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RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation

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Abstract

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A2 (cPLA2), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA2. Inhibition of RhoA, Rho kinase and cPLA2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA2. The immunofluorescence staining showed that ROCK1 or ROCK2, two isoforms of Rho kinase, was co-localized with cPLA2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK1 or ROCK2 bonded directly with cPLA2 and phospho-cPLA2. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA2 activation.

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Acknowledgments

This work was supported in part by NIH NS059622, NS073636, DOD CDMRP W81XWH-12-1-0562, Merit Review Award I01 BX002356 from the US Department of Veterans Affairs, Craig H Neilsen Foundation 296749, Indiana Spinal Cord and Brain Injury Research Foundation and Mari Hulman George Endowment Funds (XMX), and by the State of Indiana ISDH, Grant # 13679 (XW).

Author Contributions

XW designed, performed experiments, analyzed data and wrote manuscript; CW designed and wrote manuscript; QL, WW, DE, JP performed experiments; XMX designed experiments and wrote the manuscript.

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Authors and Affiliations

  1. Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 W. 15th Street, NB 500E, Indianapolis, IN, 46202, USA

    Xiangbing Wu, Chandler L. Walker, Qingbo Lu, Wei Wu & Xiao-Ming Xu

  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Xiangbing Wu, Chandler L. Walker & Xiao-Ming Xu

  3. Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Xiangbing Wu, Chandler L. Walker, Qingbo Lu, Wei Wu, Daniel B. Eddelman, Jonathan M. Parish & Xiao-Ming Xu

  4. Goodman Campbell Brain and Spine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Xiangbing Wu, Chandler L. Walker, Qingbo Lu, Wei Wu & Xiao-Ming Xu

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  1. Xiangbing Wu
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Wu, X., Walker, C.L., Lu, Q. et al. RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation. Mol Neurobiol 54, 6885–6895 (2017). https://doi.org/10.1007/s12035-016-0187-6

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