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Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs

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Abstract

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.

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References

  • Andrews PW (1984) Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol 103:285–293

    Article  CAS  PubMed  Google Scholar 

  • Bishop A, Hall A (2000) Rho GTPases and their effector proteins. Biochem J 348:241–255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F (1990) Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 27:229–237

    Article  CAS  PubMed  Google Scholar 

  • Block ML, Hong J-S (2005) Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol 76:77–98

    Article  CAS  PubMed  Google Scholar 

  • Block ML, Zecca L, Hong J-S (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69

    Article  CAS  PubMed  Google Scholar 

  • Brown GC, Neher JJ (2014) Microglial phagocytosis of live neurons. Nat Rev Neurosci 15:209–216

    Article  CAS  PubMed  Google Scholar 

  • Caron E, Hall A (1998) Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282:1717–1721

    Article  CAS  PubMed  Google Scholar 

  • Chimini G, Chavrier P (2000) Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat Cell Biol 2:E191–E196

    Article  CAS  PubMed  Google Scholar 

  • Cui G, Zuo T, Zhao Q, Hu J, Jin P, Zhao H, Jing J, Zhu J, Chen H, Liu B (2013) ROCK mediates the inflammatory response in thrombin induced microglia. Neurosci Lett 554:82–87

    Article  CAS  PubMed  Google Scholar 

  • Degasperi A, Birtwistle MR, Volinsky N, Rauch J, Kolch W, Kholodenko BN (2014) Evaluating strategies to normalise biological replicates of western blot data. PLoS ONE 9(1):e87293

    Article  PubMed  PubMed Central  Google Scholar 

  • Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140:918–934

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hackam DJ, Rotstein OD, Schreiber A, Zhang W, Grinstein S (1997) Rho is required for the initiation of calcium signaling and phagocytosis by Fcγ receptors in macrophages. J Exp Med 186:955–966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279:509–514

    Article  CAS  PubMed  Google Scholar 

  • Hanisch U-K, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394

    Article  CAS  PubMed  Google Scholar 

  • Hensel N, Rademacher S, Claus P (2015) Chatting with the neighbors: crosstalk between Rho-kinase (ROCK) and other signaling pathways for treatment of neurological disorders. Front Neurosci 9:198

    Article  PubMed  PubMed Central  Google Scholar 

  • Hiroki J, Shimokawa H, Higashi M, Morikawa K, Kandabashi T, Kawamura N, Kubota T, Ichiki T, Amano M, Kaibuchi K (2004) Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells. J Mol Cell Cardiol 37:537–546

    Article  CAS  PubMed  Google Scholar 

  • Kopp M, Liebscher T, Niedeggen A, Laufer S, Brommer B, Jungehulsing G, Strittmatter S, Dirnagl U, Schwab J (2012) Small-molecule-induced Rho-inhibition: NSAIDs after spinal cord injury. Cell Tissue Res 349:119–132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19:312–318

    Article  CAS  PubMed  Google Scholar 

  • Kubo T, Yamaguchi A, Iwata N, Yamashita T (2008) The therapeutic effects of Rho-ROCK inhibitors on CNS disorders. Ther Clin Risk Manag 4:605–615

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laurenzi MA, Arcuri C, Rossi R, Marconi P, Bocchini V (2001) Effects of microenvironment on morphology and function of the microglial cell line BV-2. Neurochem Res 26:1209–1216

    Article  CAS  PubMed  Google Scholar 

  • Lucin KM, Wyss-Coray T (2009) Immune activation in brain aging and neurodegeneration: too much or too little? Neuron 64:110–122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Minghetti L, Levi G (1998) Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide. Prog Neurobiol 54:99–125

    Article  CAS  PubMed  Google Scholar 

  • Mueller BK, Mack H, Teusch N (2005) Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov 4:387–398

    Article  CAS  PubMed  Google Scholar 

  • Neher JJ, Neniskyte U, Zhao J-W, Bal-Price A, Tolkovsky AM, Brown GC (2011) Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 186:4973–4983

    Article  CAS  PubMed  Google Scholar 

  • Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318

    Article  CAS  PubMed  Google Scholar 

  • Olazabal IM, Caron E, May RC, Schilling K, Knecht DA, Machesky LM (2002) Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcγR, phagocytosis. Curr Biol 12:1413–1418

    Article  CAS  PubMed  Google Scholar 

  • Orlando KA, Stone NL, Pittman RN (2006) Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Exp Cell Res 312:5–15

