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Rho mediates calcium-dependent activation of p38α and subsequent excitotoxic cell death

Nature Neuroscience volume 10, pages 436–443 (2007)Cite this article

Abstract

Excitotoxic neuronal death contributes to many neurological disorders, and involves calcium influx and stress-activated protein kinases (SAPKs) such as p38α. There is indirect evidence that the small Rho-family GTPases Rac and cdc42 are involved in neuronal death subsequent to the withdrawal of nerve growth factor (NGF), whereas Rho is involved in the inhibition of neurite regeneration and the release of the amyloidogenic Aβ42 peptide. Here we show that Rho is activated in rat neurons by conditions that elevate intracellular calcium and in the mouse cerebral cortex during ischemia. Rho is required for the rapid glutamate-induced activation of p38α and ensuing neuronal death. The ability of RhoA to activate p38α was not expected, and it was specific to primary neuronal cultures. The expression of active RhoA alone not only activated p38α but also induced neuronal death that was sensitive to the anti-apoptotic protein Bcl-2, showing that RhoA was sufficient to induce the excitotoxic pathway. Therefore, Rho is an essential component of the excitotoxic cell death pathway.

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Figure 1: Glutamate-evoked activation of p38α SAPK is inhibited by the Rho inhibitor C3-exoenzyme.
Figure 2: Excitotoxic stress activates Rho in cerebellar granule neuron cultures and in mouse brain.
Figure 3: Rho activates p38α in cerebellar granule neurons but not Neuro2A neuroblastoma cells.
Figure 4: The Rho inhibitor C3-exoenzyme inhibits glutamate-evoked neuronal cell death.
Figure 5: Involvement of Rho in excitotoxic signaling and cell death in hippocampal and cortical neurons.
Figure 6: FRET-based reporter of Rho activity reveals rapid glutamate-evoked activation of Rho that depends on NMDA receptors.
Figure 7: Concentration dependence of glutamate-evoked changes in cytoplasmic free calcium, Rho response, p38 activation and pyknosis.
Figure 8: Depolarization activates Rho in a manner that depends on extracellular calcium.

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Acknowledgements

We thank M. Matsuda (University of Tokyo), B.J. Mayer (University of Connecticut Health Center), A. Miyawaki, L.A. Quilliam (Indiana University School of Medicine), J.M. Kyriakis (Tufts University School of Medicine), A. Hall (University College London), S. van den Heuvel (Massachusetts General Hospital Cancer Center), T. Wieland (Universitätsklinikum Hamburg-Eppendorf), M. Negishi (US National Institute of Environmental Health Sciences), H.A. Singer (Albany Medical College), M. Jäättelä (Danish Cancer Society) and J. Ellenberg (European Molecular Biology Laboratory) for providing plasmids used in this study. This work was funded by grants from the Academy of Finland (grants 72446, 78232, 203520, 206903 and 110445), the EU 6th Framework programme, the AIVI graduate school, the K. Albin Johansson Foundation and the University of Kuopio. M.J.C. is an Academy of Finland researcher.

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  1. Maria M Semenova, Anu M J Mäki-Hokkonen and Jiong Cao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, A.I. Virtanen Institute, University of Kuopio, Kuopio, FIN 70211, Finland

    Maria M Semenova, Anu M J Mäki-Hokkonen, Jiong Cao, Vladislav Komarovski, K Marjut Forsberg, Milla Koistinaho & Michael J Courtney

  2. Medeia Therapeutics Ltd, Microkatu 1, Kuopio, FI-70211, Finland

    Milla Koistinaho

  3. Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, Turku, FIN-20521, Finland

    Eleanor T Coffey & Michael J Courtney

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All authors conducted experiments for this work and M.J.C. also wrote the manuscript.

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Supplementary information

Supplementary Fig. 1

Glutamate-induced calcium increase is not reduced by C3 exoenzyme treatment. (PDF 101 kb)

Supplementary Fig. 2

Glutamate leads to a slow reduction of GTP-loaded Rac1. (PDF 101 kb)

Supplementary Fig. 3

Activation of the p38 pathway induces neuronal pyknosis. (PDF 85 kb)

Supplementary Fig. 4

p115RhoGEF and CamKII pathways are not responisble for glutamate-evoked Rho activation and pyknosis. (PDF 150 kb)

Supplementary Fig. 5

Rho-dependent pyknosis requires the effector-interacting sequences in Rho loop6. (PDF 79 kb)

Supplementary Fig. 6

Scheme depicting the proposed role of Rho in regulation of p38α by glutamate. (PDF 80 kb)

Supplementary Table 1

The number of cell used in all cell counting and single cell FRET assays is shown for all figures. (PDF 9 kb)

Supplementary Methods (PDF 150 kb)

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Semenova, M., Mäki-Hokkonen, A., Cao, J. et al. Rho mediates calcium-dependent activation of p38α and subsequent excitotoxic cell death. Nat Neurosci 10, 436–443 (2007). https://doi.org/10.1038/nn1869

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