospho-peptide with corresponding Pyronin Y staining of total peptide is shown. (e) Immunoblot of lysates from striatal slices incubated in the absence (Control) or presence of forskolin (10 µM, 10 min) probed with pPDE4 (PKA) antibody. (f) Validation of phosphorylation state-specific antisera to phospho-Ser145 PDE4B1 [pPDE4 (Cdk5)] phosphorylated by Cdk5. Immunoblot analysis of recombinant PDE4B1 phosphorylated (+) or mock phosphorylated (–) in vitro with Cdk5 using pPDE4 (Cdk5) versus total PDE4 antibodies (top panel). Blots of PC12 cells transfected with PDE4B1 treated with control buffer or Indo A (10 µM, 60 min) are shown in the bottom panel. (g) Cdk5 inhibition with indolinone A (IndoA, 10 µM) does not affect cGMP levels in the absence or presence of sodium nitroprusside (SNP), an activator of guanylate cyclases, in striatal slices (n = 4–8. All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 2 Reciprocal relationship of Cdk5 and PKA activity in striatum.

(a) Evaluation of PKA-mediated phosphorylation in response to treatment of striatal slices with 50 µM roscovitine. Representative blots for PKA-mediated phospho-Ser133 (pS133) CREB, phospho-Ser9 (pS9) synapsin, phospho-Thr34 (P-T34) DARPP-32, phospho-Ser845 (pS845) GluR1, and respective total protein are shown with quantification (n = 3–6). (b) The dose-dependent, reciprocal relationship between PKA- and Cdk5-dependent phosphorylation of DARPP-32. Immunoblots (left) of lysates from mouse striatal slices treated with the Cdk5 inhibitor roscovitine (0–50 µM, 60 min) for phospho-Thr75 (P-T75), phospho-Thr34 (P-T34), and total DARPP-32 (D-32) are shown with quantification (right). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 3 Decreased Cdk5 activity and PDE4 phosphorylation in Cdk5 cKO.

(a) Immunoprecipitation Cdk5 kinase activity assay using striatal lysate from WT versus Cdk5 cKO. The effect of treatment of immunoprecipitate from control samples with Cdk5 inhibitor roscovitine (Ros, 10 μM) is also shown. Radiographic and Coomassie stained gels with quantification are shown (n = 6–9). (b, c) PDE4 phosphorylation levels at the Cdk5 (b) and PKA sites (c) in lysates of ventral striatum from Cdk5 cKO and control WT mice (n = 4). (d) Levels of cGMP are unaltered in striatum from Cdk5 cKO and control mice (n = 4-5). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 4 PKA regulation of NMDA receptor currents is altered in Cdk5 cKO mice.

(a) Plot of normalized peak NMDA receptor currents showing the effect of PKI (0.2 µM, myristoylated) in striatal neurons isolated from WT and Cdk5 cKO mice. (b) Representative NMDA receptor current traces from data of panel a). (c) Cumulative data showing the percentage reduction of NMDA receptor currents by PKI in neurons from WT and Cdk5 cKO mice (n = 9–10). (d-f) Dot plots showing the NMDA receptor current amplitudes (d), capacitance (e), and current densities (f) in neurons from WT and Cdk5 cKO mice (n = 12). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 5 Cdk5 cKO exhibit reduced spine density in medium spiny neurons and altered behavioral responses to stress.

(a) Dendritic spine density of medium spiny neurons in the ventral striatum is decreased in Cdk5 KO mice as compared to controls (n = 14–15 cells from four mice per genotype). (b) No change in spine types, such as thin, mushroom, stubby was observed between genotypes. (c) The effect of Cdk5 cKO on social defeat. Social avoidance was assessed by time spent interacting with the social interaction target. Graphs depict time spent in interaction zone (left) and corner zones (right) in present (Target) or absence (No Target) of interactor mouse (n = 7–13). (d) Assessment of cage activity in Cdk5 cKO and control mice (n = 13–14). (e) The effect of Cdk5 cKO on social interaction. Graphs depict time spent in interaction zone (left) and corner zones (right) in presence (Target) or absence (No Target) of interactor mouse (n = 17–24). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 6 Forced swim test, social defeat and chronic unpredictable stress induce changes in PDE4 phosphorylation

(a,b) Quantitative immunoblot analysis of ventral striatum lysates from mice 1 h after FST (n = 4) compared to non-swim controls (n = 4) for pPDE4 (PKA) and pPDE4 (Cdk5). (c,d) Analysis of ventral striatum lysates from chronic socially defeated mice (n = 10) and non-defeated controls (n = 6) 1 h after social interaction testing for pPDE4 (PKA) and pPDE4 (Cdk5). (e,f) Analysis of ventral striatum lysates mice undergoing chronic unpredictable stress (n = 6) and non-stressed controls (n = 5) for pPDE4 (PKA) and pPDE4 (Cdk5). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 7 Behavioral characterization of virus-mediated Cdk5 knockout in ventral striatum.