    Article  CAS  PubMed  Google Scholar 

  • Paquet-Durand F, Bicker G (2007) Human model neurons in studies of brain cell damage and neural repair. Curr Mol Med 7:541–554

    Article  CAS  PubMed  Google Scholar 

  • Paquet-Durand F, Tan S, Bicker G (2003) Turning teratocarcinoma cells into neurons: rapid differentiation of NT-2 cells in floating spheres. Dev Brain Res 142:161–167

    Article  CAS  Google Scholar 

  • Podrygajlo G, Tegenge MA, Gierse A, Paquet-Durand F, Tan S, Bicker G, Stern M (2009) Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell Tissue Res 336:439–452

    Article  CAS  PubMed  Google Scholar 

  • Raad M, El Tal T, Gul R, Mondello S, Zhang Z, Boustany R, Guingab J, Wang KK, Kobeissy F (2012) Neuroproteomics approach and neurosystems biology analysis: ROCK inhibitors as promising therapeutic targets in neurodegeneration and neurotrauma. Electrophoresis 33:3659–3668

    Article  CAS  PubMed  Google Scholar 

  • Ren X, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18:578–585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roloff F, Scheiblich H, Dewitz C, Dempewolf S, Stern M, Bicker G (2015) Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling. PLoS ONE 10:e0118536

    Article  PubMed  PubMed Central  Google Scholar 

  • Scheiblich H, Bicker G (2015a) Regulation of microglial migration, phagocytosis, and neurite outgrowth by HO-1/CO signaling. Dev Neurobiol 75:854–876

    Article  CAS  PubMed  Google Scholar 

  • Scheiblich H, Bicker G (2015b) Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia. Dev Neurobiol. doi:10.1002/dneu.22333 in press

    Google Scholar 

  • Schmandke A, Schmandke A, Strittmatter SM (2007) ROCK and Rho: biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist 13:454–469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Streit WJ, Walter SA, Pennell NA (1999) Reactive microgliosis. Prog Neurobiol 57:563–581

    Article  CAS  PubMed  Google Scholar 

  • Takeuchi H (2010) Neurotoxicity by microglia: mechanisms and potential therapeutic strategy. Clin Exp Neuroimmunol 1:12–21

    Article  CAS  Google Scholar 

  • Tegenge M, Roloff F, Bicker G (2011) Rapid differentiation of human embryonal carcinoma stem cells (NT2) into neurons for neurite outgrowth analysis. Cell Mol Neurobiol 31:635–643

    Article  CAS  PubMed  Google Scholar 

  • Tönges L, Koch J-C, Bähr M, Lingor P (2011) ROCKing regeneration: Rho kinase inhibition as molecular target for neurorestoration. Front Mol Neurosci 4:39

    Article  PubMed  PubMed Central  Google Scholar 

  • Vicente AM, Guillén MI, Alcaraz MJ (2001) Modulation of haem oxygenase-1 expression by nitric oxide and leukotrienes in zymosan-activated macrophages. Br J Pharmacol 133:920–926

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang C, Song S, Zhang Y, Ge Y, Fang X, Huang T, Du J, Gao J (2015) Inhibition of the Rho/Rho kinase pathway prevents lipopolysaccharide-induced hyperalgesia and the release of TNF-α and IL-1β in the mouse spinal cord. Sci Rep. 5:14553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zielasek J, Hartung H-P (1996) Molecular mechanisms of microglial activation. Adv Neuroimmunol 6:191–222

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant of the German Research Foundation: BI 262/16-2. The authors would like to thank Saime Tan for generating the human NT2 neurons and our colleagues from the Forschergruppe 1103 for their contributions regarding glial culture. We are grateful to Frank Roloff and Michael Stern for helpful discussions.

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Authors and Affiliations

  1. Division of Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, 30173, Hannover, Germany

    Hannah Scheiblich & Gerd Bicker

  2. Center for Systems Neuroscience, Hannover, Germany

    Gerd Bicker

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  1. Hannah Scheiblich
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  2. Gerd Bicker
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Correspondence to Gerd Bicker.

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Scheiblich, H., Bicker, G. Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs. Cell Mol Neurobiol 37, 461–473 (2017). https://doi.org/10.1007/s10571-016-0379-7

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  • DOI: https://doi.org/10.1007/s10571-016-0379-7

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