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Rho–Rho-Kinase Regulates Ras-ERK Signaling Through SynGAP1 for Dendritic Spine Morphology

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Abstract

The structural plasticity of dendritic spines plays a critical role in NMDA-induced long-term potentiation (LTP) in the brain. The small GTPases RhoA and Ras are considered key regulators of spine morphology and enlargement. However, the regulatory interaction between RhoA and Ras underlying NMDA-induced spine enlargement is largely unknown. In this study, we found that Rho-kinase/ROCK, an effector of RhoA, phosphorylated SynGAP1 (a synaptic Ras-GTPase activating protein) at Ser842 and increased its interaction with 14-3-3ζ, thereby activating Ras-ERK signaling in a reconstitution system in HeLa cells. We also found that the stimulation of NMDA receptor by glycine treatment for LTP induction stimulated SynGAP1 phosphorylation, Ras-ERK activation, spine enlargement and SynGAP1 delocalization from the spines in striatal neurons, and these effects were prevented by Rho-kinase inhibition. Rho-kinase-mediated phosphorylation of SynGAP1 appeared to increase its dissociation from PSD95, a postsynaptic scaffolding protein located at postsynaptic density, by forming a complex with 14-3-3ζ. These results suggest that Rho-kinase phosphorylates SynGAP1 at Ser842, thereby activating the Ras-ERK pathway for NMDA-induced morphological changes in dendritic spines.

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Data Availability

The datasets generated during this study are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful to T. Watanabe, T. Nishioka, K. Kuroda, S. Kozawa and other Kaibuchi laboratory members for helpful discussions and preparation of some materials, T. Ishii for secretarial assistance, and S. Furuta for life support. We also thank the Division for Research on Laboratory Animals, the Radioisotope Center Medical Branch and Medical Research Engineering of Nagoya University Graduate School of Medicine and the Education and Research Center of Animal Models for Human Diseases in Fujita Health University.

Funding

This work was supported by the following funding sources: ‘‘Bioinformatics for Brain Sciences’’ performed under the SRPBS from MEXT and AMED (KK); AMED Grant Nos. JP21dm0207075 (KK), JP21wm0425017 (YF); JSPS KAKENHI Grant Nos. JP17H01380 (KK), JP17J09461 (MW), JP17K07383 (MA), JP18K14849 (YF), JP21K06428 (YF), JP21K06427 (DT); MEXT KAKENHI Grant Nos. JP19H05209 (KK), JP21H00196 (KK); the Uehara Science Foundation (KK, YF), the Takeda Science Foundation (KK, YF), and the Hori Sciences & Arts Foundation (KK, YF).

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  1. Mengya Wu and Yasuhiro Funahashi have contributed equally.

Authors and Affiliations

  1. Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan

    Mengya Wu, Mutsuki Amano & Kozo Kaibuchi

  2. International Center for Brain Science, Fujita Health University, Toyoake, Aichi, 470-1129, Japan

    Yasuhiro Funahashi, Emran Hossen, Rijwan Uddin Ahammad, Daisuke Tsuboi & Kozo Kaibuchi

  3. Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan

    Tetsuya Takano

  4. PRESTO-JST, Kawaguchi, Saitama, 332-0012, Japan

    Tetsuya Takano

  5. Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

    Kiyofumi Yamada

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  1. Mengya Wu
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Contributions

Conceptualization: [MW, YF, TT, KK]; Methodology: [MW, YF, DT, MA]; Formal analysis and investigation: [MW, YF, EH, RUA, MA]; Writing—original draft preparation: [MW]; Writing—review and editing: [YF, KK]; Funding acquisition: [MW, YF, DT, MA, KK]; Resources: [YF, TT, DT, MA]; Supervision: [KY, KK]. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors approved the final version submitted.

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Correspondence to Kozo Kaibuchi.

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The authors have no relevant financial or non-financial interest to disclose.

Ethical Approval

All animal experiments were approved and performed in accordance with the guidelines for the care and use of laboratory animals established by the Animal Experiments Committee of Nagoya University Graduate School of Medicine (Approval Number: 20094) and Fujita Health University (Approval Number: AP20037). All experiments were conducted in compliance with the ARRIVE guidelines.

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11064_2022_3623_MOESM1_ESM.eps

Supplementary file1 (EPS 1653 kb)—Interaction of SynGAP1 with 14-3-3 proteins. Extracts of mouse striatum were incubated with rabbit IgG or anti-SynGAP1 antibody for 1 hour, and then with Protein A sepharose 4 Fast Flow beads for 1 hour. The immunoprecipitates were analyzed by immunoblotting with anti-SynGAP1, anti-14-3-3 (pan), anti-14-3-3β, anti-14-3-3γ, anti-14-3-3ε, 14-3-3η, 14-3-3σ, anti-14-3-3θ, and anti-14-3-3ζ, antibodies. Data represent the mean ± SEM of three independent experiments and were analyzed by Student's t-test. *P < 0.05, **P < 0.01, ns: not significant.

11064_2022_3623_MOESM2_ESM.eps

Supplementary file2 (EPS 1912 kb)—The effect of CaMKII on the interaction of SynGAP1 with PSD95. COS7 cell lysates expressing the indicated proteins were incubated with glutathione sepharose 4B beads. The bound proteins and cell lysates were subjected to immunoblot analysis using an anti-Myc, anti-GST, anti-GFP antibodies. The data represent the mean±SEM of three independent experiments and were analyzed by one-way ANOVA with Tukey's post hoc test. **p<0.01, ***p<0.001, ****p<0.0001.

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Wu, M., Funahashi, Y., Takano, T. et al. Rho–Rho-Kinase Regulates Ras-ERK Signaling Through SynGAP1 for Dendritic Spine Morphology. Neurochem Res 47, 2757–2772 (2022). https://doi.org/10.1007/s11064-022-03623-y

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