(a) Immunostain showing Cdk5 knockout in the ventral striatum induced by stereotactic bilateral infusion of AAV2-Cre into the NAc of homozygous floxed Cdk5 (fl/fl AAV) and WT mice (WT AAV). Arrowheads at the border of viral transduction fields indicate locations of inserts (right). (b) Assessment of cage activity in fl/fl AAV and WT AAV (n = 7–8). (c,d) Elevated plus maze performance of fl/fl AAV and WT AAV (n = 7–8) indicating time in zones (c) and locomotor activity (d). (e) Social interaction (SI) in fl/fl AAV and WT AAV (n = 10). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 8 Reduced PDE4 phosphorylation in striatum of D1R-Cdk5-KO.

(a) Immunostain showing D1 dopamine receptor promoter-driven Cre expression via a GFP reporter (top) and Cdk5 loss in medium spiny neurons of ventral striatum (encircled; bottom). (b) Reduced Cdk5 expression in striatal lysates from D1R-Cdk5-KO (n = 6). (c,d) Quantitative immunoblot analysis of ventral striatum lysates from D1R-Cdk5-KO mice and controls (n = 6) for pPDE4 (PKA) (c) and (Cdk5) (d). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 9 Cdk5 knockout in D1 dopamine receptor neurons alters stress-induced behavioral responses.

(a,b) D1R-Cdk5-KO exhibit reduced immobility and increased latency to initiation of floating in the FST (n = 9–16) (a) and increased time struggling in the TST (n = 12–14) (b). (c) Effect of D1R-Cdk5-KO on social defeat. Social avoidance was assessed by time spent interacting with the social target. Graphs depict time spent in interaction zone (left) and corner zones (right) in presence (Target) or absence (No Target) of interactor mouse (n = 8–10). (d) Locomotor activity during social interaction testing (n = 8–10). (e) Social interaction (SI) in D1R-Cdk5-KO (n = 10). (f,g) Elevated plus maze performance of D1R-Cdk5-KO and control mice (n = 7) indicating time in zones (f) and locomotor activity (g). (h) Assessment of cage activity in D1R-Cdk5-KO and control mice (n = 12–13). (i) Evaluation of locomotor activity and center times in the open field assay for D1R-Cdk5-KO and control mice (n = 9–10). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 10 Increased water intake in D1R-Cdk5-KO mice.

(a) Effect of Cdk5 knockout in the D1R-Cdk5-KO mouse lines on sucrose preference test (SPT), a test of anhedonia that does not rely on locomotor activity. Graph depicts sucrose preference for D1R-Cdk5-KO (n = 10) and control mice (n = 15). (b) Total liquid consumption per day measured during water, sucrose and choice (water and sucrose) phase of the SPT. (c) Normalized intake volumes for each bottle during water and sucrose phase show no bias. (d) D1R-Cdk5-KO mice exhibit normal water intake during the night cycle, but have increased water intake during the day cycle (n = 5–6). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 11 Reversal of forced swim test phenotype by PKA inhibition in D1R-Cdk5-KO and Cdk5 cKO.

(a,b) Effect of infusion of the PKA inhibitor Rp-cAMPs into ventral striatum of D1R-Cdk5-KO (a) and Cdk5 cKO (b) as assessed by the two-trial FST (n = 6–10). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 12 In vitro and in vivo assessment of PDE4-siP specificity and its effect on PDE4 phosphorylation.

(a) Effect of the D1 dopamine receptor agonist SKF81297 (SKF), PDE4-siP, or both on the defined Cdk5 site, phospho-Thr75 DARPP-32 (pT75 D-32). †P < 0.05 compared to SKF81297 only; one-way ANOVA and Newman-Keuls test, (n = 4–8). (b) Evaluation of cage activity in mice infused with PDE4-siP (n = 5) and scrambled peptide (n = 9) before (40 min) and after (30 min) FST. (c) Effect of infusion of the PDE4 peptide with an N-terminal poly-arginine (R7) cell membrane permeablizing tag into NAc on the two-trial FST (n = 5–7). (d) Effect of infusion of peptides, in which the PKA (S133A; n = 8), Cdk5 (S145A; n = 8), or both serine sites (S133A/S145A; n = 8) were mutated to alanine, into NAc on the two-trial FST (n = 5–10) and compared to infusions with PDE4-siP (n = 9) and scrambled peptide (n = 7). (e) Infusion of the PDE4-siP into the ventral striatum has no impact on PDE4 phosphorylation levels at the Cdk5 and PKA sites within the dorsal striatum (n = 5–6). All data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 13 Whole blot images of the immunoblots presented in the main figures

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Plattner, F., Hayashi, K., Hernández, A. et al. The role of ventral striatal cAMP signaling in stress-induced behaviors. Nat Neurosci 18, 1094–1100 (2015). https://doi.org/10.1038/nn.4066

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